[3-11C]Pyruvate PET detects alterations in cardiac pyruvate metabolism induced by doxorubicin chemotherapy

[3-11C]Pyruvate PET detects alterations in cardiac pyruvate metabolism induced by doxorubicin chemotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article [3- 11 C]Pyruvate PET detects alterations in cardiac pyruvate metabolism induced by doxorubicin chemotherapy Chul-Hee Lee, Thomas Ruan, Shuvra Debnath, Anja S. Wacker, Grace Figlioli, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7465913/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Changes in cardiac metabolism typically precede cardiac dysfunction and therefore represent an important target for diagnosis and treatment designed to prevent progression to heart failure, a leading cause of death. Profound changes in pyruvate metabolism, including reduced expression of the mitochondrial pyruvate carrier (MPC), are increasingly recognized as early maladaptive alterations in cardiomyopathies, but no methods currently exist to determine MPC expression in vivo. We exposed mice to doxorubicin (DOX), an anthracycline chemotherapeutic known to induce cardiotoxicity, and demonstrated that cardiac tissue levels of MPC decrease within 4 weeks of initial DOX exposure. Using a combination of stable isotope tracing metabolomics, hyperpolarized [1- 13 C]pyruvate magnetic resonance imaging (MRI), and [3- 11 C]pyruvate positron emission tomography (PET), we found that loss of MPC and monocarboxylate transporter 1 (MCT1) resulted in decreased utilization of pyruvate for mitochondrial oxidative metabolism and resulted in decreased cardiac carbon-11 flux. Significantly, cardiac [3- 11 C]pyruvate flux was sensitive to MPC expression levels and was restored when expression rebounded 16 weeks after DOX exposure. [3- 11 C]Pyruvate PET is therefore a promising approach to imaging cardiac pyruvate transport with potential application to the identification of maladaptive changes in MPC expression and monitoring response to therapy. Health sciences/Cardiology Biological sciences/Physiology cardiotoxicity cardiac pyruvate metabolism doxorubicin mitochondrial pyruvate carrier monocarboxylate transporter positron emission tomography Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Heart disease is a leading cause of death worldwide 1 . Although the etiology of heart failure is diverse, metabolic perturbation is a common feature. Given the prevalence of this condition, methods of imaging cardiac metabolism could play a critical role in the treatment of millions of patients. Significantly, metabolic dysfunction precedes functional and structural changes, thereby identifying these processes as diagnostic and therapeutic targets of great potential significance. In this context, molecular and cellular imaging using probes targeting cardiac metabolism potentially offers a powerful approach to advancing precision medicine in cardiology 2 . Heart failure may present with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF). One important subset of HFrEF is anthracycline cardiotoxicity. Anthracycline chemotherapeutics (e.g., doxorubicin, DOX) are widely used and effective against numerous cancers 3 . However, their use is compromised by the development of cardiotoxicity. This condition may present as acute, early (within 1 year of treatment), or late onset (several years after treatment). There are no established strategies for distinguishing between reversible and progressive cardiac dysfunction 4 . Furthermore, predicting late onset disease is challenging due to the combination of treatments that patients may receive and the lack of biomarkers of cardiac damage that precede dysfunction. These observations highlight the critical need to develop methods of early detection of anthracycline-induced cardiac injury that are independent of assessments of cardiac structure and function to enable timely intervention that may prevent irreversible cardiac damage and long-term heart failure. The critical role of metabolic reprogramming in promoting cardiac functional and structural changes after DOX exposure is increasingly recognized 5 – 7 , but there remains a lack of extensive longitudinal studies examining the key molecular mediators that regulate these adaptations. Mitochondria are known targets of DOX-induced toxicity in cardiomyocytes. These cells have the highest mitochondria content of any cell type 8 , highlighting the value of targeting biochemical processes that occur in mitochondria as a means of imaging pathological cardiac metabolic reprogramming. Under normal conditions, the heart derives more than 95% of its ATP from oxidative phosphorylation (OXPHOS) 9 , 10 , which relies on oxidation of substrates in the tricarboxylic acid (TCA) cycle. In HFrEF, glucose consumption by the TCA cycle decreases as glycolysis becomes uncoupled from OXPHOS 10 , 11 . Pyruvate is a key metabolite that links glycolysis to mitochondrial oxidative metabolism. The potential for using this metabolite to image physiological cardiac metabolism and disease-related changes has been demonstrated by hyperpolarized (HP) [1- 13 C]pyruvate magnetic resonance imaging (MRI) in both animals and human subjects 12 – 16 . Despite the encouraging developments in HP [1- 13 C]pyruvate cardiac MRI, the short half-life of the hyperpolarized species (T 1 relaxation time = 1 min) and the requirement for supra-physiological masses of pyruvate may ultimately limit some applications of this technology for cardiac imaging 17 . In this context, it is notable that 11 C-labeled pyruvate positron emission tomography (PET) has not been widely pursued as a complementary or alternative imaging strategy. The physical properties of carbon-11 (t 1/2 = 20.4 min), which decays almost entirely by positron emission (β + = 99.8%, E avg = 385.7 keV), render 11 C-labeled pyruvate an intriguing candidate for imaging changes in cardiac pyruvate metabolism. Two isotopologues of 11 C-labeled pyruvate, [1- 11 C]pyruvate and [3- 11 C]pyruvate, are available in sufficient activities and purities for PET imaging 18 – 20 . Pyruvate requires monocarboxylate transporters (MCTs) for transport into cells and the mitochondrial pyruvate carriers 1 and 2 (MPC1/2) for transport across the mitochondrial membrane 21 , 22 . Since the discovery of MPC1/2 as essential transporters for pyruvate across the mitochondrial membrane, their role in cardiac metabolic changes associated with heart failure has been increasingly recognized 23 – 27 . Inhibition of pyruvate transport by DOX in rat cardiomyocytes was previously reported 28 , suggesting that MPC1/2 might contribute to DOX-induced metabolic disruption in cardiomyocytes. In this study, we hypothesize that DOX exposure compromises murine cardiac pyruvate metabolism by disrupting its transport and reducing expression of MCT and MPC1/2. Most notably, we hypothesize that DOX-induced changes in cardiac MPC1/2 expression can be detected and quantified through dynamic cardiac PET imaging using [3- 11 C]pyruvate. We demonstrate that cardiac [3- 11 C]pyruvate flux is inversely related to MPC expression and use stable isotope tracing and HP [1- 13 C]pyruvate MRI to confirm that increased flux corresponds to decreased utilization of pyruvate by the TCA cycle. Results MPC1/2 expression decreases in the murine heart after exposure to doxorubicin To assess the initial effect of DOX exposure on cardiac metabolism, we analyzed cardiac tissue samples collected from mice 4 weeks after the first administration of DOX. At this point, we previously observed cardiac atrophy in the mice exposed to DOX 29 , a finding that we confirmed in this study (Supplemental Fig. 1a). We performed bulk RNA sequencing analysis on these tissues and identified 1618 differentially expressed genes (DEGs) between the DOX and control groups (p |1|). The DEGs were used for Gene Ontology (GO) and KEGG pathway enrichment using the DAVID and STRING databases. Among the most significantly downregulated pathways were those involving pyruvate metabolism and processes supporting mitochondrial function (Fig. 1 a, b, and Supplementary Tables 1, 2). These findings are consistent with prior observations of mitochondrial oxidative stress induced by DOX 30 . From the EnhancedVolcano plot of the DEGs, we identified 9 significantly downregulated genes associated with pyruvate transport (Fig. 1 c). Two of these genes, Mpc1 and Mpc2, are highly expressed in the hearts of mice and humans (Supplemental Fig. 1b, c, and Supplementary Table 3) and encode subunits of the mitochondrial pyruvate carrier (MPC), which assembles as a heterodimer to facilitate pyruvate import into the mitochondrial matrix 21 , 22 . Given prior reports of MPC expression deficits in other cardiac pathologies 23 – 25 , 27 and the interaction between MPC1/2 and the pyruvate dehydrogenase complex, which catalyzes the irreversible conversion of pyruvate to acetyl-CoA (Supplemental Fig. 1d and Supplementary Table 4), we evaluated the protein expression levels of MPC1 and MPC2 in the heart tissue samples. There was a significant reduction in MPC1 and MPC2 expression in the tissue taken from the mice exposed to DOX compared to the age-matched controls (p = 0.0248 for MPC1; p = 0.0028 for MPC2; Fig. 1 d, e). In parallel, we observed a significant reduction in MCT1 transcripts (Slc16a1) and expression in these tissues (p = 0.0300; Fig. 1 c and Supplemental Fig. 1e). By contrast, expression of MCT4 (Slc16a3) was not significantly different in the mice exposed to DOX compared to controls. Next, we evaluated myocardial carbohydrate metabolism and tricarboxylic acid (TCA) cycle flux by stable isotope tracing in mice using [3- 13 C]pyruvate at the 4-week time point (Fig. 1 f). We chose this time point for the experiment because prior studies in healthy mouse hearts indicated that incorporation of multiple isotopic labels into TCA cycle intermediates, corresponding to multiple iterations of the TCA cycle, was possible when samples were collected 10 min post-injection (p.i.; Supplemental Fig. 1f). These experiments revealed no significant changes in the total cardiac lactate, alanine, citrate, and glutamate pools in hearts collected from the animals exposed to DOX at 10 minutes p.i. compared to controls (Fig. 1 g-j), although the total metabolite pool size was significantly lower (p = 0.0079) in the mice exposed to DOX (Supplemental Fig. 1g). Fractional enrichment of the M + 1 peak in lactate was slightly, but not statistically significantly, increased in the hearts from the DOX animals (n = 5) compared to the controls (0.19 vs . 0.12; Fig. 1 g), although we observed considerable variability within this group. We additionally observed reduced expression of lactate dehydrogenase (LDH) A and B, while there was no change in alanine aminotransferase (ALT) at the 4-week time point in mice exposed to DOX (Supplemental Fig. 1e). In agreement with the lactate data, M + 1 fractional enrichment of alanine increased in the hearts exposed to DOX, although the variability within this group ensured that the difference to the control hearts was not statistically significant (Fig. 1 i). By contrast, M + 1 fractional enrichment of citrate (p = 0.0230) and glutamate (p = 0.0052) was significantly lower in the hearts extracted from the mice exposed to DOX (Fig. 1 i and j). Similarly, M + 1 fractional enrichment of other TCA cycle intermediates, including aconitate, fumarate, malate, oxoglutarate, and succinate, was significantly lower in the DOX hearts (Supplementary Fig. 1h). The pool size of these TCA cycle metabolites was generally comparable between the two groups, with only deficits in cardiac fumarate and malate in the DOX group reaching statistical significance. Given the reduced MPC1 and MPC2 expression in DOX-treated hearts and the corresponding decrease in M + 1 enrichment of TCA cycle metabolites, our findings suggest that DOX exposure impairs pyruvate entry into mitochondria and promotes its conversion to lactate and alanine in the cytosol. MPC1 and MPC2 are key mediators of pyruvate metabolism in human cardiomyocytes exposed to DOX To test our hypothesis that DOX impairs mitochondrial pyruvate oxidation by targeting MPC1/2, we investigated whether DOX contributes to alterations in MPC1/2-mediated pyruvate metabolism in human cardiomyocytes. Consistent with previously established conditions 29 , 31 , human cardiomyocytes (HCM) were treated with 0.1 µM DOX for two consecutive 48-hour periods (Supplemental Fig. 2a). There was no difference in cell viability between groups (Supplemental Fig. 2b). We incubated control and DOX-treated HCM with [3- 14 C]pyruvate for up to 8 minutes and observed significantly decreased time-dependent retention of signal in the DOX-treated HCMs (p = 0.0137; Fig. 2 a). The difference in uptake in the DOX-treated HCMs was then compared to HCM treated with UK5099, a selective pharmacological inhibitor of MPC, 32 for 72 hours before the addition of [3- 14 C]pyruvate, as shown in Supplemental Fig. 2a. These conditions were previously demonstrated to downregulate MPC1/2 33 . We confirmed downregulation of MPC1/2 in the UK5099- and DOX-treated cells and also determined by Western blotting that these treatments decreased expression of MCT1 (Fig. 2 b). These experiments revealed that [3- 14 C]pyruvate uptake after 8 minutes was reduced by 2.3-fold in UK5099-treated HCM compared to the untreated control cells (p < 0.0001 for CTRL versus UK5099 or DOX; Fig. 2 c). By contrast, [3- 14 C]pyruvate uptake was comparable between the UK5099-treated HCM and the DOX-treated HCM. These results indicate that DOX-induced changes in pyruvate metabolism are comparable to those induced by UK5099. Treatment with DOX or UK5099 did not affect cell viability but did result in a lower pH (6.8 vs. 7.5) in the media (Supplemental Fig. 2c, d). We attributed this acidification to increased lactate production resulting from impaired mitochondrial import of pyruvate. To disentangle the contributions of MCT1 and MPC1/2 to reduced pyruvate metabolism in DOX-treated cardiomyocytes, we used MPC1- and MPC2-specific siRNA to prepare HCM with selective inhibition of MPC1/2 (Supplemental Fig. 2a, e). These cells demonstrated a 25% reduction in [3- 14 C]pyruvate uptake compared to the scrambled control cells (p < 0.0001; Fig. 2 d), although there were no changes in the expression of MCT1, MCT4, LDHA, LDHB, ALT, or the PDH complex (Supplemental Fig. 2f). A comparison these results with the changes induced by DOX or UK5099 (Fig. 2 c) suggests that deficits in DOX-induced pyruvate flux in cardiomyocytes are attributable to both impaired transport of pyruvate across the cell membrane (loss of MCT1 expression) and impaired transport across the mitochondrial membrane (loss of MPC1/2 expression). One limitation of the uptake assay is that it is not possible to determine the chemical form of the 14 C label after its addition to the well. There are three fates of the 14 C label: it can be retained in the cell (as [3- 14 C]pyruvate, a TCA cycle intermediate, or another metabolite), released into the media (as [3- 14 C]lactate or [3- 14 C]pyruvate), or released as [ 14 C]CO 2 , as shown in Fig. 2 e. We hypothesized that one major difference between HCM consuming [3- 14 C]pyruvate in the TCA cycle and HCM deficient in MPC would be the production of [ 14 C]CO 2 , which could be formed after multiple iterations of the TCA cycle (Supplemental Fig. 2g). Consequently, we performed a [ 14 C]CO 2 capture assay 34 following incubation of HCM with [3- 14 C]pyruvate for 8 min. This experiment confirmed that lower activities of [3- 14 C]CO 2 were evolved in the HCM with reduced MPC1/2 levels (p = 0.0002 for CTRL versus MPC1/2 siRNA; p = 0.0004 for CTRL versus UK5099; p = 0.0137 for CTRL versus DOX; Fig. 2 f). The total 14 C counts, representing the sum of the [ 14 C]CO 2 captured, the cell-associated activity, and the remaining activity in the media, did not change (Supplemental Fig. 2h). Taken together, our findings indicate that the DOX-induced reduction in pyruvate metabolism in cardiomyocytes reflects impaired pyruvate transport and is partially mediated by MPC1/2. [3-C]Pyruvate PET detects DOX-induced changes in cardiac pyruvate flux at 4 weeks Given the potential significance of cardiac MPC as a target of DOX, we aimed to develop a method for assessing MPC levels in vivo. We hypothesized that measurements of the cardiac flux of [3- 11 C]pyruvate by positron emission tomography (PET) (Fig. 3 a) would highlight deficiencies in pyruvate transport and thereby permit MPC expression to be inferred. The rationale for this hypothesis was the difference in [ 11 C]CO 2 production between cells that utilize pyruvate in the TCA cycle and cells that do not. Based on our in vitro studies, we anticipated rapid clearance of [ 11 C]CO 2 from cardiac tissue and a correspondingly rapid activity flux in hearts without impaired transport. To test our hypothesis, we acquired 30 minute dynamic PET images upon intravenous administration of [3- 11 C]pyruvate to mice exposed to DOX (n = 10) and age-matched controls (n = 10) at the 4-week time point (Supplemental Fig. 3a). Summed sagittal-axis PET images over the first 10 minutes post-injection showed a markedly enhanced [3- 11 C]pyruvate signal in the DOX group at 4 weeks compared to the control group (Fig. 3 b). Consistent with PET images and [3- 13 C]pyruvate metabolomics, the time-activity curves (TACs) drawn using regions-of-interest (ROIs) over the whole heart from the 10-minute acquisitions—normalized to peak uptake—highlighted a slower clearance of activity from the hearts of mice in the DOX group (Fig. 3 c). Normalization to peak uptake allows us to account for differences in injected activity and heart size. This translated to a significantly increased transit time of 320 ± 52 s in the animals exposed to DOX (n = 9) compared to 272 ± 36 s in the control group (n = 8; p = 0.0355). Three mice, 1 from the DOX group and 2 from the control group, were excluded from the analysis because the cardiac transit time in these animals was greater than 2 standard deviations from the mean. The transit time is proportional to the clearance rate from cardiac tissue and supports the conclusion that [ 11 C]CO 2 is not generated as quickly in the hearts exposed to DOX. As a further test of our hypothesis, we performed magnetic resonance spectroscopy (MRS) of hyperpolarized (HP) [1- 13 C]pyruvate MRI in the two groups at 4 weeks. In contrast to PET, MRS of HP pyruvate allows the metabolism of pyruvate to be directly observed in the mouse heart 35 . Although the HP tracer bore the isotopic label at the C-1 position rather than the C-3 position, the metabolism of these two isotopologues would be expected to be identical through PDH-catalyzed entry into the TCA cycle. We observed a significant increase in the cardiac lactate/pyruvate ratio in the mice exposed to DOX (p < 0.0001; Fig. 3 d). This is consistent with enhanced lactate production resulting from decreased utilization of pyruvate by the TCA cycle. Indeed, in a separate experiment using 1 H NMR, we measured a higher lactate pool in cardiac tissues from mice in the DOX group compared to the control group (Supplementary Fig. 3b). In addition, the bicarbonate/lactate ratio was significantly lower for the mice from the DOX group (p = 0.0260; Fig. 3 e). The alanine/lactate ratio did not change between groups (Supplementary Fig. 3c). Collectively, these studies support the interpretation that slower cardiac pyruvate flux corresponds to lower pyruvate oxidation and higher conversion to lactate, and that flux measurements can be used to infer MPC expression in the heart. Recovery of MPC1/2 expression is coincident with accelerated cardiac growth Exposure to DOX is known to induce cardiac atrophy followed by a period of accelerated growth 29 , 36 . Therefore, we sought to investigate whether MPC1/2 expression changes during the growth phase. For this purpose, we collected heart tissue from mice 16 weeks after the first administration of DOX. The heart-weight-to-tibia-length (HW/TL) ratio was 5.94 for these mice, compared to 7.55 for the controls (p < 0.0001, Fig. 4 a). At 4 weeks, the ratios were 7.69 and 5.13, respectively. As such, the HW/TL ratio in DOX-treated tissues at 16 weeks demonstrated a 1.2-fold increase relative to those at 4 weeks of DOX exposure (p = 0.0023; Supplementary Fig. 4a). We correspondingly observed increased expression of markers of cardiomyocyte growth and cardiomyopathy, such as p53 37 and p21 38 , as shown in Supplementary Fig. 4b. Bulk RNA sequencing analysis conducted at 16 weeks (false discovery rate; FDR < 0.05) for KEGG pathways reinforced the growth profile of the heart, as shown in Supplementary Fig. 4c and Supplemental Table 5. Additionally, the EnhancedVolcano plot of the DEGs revealed neither Mpc1 (Log2FC = -0.00012) and Mpc2 (Log2FC = -0.02665) nor Mct1 (Log2FC = 0.00508) and Mct4 (Log2FC = -0.04813) to be significantly different between the mice exposed to DOX and the control animals (Fig. 4 b). Similarly, and in contrast to the expression profile in the samples collected at 4 weeks, there were neither significant differences in MPC1 and MPC2 protein expression nor MCT1 and MCT4 expression between the mice in the DOX and control groups at 16 weeks (Fig. 4 c, d, Supplemental Fig. 4d). Collectively, these observations suggest that recovery of pyruvate transport, including MCT1 and MPC1/2 expression, after DOX exposure is associated with accelerated cardiac growth. Cardiac [3-C]pyruvate flux reflects longitudinal changes in MPC1/2 expression Having observed the recovery of MCT1 and MPC1/2 expression at 16 weeks, we sought to determine how these changes affected metabolism. We conducted stable isotope tracing metabolomics at 16 weeks using the same methodology as at 4 weeks. In contrast to the 4-week time point, we detected significantly increased M + 1 fractional enhancement of lactate (p < 0.0001), alanine (p < 0.0001), and glutamate (p = 0.0002) in the animals exposed to DOX, while fractional enhancement of citrate was comparable between the two groups (Fig. 5 a-d). The total pool of lactate was comparable to the control samples, but the alanine pool was significantly greater (p = 0.0295), and the glutamate and citrate pools were significantly lower (p = 0.0157 and p = 0.0086, respectively) in the hearts exposed to DOX (Fig. 5 a-d). Notably, we observed substantially increased incorporation of the 13 C label into metabolites associated with the TCA cycle, with M + 1 fractional enhancement of key metabolites for TCA cycle, including aconitate, fumarate, malate, oxoglutarate, and succinate, comparable or significantly greater than the corresponding controls (Supplemental Fig. 5a). However, the total pool size of these metabolites – and the total of all metabolites detected – was significantly lower (p = 0.0035) in the mice exposed to DOX than the controls (Supplemental Fig. 5b), evidencing metabolic reprogramming even 16 weeks after the initial DOX exposure. Expression of other key components of pyruvate metabolism, such as LDHA, LDHB, and ALT, as well as MCT1 and MCT4, was comparable between the groups, as shown in Supplemental Fig. 4d, suggesting recovery of pyruvate transport. A similar trend was evident in genes encoding key proteins involved in β-oxidation, whose expression was significantly increased at 16 weeks compared to 4 weeks (Supplemental Fig. 5c), albeit without recovering to the expression levels in the control hearts. These findings highlight the restoration of pyruvate flux through mitochondria at 16 weeks. When these mice were imaged by [3- 11 C]pyruvate PET, we observed no significant difference in cardiac flux between the groups (Fig. 5 e). In contrast to the 4-week time point, [3- 11 C]pyruvate flux in the DOX-treated mice (n = 7) was more rapid than in the control animals (n = 4) during the first 10 minutes (p = 0.0426; Fig. 5 f). This was reflected in a transit time of 315 ± 42 s for the DOX mice and 323 ± 27 s for the control mice. Notably, the accelerated cardiac flux observed in the DOX group is consistent with restored pyruvate utilization via the TCA cycle. However, as in week 4, the cardiac time-activity curves for [3- 11 C]pyruvate trended towards convergence after 30 minutes (Supplemental Fig. 5d). We next performed cardiac segmentation using the Carimas software package 39 and performed compartmental modeling using the image-derived left ventricular input function. Using Akaike’s information criterion, we identified the two-tissue, four-parameter compartmental model as the best fit for 93% of the images (n = 27/29), as shown in Supplemental Fig. 6. The two remaining curves were best modeled by a one-tissue compartment model. No compartment model fit the curves obtained from any of the three outliers that we excluded in our analysis at 4, highlighting the discrepancy between these animals and the bulk of our subjects. The two-tissue, four-parameter model is in agreement with that derived for the utilization of [3- 11 C]lactate by the porcine heart 40 . In this model, k4 represents the oxidation of [3- 11 C]pyruvate to [ 11 C]CO 2 . We compared the k4 for each of the four groups, as shown in Supplemental Table 6 and found a trend towards lower k4 values in the DOX mice at 4 weeks compared to the other groups, although these differences were not statistically significant. We excluded 2 additional mice (n = 1 for DOX 4W and control 4W) due to k4 values that were two standard deviations from the mean. These mice received lower activities of [3- 11 C]pyruvate upon injection, which likely resulted in increased noise in the early time points of the TAC. There were no statistically significant differences between groups for kinetic constants VA, K1, k2, or k3 (Supplemental Table 7). Overall, our findings support the hypothesis that exposure to DOX causes rapid changes in cardiac pyruvate metabolism by modulating pyruvate transport in cardiomyocytes. The deficits in MCT1 and MPC1/2 expression contribute to decreased utilization of pyruvate by the TCA cycle. After the initial decrease, MPC1/2 expression (and MCT1 expression) is restored in concert with pyruvate oxidation and accelerated cardiac growth. Significantly, this dynamic process can be imaged in vivo using PET-based measurements of [3- 11 C]pyruvate cardiac flux. Discussion Healthy cardiac tissue generates the bulk of its energy from oxidizable substrates via processes that take place in mitochondria, which comprise a large part of the cardiomyocytes 41 . However, during the onset and progression of doxorubicin-induced cardiotoxicity, cardiac tissue undergoes significant molecular and metabolic remodeling necessitated by the emergence of mitochondrial dysfunction. Impaired mitochondrial function may ultimately lead to cardiomyocyte apoptosis and cell death 42 , 43 . In this context, we identified pyruvate to potentially be a key metabolite in a rodent model of cardiotoxicity. Pyruvate, the end-product of glycolysis, plays a pivotal role in mitochondrial ATP production after its entry into the TCA cycle via PDH or its conversion to oxaloacetate via pyruvate carboxylase. One of the major regulators of this process is the MPC, which is exclusively responsible for mitochondrial pyruvate import 44 and reported by some groups to be a rate-limiting step in pyruvate oxidation in the heart 45 . As MPC can regulate both pyruvate oxidation and anaplerosis, it may contribute more significantly to cardiac metabolic reprogramming than PDH 23 . To this end, reduced expression or genetic knockout of MPC1/2 was recently shown to promote hypertrophic cardiomyopathy in murine models of heart failure 23 – 25 while MPC1/2 abundance promoted survival of porcine cardiac tissue following ischemia-reperfusion 46 . Similarly, reduced MPC1/2 expression has been observed in failing hypertrophic human hearts 24 and patients that failed to respond to left ventricular assist device implantation 26 . Despite this emerging evidence of the importance of MPC in hypertrophic cardiomyopathy, we are not aware of any studies that have investigated its expression in hearts at risk of cardiotoxicity. Although doxorubicin-induced cardiotoxicity shares some characteristics, such as reduced left ventricular ejection fraction (LVEF), of these cardiomyopathies, in murine models it is associated with an initial phase of cardiac atrophy rather than hypertrophy 7 , 47 , 48 . In this context, it is significant that we observed decreased MPC expression and activity when atrophy, as assessed by HW/TL ratio, was more pronounced, while expression and activity recovered during a phase of accelerated cardiac growth. The stable isotope tracing and PET imaging experiments support decreased flux through MPC in the atrophy phase and increased flux through MPC when expression is higher, although the possibility that pyruvate uptake through MCT1 is rate-limiting cannot be discounted. Post-translational modification of MPC1 and MPC2 has been reported in other animal models 27 , but the agreement between MPC expression and flux through MPC in our model suggests that MPC activity is not modulated by post-translational modification in our model. One plausible explanation for the increased MPC expression that we observed in the cardiac tissue samples collected from the DOX group at 16 weeks is that MPC promotes fibrosis. Inhibition of MPC reduced fibrosis in models of nonalcoholic steatohepatitis 49 and corneal fibrosis 50 . This outcome was proposed to reflect the diversion of glutamine into the TCA cycle to compensate for the shortfall of mitochondrial acetyl-CoA derived from pyruvate 50 . Glutamine stimulates collagen biosynthesis in activated fibroblasts through its conversion to glutamate and proline 51 , but these pathways are suppressed when glutamine is consumed by the TCA cycle. Our prior work with this model demonstrated increased cardiac fibrosis in mice exposed to DOX after 16 weeks compared to 4 weeks or control populations 29 . The relationship between MPC and cardiac fibrosis requires further investigation. Similarly, stable isotope tracing analyses in cultured cardiomyocytes revealed that synthesis of aspartate from glucose supports hypertrophy, likely due to the contribution of the amine group of aspartate to nucleotide biosynthesis 52 . Aspartate is formed by transamination of oxaloacetate produced by the TCA cycle. In the heart samples collected at 16 weeks, M + 1 fractional enhancement of aspartate is 43% higher in the hearts exposed to DOX (39.1 ± 4.3% vs. 27.3 ± 3.5% abundance, respectively). Our studies do not report on the metabolic fate of aspartate but do suggest another mechanism by which the recovery of MPC expression in the hearts exposed to DOX between 4 weeks and 16 weeks is associated with accelerated cardiac growth. In contrast to [1- 11 C]pyruvate, which rapidly produces [ 11 C]CO 2 during PDH-catalyzed conversion to acetyl-CoA, [3- 11 C]pyruvate can only produce [ 11 C]CO 2 by undergoing multiple rounds of the TCA cycle, as shown in Supplemental Fig. 2f. The rapid decarboxylation of [1- 11 C]pyruvate to [ 11 C]CO 2 , as would be expected in the healthy heart, results in a lack of tissue signal retention 19 . By contrast, administration of [3- 11 C]pyruvate results in longer retention. We elected to use [3- 11 C]pyruvate for our studies because it is readily synthesized from [ 11 C]methyl iodide and a commercially available derivative of glycine 53 , and the additional retention would enable flux measurements over a longer time interval. This rationale was previously used to support the use of [3- 11 C]lactate to measure myocardial lactate kinetics in porcine hearts 40 . [3- 11 C]Lactate was found to track the fate of lactate in the heart, where it is either oxidized or backdiffused, and its kinetics were best described using a 2-tissue, 4-compartment model. Given the interconvertibility of pyruvate and lactate and the fact that both enter the mitochondria as pyruvate, we also fitted our data to a 2-tissue, 4-compartment model. Graphical analyses confirmed this model to be superior to other compartmental or simple exponential models, suggesting that [3- 11 C]pyruvate PET imaging can similarly be used to estimate myocardial pyruvate oxidation rates. However, this hypothesis will need to be tested by blood sampling in larger animals. We interpreted the slower cardiac flux of radioactivity in the DOX group at 4 weeks evident in the PET imaging to reflect differences in the rate of formation of [ 11 C]CO 2 . Cardiomyocytes efficiently eliminate CO 2 through high CO 2 permeability and carbonic anhydrase activity 54 . The [3- 14 C]pyruvate uptake studies in cultured human cardiomyocytes (Fig. 2 ), confirm that evolution of [ 14 C]CO 2 is related to MPC expression and consistent with our hypothesis. To reinforce this interpretation, we measured the cardiac bicarbonate-to-lactate (Bic/Lac) ratios in mice exposed to DOX and control animals at the 4-week time point by HP [1- 13 C]pyruvate MRI. This substrate bears the isotopic label on a different carbon atom to our PET probe, but the rapid release of [ 13 C]CO 2 upon entry of [1- 13 C]pyruvate into the TCA cycle allowed use the formation of this metabolite as a means to distinguish between mitochondrial pyruvate oxidation and its metabolism by other pathways in a time course that is compatible with the hyperpolarization of [1- 13 C]pyruvate (T1 = 90 s in D 2 O 55 , 56 ). Hyperpolarized [2- 13 C]pyruvate MRI has previously been used to measure labeling of downstream mitochondrial metabolites in rat hearts 12 and could represent a valuable follow up study for this work. Bicarbonate is formed when CO 2 is hydrated by reaction with carbonic anhydrase. Bic/Lac ratios were typically lower in the mice exposed to DOX, as shown in Fig. 3 e. Intriguingly, lower Bic/Lac ratios were associated with lower LVEF in human patients 16 , highlighting a possible relationship between pyruvate metabolism through MPC and cardiac function. The statistically significant increase in Lac/Pyr ratio in the hearts exposed to DOX (Fig. 3 d) agrees with decreased flux of pyruvate through the TCA cycle and points toward a glycolytic phenotype in these hearts. Although we did not specifically evaluate [3- 11 C]pyruvate PET as an imaging biomarker of cardiotoxicity, reports of cardiomyocyte metabolic reprogramming preceding changes in cardiac structure and function support its use for this application 5 , 7 . Cardiotoxicity is traditionally defined as a drop in LVEF of > 10% in symptomatic patients or > 5% in asymptomatic patients 57 , 58 . Such cardiac dysfunction is detected by echocardiography and may arise many years after cessation of DOX treatment 59 . However, early detection of subacute or subclinical disease remains challenging 60 , and new methods of doing so could greatly improve management of these conditions, especially when they convey information about specific biochemical pathways that are disrupted. In our previous work with this model 29 , we demonstrated that significant changes in fractional shortening were not evident until 10 weeks after the first exposure to DOX. This timeline was comparable to other murine models in which the mice received a similar cumulative dose 7 . In this light, it is potentially significant that we observed reduced cardiac [3- 11 C]pyruvate flux at 4 weeks. Future studies are required to determine whether [3- 11 C]pyruvate PET predicts changes in LVEF and therefore represents a biomarker of cardiotoxicity. The major limitation of our approach is that non-invasive measurement of radioactivity fluxes cannot necessarily isolate the effect of individual molecular targets. Our goal was to identify a method of non-invasively assessing MPC protein expression because decreased protein, but not gene expression, has been detected in cardiac tissue samples taken from patients with HFrEF 61 . We present evidence that the decreased flux in the 4-week DOX group reflects decreased expression of MPC1 and MPC2, but we cannot definitively rule out the contribution of other enzymes and transporters to clearance patterns. Most significantly, expression of MCT1 mirrored that of MPC1/2 on a population level in our model, although individual differences in MCT1 expression might be responsible for the lower pool size and M + 1 fractional enrichment of lactate observed in a subset of the mice (n = 3) exposed to DOX (Fig. 1 g). Conversion of HP [1- 13 C]pyruvate to [1- 13 C]lactate and other downstream metabolites is rate-limited by MCT1-mediated transport across the plasma membrane in cancer cells 62 , and the same kinetics likely apply to cardiomyocytes. One potential advantage of PET imaging in this context is that the tracer is administered in nanomolar concentrations, rather than the supra-physiological amounts required for HP [1- 13 C]pyruvate MR imaging and stable isotope tracing. As such, pyruvate transport through MCT1 may not influence radioactivity flux to the same extent and the PET time-activity curves might more closely reflect MPC expression levels. In addition, despite significantly lower levels of LDHB, the more prevalent isoform of LDH in the heart, we did not observe any impairment of pyruvate and lactate interconversion. This apparent contradiction can be explained by the observation that LDHB – and many of the enzymes involved in pyruvate metabolism – are subject to post-translational modification and substrate or product inhibition 63 – 65 . It is noteworthy that [3- 11 C]pyruvate flux was faster in the DOX-exposed hearts at 16 weeks compared to the controls and incorporation of the 13 C label into TCA cycle metabolites was similarly elevated even though neither MCT1 nor MPC1/2 expression was not significantly higher in the hearts exposed to DOX. This may indicate a compensatory increase in pyruvate oxidation following decreased fatty acid β-oxidation, a phenomenon previously reported for a rat model of sub-chronic DOX-induced cardiotoxicity 5 . Indeed, although we observed increased expression of genes involved in β-oxidation in the hearts exposed to DOX at 16 weeks compared to 4 weeks, expression levels remained significantly lower than the age-matched controls (Supplemental Fig. 5b). This example highlights the challenge of relating flux measurements to changes in MPC protein expression levels alone, although development of a probe that selectively binds to MPC could further clarify this relationship. These efforts are ongoing in our laboratory. Collectively, our findings support dynamic [3- 11 C]pyruvate PET as a method for imaging cardiac pyruvate metabolism in vivo that conveys information about MPC expression levels in this tissue. Exposure of cardiomyocytes to doxorubicin induces atrophy and loss of MPC1/2 and MCT1 expression in vitro and in a mouse model. At later time points, expression of these markers returns to baseline, where it appears to be related to rates of heart growth. Loss of MPC results in reduced oxidation of pyruvate in mitochondria and decreased flux of [3- 11 C]pyruvate in the heart. Restoration of MPC expression levels are evident through increased [3- 11 C]pyruvate flux. Although further work is required, this study supports the potential of [3- 11 C]pyruvate PET for detecting impaired pyruvate transport, which could serve as an early indicator of cardiotoxicity in cancer patients undergoing doxorubicin treatment. Methods Ethics Statement All animal experiments carried out in this protocol were approved by the Institutional Animal Care and Use Committees (IACUC) of Weill Cornell Medicine and Memorial-Sloan Kettering Cancer Center (protocol 2019-0043). General Doxorubicin (DOX) hydrochloride was purchased from Tocris Bioscience, USA and used without further purification. It was dissolved at a concentration of 0.75 mg/mL in sterile saline for injection (Hospira, USA) with the aid of sonication. The solution was stored in the dark at -20°C for up to 24 hours (h) before use. Stable isotope tracing Four or 16 weeks after initial DOX exposure, DOX-treated mice (n = 3 and n = 5, respectively) and age-matched control mice (n = 4 and n = 5, respectively) received an intravenous bolus injection of 1 M sodium [3- 13 C]pyruvate (Millipore Sigma). The total injection volume was 100 ± 5 µL. At 10 min post-injection, blood was removed by cardiac puncture and the mice were sacrificed by cervical dislocation. The heart was excised, dried, weighed, and flash frozen in liquid nitrogen. Metabolites were extracted using 80% methanol. The extracts were dried down and then re-dissolved in water. Targeted LC/MS analyses were performed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled to a Vanquish UPLC system (Thermo Scientific). The Q Exactive operated in polarity-switching mode. A Sequant ZIC-pHILIC column (2.1 mm i.d. × 150 mm, particle size of 5 µm, Millipore Sigma) was used for the separation of metabolites. A 2.1 × 20 mm guard column with the same packing material was used for the protection of the analytical column. The flow rate was set at 150 µL/min. The mobile phases consisted of 100% acetonitrile for mobile phase A and 0.1% NH 4 OH/20 mM CH 3 COONH 4 in water for mobile phase B. The chromatographic gradient ran from 85–30% A in 20 minutes, followed by a wash with 30% A and re-equilibration at 85% A. The raw data was processed using El-MAVEN (v0.12.0). Metabolites and their 13 C isotopologues were identified on the basis of exact mass within 5 ppm and standard retention times. The fractional abundance of isotopically labeled metabolites was determined by determining the ratio of peak intensity of 13 C-labeled species to the peak intensity of the corresponding natural 12 C metabolite. The fractional labeling of each metabolite was compared by two-tailed, unpaired t-test, with p -values < 0.05 considered statistically significant. Radiosynthesis General A 5 M KOH solution was prepared by dissolving KOH, ≥ 99.95% trace metals basis (Millipore Sigma) in a suitable volume of 18 mΩ H 2 O. Stock solutions of 5 mg/mL D-amino acid oxidase from porcine kidney (D-AAO; Millipore Sigma) and 5 mg/mL catalase from bovine liver (Millipore Sigma) were prepared in 18 mΩ H 2 O. A 1 M solution of HCl in dioxane was prepared by diluting 0.4 mL of a 4 M HCl/dioxane solution (Millipore Sigma) with 1.2 mL anhydrous dioxane (VWR). Stock solutions of 0.5 M and 0.05 M Tris-HCl, pH 8.5, was prepared by diluting 1 M Tris-HCl, pH 8.5 (VWR) with 18 mΩ H 2 O. Production of [ 11 C]CH 3 I [ 11 C]CO 2 was produced by a [ 14 N(p,α) 11 C] transformation on a TR19 cyclotron (Advanced Cyclotron Systems, Inc.). The [ 11 C]CO 2 was converted to [ 11 C]CH 3 I using a TracerLab FX C Pro (GE Healthcare). Conversion of [ 11 C]CO 2 to [ 11 C]CH 3 I took approximately 14 min. [ 11 C]CH 3 I was trapped on an ascarite column and distilled into the reaction vial in a stream of N 2 gas by heating the column to 250°C. Synthesis of [3- 11 C]Pyruvate [3- 11 C]Pyruvate was synthesized following published methods 53 , 66 , with small modifications. Briefly, N -(diphenylmethylene)-glycine tert -butyl ester (3.0 ± 0.1 mg, Millipore Sigma) was dissolved in 350 µL N,N-dimethylformamide (ThermoFisher) in a glass vial. Next, 10 µL 5 M KOH was added, and [ 11 C]CH 3 I (approximately 6 GBq) was transferred to the vial. After the [ 11 C]CH 3 I was trapped, the reaction was heated at 85°C for 5 min. The intermediate was diluted with 10 mL H 2 O and passed through a pre-conditioned Sep-Pak C18 plus short cartridge (Waters). The cartridge was washed with 5 mL H 2 O and eluted into a clean glass vial with 1.6 mL 1 M HCl in dioxane. Deprotection was effected by heating the reaction at 130°C for 5 min. The contents of the vial were taken up in 20 mL H 2 O and passed through a Bond Elut Jr SCX 1000 mg cartridge (Agilent Technologies) pre-conditioned with 10 mL H 2 O. The cartridge was washed successively with 5 mL H 2 O and 2 mL 0.5 M Tris-HCl, and D/L-[3- 11 C]alanine was eluted in 2 mL 0.05 M Tris-HCl, pH 8.5. The mixture was transferred to a ThermoMixer® C (Eppendorf) and 75 µL of the D-AAO solution and 5 µL of the catalase solution was added. The reaction was shaken for 6 min at 40°C before acidification to pH < 2 with addition of 1 M HCl. The contents were passed through a pre-conditioned Bond Elut Jr SCX 1000 mg cartridge (Agilent Technologies). The filtrate was adjusted to pH 5–6 by addition of 1 M NaOH. The radiochemical purity of the product was determined by analytical radioHPLC by injection onto a Chirex 3126 (D)-penicillamine, 4.6 x 150 mm column (Phenomenex). The isocratic mobile phase was 1 mM CuSO 4 set to a flow rate of 1 mL/min. The retention time, t R , of the final product was compared to the t R of sodium pyruvate (11.5 min). Small animal microPET/CT imaging experiments Prior to administration of [3- 11 C]pyruvate, the lateral tail vein of each mouse was cannulated for intravenous (i.v.) administration. A 27G × ½ inch, 8 cm catheter (SURFLO® Winged Infusion Set, USA) was utilized for the cannulation procedure. Following confirmation of proper catheter insertion using a small saline flush, the tubing was prefilled with sterile saline and capped. Mice were imaged at 4 weeks after initial DOX exposure (n = 10 per group) or 16 weeks after initial DOX exposure (n = 8 treated animals and n = 4 controls). Cannulated mice were anesthetized under isoflurane (3.5% for induction, 1.5% for maintenance) and placed in pairs on the imaging bed. Prior to radiotracer administration, a CT acquisition was performed for anatomic co-registration and scatter and attenuation correction. The mice were administered 3.7–11.1 MBq [3- 11 C]pyruvate in a total volume of 100–150 µL. Imaging was performed using small animal microPET/CT (Siemens Inveon™, USA), and the 30-min acquisition began immediately upon tracer injection. The data were collected in list mode, histogrammed into 54 dynamic frames (12x5s, 12x10s, 8x15s, 10x30s, 10x60s, and 2x300s) and reconstructed using the OSEM-MAP algorithm. microPET/CT image analysis Cardiac segmentation was performed using Carimas 39 . The global time-activity curve (TAC) was plotted, and compartmental modeling was performed using the built-in software models. The input function was derived from the left ventricle. The TAC and compartmental modeling curves were plotted using GraphPad Prism. Using Akaike’s information criterion, we identified the two-tissue, four parameter compartmental model as the best fit for 93% of the images (n = 27/29). The remaining curves were best modeled by a one tissue compartment model. Statistical comparison of each kinetic constant, VA, K1, k2, k3, and k4, was performed between groups by one-way ANOVA. P-values < 0.05 were considered statistically significant. Tissue transit time (τ) in the heart was determined by dividing the area under the curve (AUC) of the TAC by its highest activity. The mean τ values and standard deviations for each of the four groups were determined. Three mice (n = 1, DOX 4 weeks and n = 2, control 4 weeks) were excluded from the comparison because their transit time was greater than 2 standard deviations below (DOX and control, n = 1) or above (control, n = 1). Statistical comparisons of transit times between DOX and control groups at each time point were performed by two-tailed, unpaired t-test. Hyperpolarization of [1- 13 C]pyruvate 35 µL neat [1- 13 C]pyruvic acid doped with 15 mM AH111501 trityl radical was polarized in a SpinLab hyperpolarizer (GE Healthcare, USA) at 5 T and 0.8 K with 139.88 GHz microwave irradiation. After at least 45 min of solid-state polarization buildup, the frozen sample was dissolved with 10 mL superheated (~ 400 K) D 2 O buffer containing 100 mM Tris and 1 mM EDTA for a final [1- 13 C]pyruvate concentration of 100 mM. The HP pyruvate solution was pH neutralized by a stoichiometric quantity of 10 N NaOH to the receiver flask. Dissolution polarization levels were estimated by measuring T1 decay on an aliquot of HP dissolution with a 1 T Spinsolve spectrometer (Magritek). The molar concentration of the pyruvate solution was measured by 13 C NMR at 11.7 T (Bruker; MSKCC NMR Core) with reference to a 100 mM [ 13 C]urea standard. 13 C Magnetic resonance spectroscopy Animal experiments were performed in a 3T Biospec MRI scanner (Bruker, USA) with a 13 C/ 1 H volume quadrature coil (RAPID MR). A T1-weighted FLASH sequence was used for 1 H anatomic imaging of the heart. Pre-scans were performed while mice were under ~ 1.5% isoflurane anesthesia. Immediately before initiating the hyperpolarized [1- 13 C]pyruvate dissolution process, the isoflurane flow was turned off and the mouse was allowed to wake up in the scanner. Mice (n = 3 for the control group, n = 2 for the DOX group) were restrained with three pieces of double-backed tape to prevent movement during the experiment and were not left awake under restraint for more than five min. The total time under isoflurane anesthesia was less than 30 min. For the 13 C spectroscopy experiment, the scanner was set to continuously acquire slice-selective spectra with a 60° flip angle, 2048 spectral points, and 1280 Hz (39.9 ppm) bandwidth. The HP pyruvate bolus was split into multiple injections to enhance the number of replicates measured per HP pyruvate dissolution 67 . 100 mM HP pyruvate was injected via tail vein catheter four times separated by delays of at least 30 s to allow complete relaxation of the preceding HP pyruvate injection. MR image analysis Dynamic spectra were zero-filled, phased, apodized with 5 Hz exponential decay, and baseline-corrected in MNova software (Mestrelab Research). Pyruvate (171 ppm), lactate (183 ppm), alanine (176 ppm), and bicarbonate (161 ppm) signals were then quantified by integration. Treating each injection of HP pyruvate substrate as a distinct measurement, dynamic area under curve (AUC) values were calculated by summing the metabolite integral intensities over the time-course of the pyruvate bolus. Metabolite AUCs were normalized by calculating lactate-to-pyruvate ratios and bicarbonate- or alanine-to-lactate ratios. Mean values were statistically compared between groups by two-tailed, unpaired t-test, with p -values < 0.05 considered statistically significant. 1 H NMR measurement of tissue lactate pool size Four weeks after initial exposure to DOX, mouse hearts (n = 4 per group) were dissected and snap frozen in liquid nitrogen. At least 100 mg tissue was transferred to pre-filled bead mill tubes (Fisher Scientific, USA) containing 400 µL 4% (w/v) perchloric acid) and finely ground by a Fisherbrand™ Bead Mill 24 Homogenizer (Fisher Scientific, USA). Homogenates were centrifuged for 15 minutes at 14000 rpm and 4°C, and the supernatant was transferred to a new tube containing 1 mL chloroform/tri-n-octylamine (78/22 v/v), followed by centrifugation for 15 minutes at 4000 rpm and 4°C. The aqueous phase was transferred to a new tube and lyophilized overnight. Dried samples were reconstituted in D 2 O solvent containing 1 mM sodium trimethylsilylpropanesulfonate standard and transferred to 5 mm NMR tubes. 1 H NMR spectra of metabolite extract samples were acquired in a 14.1 T spectrometer (Bruker; MSKCC NMR core). Spectra were processed and metabolite peaks were quantified with Chenomx NMR suite software (Chenomx, Canada). Preparation of cardiac tissue for analysis The mice were anesthetized by i.p. ketamine injection and perfused with phosphate-buffered saline (PBS) via the left ventricle at a constant pressure of 80 mmHg. To perform the molecular analysis, the whole hearts were homogenized by using liquid nitrogen and a mortar and pestle. The homogenized tissues were separated for RNA and protein extraction. The extracts were flash frozen in liquid nitrogen and stored at -78°C until further use. RNA isolation Frozen heart tissue fractions were collected and soaked in Trizol (Invitrogen, USA), and RNeasy Fibrous tissue mini kit (Qiagen, USA) was used to isolate total RNA from heart tissues. Genomic DNA was removed by DNase I (Qiagen), and RNA was reverse transcribed using an iScript kit (Bio-Rad, USA). RNA extracts were validated prior to sequencing. Bulk RNA-seq library construction and data analysis RNA libraries were sequenced with paired-end 50 bps on the NovaSeq 6000 Sequencer (Illumina, USA). The raw sequencing reads in BCL format were processed through bcl2fastq 2.20 (Illumina) for FASTQ conversion and demultiplexing. After trimming the adaptors with cutadapt (version 1.18; https://cutadapt.readthedocs.io/en/v1.18/ ), RNA reads were aligned and mapped to the GRCm39 mouse reference genome by STAR (version 2.5.2; https://github.com/alexdobin/STAR ) 68 , and transcriptome reconstruction was performed by Cufflinks (Version 2.1.1) ( http://cole-trapnell-lab.github.io/cufflinks/ ). The abundance of transcripts was measured using Cufflinks, with fragments per kilobase of transcript per million mapped reads (FPKM) as the output 69 , 70 . Raw read counts per gene were extracted using HTSeq-count version 0.11.2 71 . Gene expression profiles were constructed for differential expression, cluster, and principal component analyses with the DESeq2 package ( https://bioconductor.org/packages/release/bioc/html/DESeq2.html ) 72 . For differential expression analysis, pairwise comparisons were performed between two or more groups using parametric tests where read counts follow a negative binomial distribution with a gene-specific dispersion parameter. Corrected p -values were calculated based on the Benjamini-Hochberg method to adjust for multiple testing. For the differentially expressed genes (DEGs) analysis in the 4-week groups, p |1| was used to distinguish upregulated (Up) and downregulated (Down) DEGs, respectively. For the 16-week groups, those differentially expressed genes (DEGs) among a total 49,135 variables with a false discovery rate (FDR) of less than 0.05 were divided into up- and down-regulated groups for analysis. The EnhancedVolcano plot was generated using R studio for the overall distribution of DEGs. DAVID classification of DEGs The Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov/ ) was employed to classify differentially expressed genes (DEGs) based on their biological functions. For the 4-week group, 389 upregulated and 1,229 downregulated genes were analyzed, whereas 86 upregulated and 107 downregulated genes were assessed in the 16-week group. These DEGs were subjected to Gene Ontology (GO) enrichment analysis, focusing on biological processes (BP), as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. GO terms with a p -value < 0.05 were considered statistically significant. STRING database analysis A protein-protein interaction (PPI) network was constructed to identify the associations between the target and related differentially expressed genes (DEGs) using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database ( http://string-db.org/ ) 73 . GO terms and PPI networks with a p-value < 0.05 were considered statistically significant. Western blot analysis Frozen heart tissue fractions were soaked in tissue protein extraction reagent (#78510, ThermoFisher, USA), supplemented with a protease inhibitor cocktail (#87786, ThermoFisher, USA) for protein extraction. Western blots were prepared and processed as previously reported 29 . The information on all primary and secondary antibodies can be found in Supplemental Table 8. The chemical luminescent signals were measured by the ChemiDoc imaging system (Bio-Rad, USA). Protein expression was quantified by drawing a region of interest (ROI) over the corresponding band using ImageJ software. In vitro [3- 14 C]pyruvate uptake in human cardiomyocytes Cell culture Human cardiomyocytes (HCMs) isolated from adult left ventricles (PromoCell, Heidelberg, Germany) were cultured according to the manufacturer’s instructions. Briefly, once the cultures reached 80–90% confluency, the cells were washed with PBS and refreshed with either control medium (DOX-free) or medium containing DOX or UK5099. Cells in the former group were incubated in media containing 0.1 µM DOX. The cells were incubated for 48 h, followed by a medium change with fresh medium. After an additional 48 h incubation, the medium was replaced again with 0.1 µM DOX-containing medium to complete the "two-hit" treatment 31 . Following a final incubation period of 48 h, the medium was removed and replaced with DOX-free control medium. All DOX-treated assays were performed on day 7. The second group of HCM was treated with 100 µM UK5099 for 72 h 33 . The control cells received an equal volume of dimethyl sulfoxide (DMSO). Silencing of MPC1 and MPC2 MPC1 and MPC2 siRNA were used to downregulate MPC1/2-specific expression in HCMs. Pre-designed siRNA products targeting MPC1 (#4392420, ID s28488) and MPC2 (#4392420, ID s24657), along with negative control siRNA (#4390843, scramble) were purchased and 50 nM of MPC1 and MPC2 siRNA and 100 nM of scramble siRNA were mixed with Lipofectamine RNAiMAX (#13778100) or Lipofectamine Stem (STEM00008) and Opti-MEM (#31985070) according to established protocols 74 . The siRNA mixture was applied to HCMs for 24 h, followed by replacement with fresh culture medium for an additional 24 h of incubation. MPC1 siRNA, sense; UGCUAUUCUUUGACAUUCAtt/ antisense; UGAAUGUCAAAGAAUAGCAac. MPC2 siRNA, sense; UCACUUGUAAUUAUUCCAAtt/ antisense; UUGGAAUAAUUACAAGUGAgt. All materials for siRNA work were purchased from ThermoFisher, USA. [3-C]Pyruvate uptake All cells were seeded in 24-well plates. Each group of cells was washed with PBS and incubated with a Hanks' Balanced Salt Solution (HBSS, 21-023-CV, Corning, USA)-based labeling medium containing sodium [3- 14 C]pyruvate (0.1 mCi/ml, ARC0220, American Radiolabeled Chemicals, USA) diluted 1:2000 and 0.5% fatty acid-free bovine serum albumin (BSA, A7030, Sigma-Aldrich, USA) in each well for 0, 2, 4, and 8 min at 37°C. The cells were then washed twice with PBS and lysed with 1% sodium dodecyl sulfate (SDS). The radioactivity of the cell lysates was measured after dilution with scintillation buffer using a Liquid Scintillation Counter (Tri-carb 2910 TR, Perkin Elmer, USA). Cell uptake was corrected for activity added and normalized to the total protein concentration at the time of the assay (% of uptake/mg). Total protein concentrations were determined using the BCA protein assay kit (ThermoFisher, USA). Statistical analysis was performed using Pearson correlation coefficients from Fig. 2 a (R 2 = 0.9728), two-way ANOVA (Fig. 2 b), and unpaired t-test (Fig. 2 d). [ 14 C]CO 2 measurement All cells were seeded in 35 mm culture plates. For the [ 14 C]CO 2 capture assay, a Whatman filter (Millipore Sigma, USA) was placed in the plate cap and moistened with 100 µL of 40% KOH, according to established procedures 34 . The cells were treated with sodium [3- 14 C]pyruvate as described above and incubated at 37°C for 8 minutes. After incubation, the cells were washed twice with PBS and lysed using 1% SDS. The filter was suspended in scintillation buffer for counting on the Liquid Scintillation Counter. In parallel, cell lysates and the remaining supernatants were mixed with a scintillation buffer for radioactivity counting. Statistical analysis was performed using one-way ANOVA (Fig. 2 f and Supplementary Fig. 2h). Cell viability The viability of the HCM treated with DOX or UK5099 was determined using the trypan blue method. Briefly, after cell harvesting by trypsinization and centrifugation, cell pellets were resuspended in PBS. An equal volume of trypan blue solution (ThermoFisher, USA) was added to the suspension and gently mixed. The mixture was incubated for 3 minutes at room temperature. Subsequently, 10 µL of the stained cell suspension was loaded onto a hemocytometer and examined under a light microscope. Viable cells (excluding dye) and non-viable cells (blue-stained) were counted manually, and the percentage of viable cells was calculated. Statistical analysis was performed using one-way ANOVA. Cell counting HCMs were seeded in 96-well plates. Seven days following DOX treatment, both control and DOX-treated groups were washed with PBS and incubated with a CCK-8 solution for 2 h. Subsequent steps were performed following the manufacturer’s instructions (#K1018, APExBIO, USA). Extracellular pH measurement All cells were seeded in 24-well plates and treated with either DOX or UK5099 as described above. Control cells received an equal volume of DMSO. Each group of cells was washed with PBS and incubated in HBSS medium for 8 min at 37°C. The supernatant was collected, and the pH was measured using pH-Test 4.5–10.0 indicator strips (VWR Chemicals, USA). Statistics Statistical analyses were performed as described using GraphPad Prism. Declarations Competing Interests Unrelated to this work, K.R.K. is co-founder of Atish Technologies and serves on the Scientific Advisory Boards of NVision Imaging Technologies, Imaginostics and Mi2. He holds patents related to imaging and leveraging cellular metabolism. The other authors declare no competing interests. Author Contribution J.M.K. secured funding for the project. J.M.K., J.W.B., and K.R.K. designed the experiments. C.H.L., T.R., S.D., A.W., G.F., and J.M.K performed the experiments and analyzed the data. S.N. and J.M.K. performed the kinetic modeling. C.H.L., T.R., and J.M.K. wrote the manuscript. All authors reviewed the manuscript. Acknowledgement The authors wish to acknowledge the assistance of the Weill Cornell Medicine Proteomics and Metabolomics Core Facility for the stable isotope metabolomics experiments, the Weill Cornell Medicine Genomics Core Facility for the bulk RNAseq experiments, and the Citigroup Biomedical Imaging Center at Weill Cornell Medicine for the PET imaging experiments. Data Availability Data is provided within the manuscript or supplementary information files. DICOM files for PET images are available from the authors upon request. References Martin, S. S. et al. 2025 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation 151 , e41-e660 (2025). https://doi.org/10.1161/CIR.0000000000001303 Lindner, J. R. & Morello, M. 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Front Oncol 11 , 719922 (2021). https://doi.org/10.3389/fonc.2021.719922 Ritterhoff, J. et al. Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis. Circ Res 126 , 182-196 (2020). https://doi.org/10.1161/CIRCRESAHA.119.315483 Bjurling, P., Watanabe, Y. & Långström, B. The synthesis of [3-11C] pyruvic acid, a useful synthon, via an enzymatic route. International Journal of Radiation Applications and Instrumentation. Part A. Applied Radiation and Isotopes 39 , 627-630 (1988). Arias-Hidalgo, M. et al. CO(2) permeability and carbonic anhydrase activity of rat cardiomyocytes. Acta Physiol (Oxf) 221 , 115-128 (2017). https://doi.org/10.1111/apha.12887 Cho, A., Eskandari, R., Miloushev, V. Z. & Keshari, K. R. A non-synthetic approach to extending the lifetime of hyperpolarized molecules using D(2)O solvation. J Magn Reson 295 , 57-62 (2018). https://doi.org/10.1016/j.jmr.2018.08.001 Deh, K. et al. First in-human evaluation of [1-(13)C]pyruvate in D(2)O for hyperpolarized MRI of the brain: A safety and feasibility study. Magn Reson Med 91 , 2559-2567 (2024). https://doi.org/10.1002/mrm.30002 Pardo Sanz, A. & Zamorano, J. L. 'Cardiotoxicity': time to define new targets? Eur Heart J 41 , 1730-1732 (2020). https://doi.org/10.1093/eurheartj/ehaa013 Lopez-Sendon, J. et al. Classification, prevalence, and outcomes of anticancer therapy-induced cardiotoxicity: the CARDIOTOX registry. Eur Heart J 41 , 1720-1729 (2020). https://doi.org/10.1093/eurheartj/ehaa006 Thavendiranathan, P. et al. Use of myocardial strain imaging by echocardiography for the early detection of cardiotoxicity in patients during and after cancer chemotherapy: a systematic review. J Am Coll Cardiol 63 , 2751-2768 (2014). https://doi.org/10.1016/j.jacc.2014.01.073 Wang, S., Wang, Y. & Wang, S. The role of stress echocardiography in identifying cardiotoxicity: an in-depth exploration. 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Nucleic Acids Res 31 , 258-261 (2003). https://doi.org/10.1093/nar/gkg034 Kim, S. H. et al. Cardiomyocyte-targeted siRNA delivery by prostaglandin E(2)-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis. Biomaterials 29 , 4439-4446 (2008). https://doi.org/10.1016/j.biomaterials.2008.07.047 Additional Declarations No competing interests reported. Supplementary Files 311CPyruvatePETMPCSupplementary.docx floatimage6.jpeg Graphical abstract Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviews received at journal 06 Oct, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers invited by journal 07 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Submission checks completed at journal 27 Aug, 2025 First submitted to journal 26 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7465913","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":511433742,"identity":"c8269480-7d1b-47f3-ad17-9c6f78d5da41","order_by":0,"name":"Chul-Hee Lee","email":"","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chul-Hee","middleName":"","lastName":"Lee","suffix":""},{"id":511433745,"identity":"f0e80752-dc01-4dc3-9be3-049160d4b21f","order_by":1,"name":"Thomas Ruan","email":"","orcid":"","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Ruan","suffix":""},{"id":511433746,"identity":"e5b264aa-eebf-43ee-b684-b2a2d645c270","order_by":2,"name":"Shuvra Debnath","email":"","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuvra","middleName":"","lastName":"Debnath","suffix":""},{"id":511433747,"identity":"53e5fb8c-8f63-4395-825c-b13fb6c12b94","order_by":3,"name":"Anja S. 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Babich","email":"","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"W.","lastName":"Babich","suffix":""},{"id":511433754,"identity":"d424552a-e85e-4b08-b26b-c38343dba2be","order_by":6,"name":"Sadek Nehmeh","email":"","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sadek","middleName":"","lastName":"Nehmeh","suffix":""},{"id":511433756,"identity":"d2686597-0491-4ff7-85af-cffec38fe830","order_by":7,"name":"Kayvan R. Keshari","email":"","orcid":"","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Kayvan","middleName":"R.","lastName":"Keshari","suffix":""},{"id":511433757,"identity":"3a81ef8d-6607-4617-9755-a07ffa20b1db","order_by":8,"name":"James M. Kelly","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACxgY2xgcJPDZyQLYBRIiduYGBgQ2vFmaDDzJpxggtzIz4tQAl2SRn2BxKbCBaC3P7sQRpnpwD6Wvbmzcw/Ki4l8cP0vKh7DBuh/WkHTDmOXMnd9uZYwWMPWeKiyWbGRsYZ5zDo6UhvSGZt+dZ7rYbOQbMjG0JiRsOMzYw87bh0dL/vOEw77/D6Wb330C07Adp+YtPy4y0g40zeA4nmN3ggdoC9AuQgU/Ls2SGDzxphtvOpBUc7DmTkDgDaMvBnnPpOLUY9qeZ/wBGpbzZ8cMbH/yoSEjsb28++OBHmTVuLQ1InAMYDGxAHp/kKBgFo2AUjAIwAADQvF4Qltov5AAAAABJRU5ErkJggg==","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"M.","lastName":"Kelly","suffix":""}],"badges":[],"createdAt":"2025-08-26 20:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7465913/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7465913/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91198281,"identity":"634e54ef-d3d8-494d-a94d-7c9671b81204","added_by":"auto","created_at":"2025-09-12 15:14:18","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":485379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDOX exposure leads to decreased cardiac pyruvate metabolism in mice. (a) \u003c/strong\u003eSelected significantly downregulated KEGG pathways at 4 weeks after DOX exposure. \u003cstrong\u003e(b)\u003c/strong\u003e Selected significantly downregulated GO:BPs at 4 weeks after DOX exposure. The number of genes in each pathway is indicated in parenthesis. \u003cstrong\u003e(c)\u003c/strong\u003e EnhancedVolcano plot from the bulk RNA sequencing performed on cardiac tissue samples collected at 4 weeks (Log2 fold change (FC) \u0026gt; |1|, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Selected genes associated with pyruvate transport and metabolism, Mpc1, Mpc2, Pdhb, Pdha1, Dlat, Dld, Vdac1, Pdhx, Slc16a1 (monocarboxylate transporter 1), and Slc16a3 (monocarboxylate transporter 4) are highlighted. \u003cstrong\u003e(d)\u003c/strong\u003e Western blot analysis of cardiac MPC1 and MPC2 expression. HSP60 was used as a reference. \u003cstrong\u003e(e)\u003c/strong\u003e ROI quantification of each protein level was performed using ImageJ.\u003cstrong\u003e (f)\u003c/strong\u003e Map of TCA cycle intermediates assessed during [3-\u003csup\u003e13\u003c/sup\u003eC]pyruvate stable isotope tracing metabolomics experiments. The illustration was created with BioRender. \u003cstrong\u003e(g-k)\u003c/strong\u003e Comparison of total pool size and M+0 and M+1 fractional enrichments of lactate (\u003cstrong\u003eg\u003c/strong\u003e), alanine (\u003cstrong\u003eh\u003c/strong\u003e), citrate (\u003cstrong\u003ei\u003c/strong\u003e), and glutamate (\u003cstrong\u003ej\u003c/strong\u003e) between the control and DOX groups in hears collected 10 min p.i. of [3-\u003csup\u003e13\u003c/sup\u003eC]pyruvate. The experiment was performed 4 weeks after first DOX exposure. Data are presented as the mean ± s.d. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. Statistical analysis was performed using multiple unpaired t-tests (\u003cstrong\u003ee\u003c/strong\u003e) or two-way ANOVA (\u003cstrong\u003eg-j\u003c/strong\u003e). DOX = doxorubicin; CTRL = control; MPC = mitochondrial pyruvate carrier; GO:BP = gene ontology:biological process; KEGG = Kyoto Encyclopedia of Genes and Genomes; TCA = tricarboxylic acid; HSP60 = heat shock protein 60; ROI = Region of interest.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/f6e8e006b7634b2f752cfa3b.jpeg"},{"id":91198283,"identity":"aae136e7-a1e2-412d-8455-8934c5dd94ba","added_by":"auto","created_at":"2025-09-12 15:14:18","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":336943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDOX exposure affects pyruvate uptake by reducing MPC1/2 in HCM. (a) \u003c/strong\u003e[3-\u003csup\u003e14\u003c/sup\u003eC]Pyruvate flux in HCMs was evaluated in a time-dependent manner (0, 2, 4, 6, and 8 min). Cells were treated with vehicle (control; CTRL) or 0.1 µM DOX for 2 x 48 h (n=6 per condition). \u003cstrong\u003e(b)\u003c/strong\u003e Western blot analysis of MPC1, MPC2, and MCT1 expression from HCM treated with vehicle (CTRL), UK5099 (100 µM for 72 h), or DOX (0.1 µM for 2 x 48 h). Vinculin was used as a reference. \u003cstrong\u003e(c) \u003c/strong\u003e[3-\u003csup\u003e14\u003c/sup\u003eC]Pyruvate uptake was assessed in HCMs treated with vehicle (CTRL), UK5099 (100 µM for 72 h), or DOX (0.1 µM for 2 x 48 h) for 8 min. \u003cstrong\u003e(d) \u003c/strong\u003e[3-\u003csup\u003e14\u003c/sup\u003eC]Pyruvate uptake in HCM treated with scramble siRNA (100 nM for 24 h) or MPC1/2 siRNA (50 nM of each MPC siRNA - total concentration of 100 nM - for 24 h) for 8 min. \u003cstrong\u003e(e) \u003c/strong\u003e\u0026nbsp;Hypothesis describing the effect of DOX treatment on the cellular fate of [3-\u003csup\u003e14\u003c/sup\u003eC]pyruvate, which can be retained within the cell as [3-\u003csup\u003e14\u003c/sup\u003eC]pyruvate or another labeled metabolite, released into the media as [3-\u003csup\u003e14\u003c/sup\u003eC]lactate, [3-\u003csup\u003e14\u003c/sup\u003eC]pyruvate, or another species, or released to the air as [\u003csup\u003e14\u003c/sup\u003eC]CO₂. The illustration was created with BioRender. \u003cstrong\u003e(f)\u003c/strong\u003e [\u003csup\u003e14\u003c/sup\u003eC]CO₂ capture assay was performed following an 8-min incubation of HCM with [3-\u003csup\u003e14\u003c/sup\u003eC]pyruvate. Data are presented as the mean ± s.d. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Statistical analysis was performed using Pearson correlation coefficients (a; R\u003csup\u003e2\u003c/sup\u003e = 0.9728), two-way ANOVA (c), unpaired t-test (d), or one-way ANOVA (f). HCM = human cardiomyocytes.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/9577c63721fd7e924b31c1df.jpeg"},{"id":91198284,"identity":"5887a0fb-4573-4e71-bd8d-0c638c7c19b1","added_by":"auto","created_at":"2025-09-12 15:14:18","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":200443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[3-\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC]Pyruvate PET reveals DOX-induced alterations in cardiac pyruvate flux at 4 weeks. (a) \u003c/strong\u003eDescription of [3-\u003csup\u003e11\u003c/sup\u003eC]pyruvate dynamic PET/CT imaging. The lateral tail vein was cannulated for intravenous (i.v.) administration before imaging. \u003cstrong\u003e(b)\u003c/strong\u003e Representative summed sagittal PET images from the first 10 min post-injection in mice at the 4-week time point. \u003cstrong\u003e(c) \u003c/strong\u003eTACs\u003cstrong\u003e \u003c/strong\u003eover the initial 10-min PET acquisition period. Individual activity measurements were normalized to maximum uptake. \u003cstrong\u003e(d)\u003c/strong\u003e Cardiac lactate/pyruvate ratio derived from HP [1-\u003csup\u003e13\u003c/sup\u003eC]pyruvate MR spectra. Mice (n=3 in the CTRL group and n=2 in the DOX group) were injected multiple times to provide a total of 10-12 independent measurements. \u003cstrong\u003e(e)\u003c/strong\u003e Cardiac bicarbonate/lactate ratios from the control (n=2) and DOX (n=2) groups. Data are presented as the mean ± s.d. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Statistical analysis was performed using the Mann-Whitney test between CTRL and DOX (c) or an unpaired t-test (d, e). PET = positron emission tomography; TAC = Time-activity curve; HP = Hyperpolarized; MR = magnetic resonance.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/53b94a05e12c56b201f7a3b0.jpeg"},{"id":91199676,"identity":"9b15978f-2c74-44aa-85f3-43dc72012a2c","added_by":"auto","created_at":"2025-09-12 15:22:18","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMPC1/2 expression rebounds 16 weeks after exposure in concert with recovery of cardiac mass. (a) \u003c/strong\u003eHW/TL ratio at 16 weeks after DOX exposure. \u003cstrong\u003e(b)\u003c/strong\u003e EnhancedVolcano plot from the bulk RNA sequencing at 16 weeks (Log2 fold change (FC) \u0026gt; |0.5|, FDR \u0026lt; 0.05). The Mpc1 and Mpc2 genes are highlighted. \u003cstrong\u003e(c)\u003c/strong\u003e Western blot analysis of MPC1 and MPC2 expression in cardiac tissue collected at 16 weeks. HSP60 was used as a reference. \u003cstrong\u003e(d)\u003c/strong\u003e ROI quantification of each protein level was performed using ImageJ.\u003cstrong\u003e \u003c/strong\u003eData are presented as the mean ± s.d. ** \u003cem\u003ep \u0026lt; 0.01; \u003c/em\u003e****\u003cem\u003e p \u0026lt; 0.0001\u003c/em\u003e. Statistical analysis was performed using a unpaired t-test (a) or multiple unpaired t-tests (d). HW/TL = heart weight-to-tibia length; FDR = false discovery rate.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/522d9e7e0b864f7fa953efa8.jpeg"},{"id":91198287,"identity":"31c75d60-3893-4298-aa99-f829522fa1fa","added_by":"auto","created_at":"2025-09-12 15:14:18","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[3-\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC]Pyruvate PET imaging captures changes in cardiac pyruvate metabolism. \u003c/strong\u003eComparison of total pool size and M+0 and M+1 fractional enrichments of \u003cstrong\u003e(a)\u003c/strong\u003e lactate, \u003cstrong\u003e(b) \u003c/strong\u003ealanine, (\u003cstrong\u003ec\u003c/strong\u003e) glutamate, and (\u003cstrong\u003ed\u003c/strong\u003e) citrate assessed during [3-\u003csup\u003e13\u003c/sup\u003eC]pyruvate stable isotope tracing metabolomics experiments. The experiment was performed 16 weeks after first DOX exposure. Heart samples were collected 10 min post-injection of [3-\u003csup\u003e13\u003c/sup\u003eC]pyruvate. \u003cstrong\u003e(e)\u003c/strong\u003e Representative summed sagittal PET images from the first 10 min post-injection of [3-\u003csup\u003e11\u003c/sup\u003eC]pyruvate in mice at the 16-week time point. \u003cstrong\u003e(f) \u003c/strong\u003eTACs\u003cstrong\u003e \u003c/strong\u003econstructed using an ROI over the whole heart\u003cstrong\u003e \u003c/strong\u003eover the initial 10-min acquisition period. Activities at each measurement were normalized to maximum uptake. Data are presented as the mean ± s.d. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u0026nbsp;Statistical analysis was performed using a two-way ANOVA (a) or the Mann-Whitney test (c). TCA = Tricarboxylic acid; PET = positron emission tomography; TAC = time-activity curve; ROI = Region-of-interest.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/1eb7f011387fef323ff22175.jpeg"},{"id":91201164,"identity":"a051a223-f9ff-47c2-a565-91d912d652c3","added_by":"auto","created_at":"2025-09-12 15:38:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3080609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/ef80c267-8ffd-4f38-b9b2-baed5da4f222.pdf"},{"id":91200642,"identity":"dc22fc70-c59d-4d5c-b014-7abb78dfea32","added_by":"auto","created_at":"2025-09-12 15:30:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1618043,"visible":true,"origin":"","legend":"","description":"","filename":"311CPyruvatePETMPCSupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/48454ab039cfcafc1501c783.docx"},{"id":91198282,"identity":"5393fdce-a71f-4ded-9309-c81369de8b0c","added_by":"auto","created_at":"2025-09-12 15:14:18","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":815554,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7465913/v1/77ae7d9d4f59c0c84e78e9e4.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e[3-\u003csup\u003e11\u003c/sup\u003eC]Pyruvate PET detects alterations in cardiac pyruvate metabolism induced by doxorubicin chemotherapy\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeart disease is a leading cause of death worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although the etiology of heart failure is diverse, metabolic perturbation is a common feature. Given the prevalence of this condition, methods of imaging cardiac metabolism could play a critical role in the treatment of millions of patients. Significantly, metabolic dysfunction precedes functional and structural changes, thereby identifying these processes as diagnostic and therapeutic targets of great potential significance. In this context, molecular and cellular imaging using probes targeting cardiac metabolism potentially offers a powerful approach to advancing precision medicine in cardiology\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHeart failure may present with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF). One important subset of HFrEF is anthracycline cardiotoxicity. Anthracycline chemotherapeutics (e.g., doxorubicin, DOX) are widely used and effective against numerous cancers\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, their use is compromised by the development of cardiotoxicity. This condition may present as acute, early (within 1 year of treatment), or late onset (several years after treatment). There are no established strategies for distinguishing between reversible and progressive cardiac dysfunction\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Furthermore, predicting late onset disease is challenging due to the combination of treatments that patients may receive and the lack of biomarkers of cardiac damage that precede dysfunction. These observations highlight the critical need to develop methods of early detection of anthracycline-induced cardiac injury that are independent of assessments of cardiac structure and function to enable timely intervention that may prevent irreversible cardiac damage and long-term heart failure.\u003c/p\u003e\u003cp\u003eThe critical role of metabolic reprogramming in promoting cardiac functional and structural changes after DOX exposure is increasingly recognized\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, but there remains a lack of extensive longitudinal studies examining the key molecular mediators that regulate these adaptations. Mitochondria are known targets of DOX-induced toxicity in cardiomyocytes. These cells have the highest mitochondria content of any cell type\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, highlighting the value of targeting biochemical processes that occur in mitochondria as a means of imaging pathological cardiac metabolic reprogramming. Under normal conditions, the heart derives more than 95% of its ATP from oxidative phosphorylation (OXPHOS)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, which relies on oxidation of substrates in the tricarboxylic acid (TCA) cycle. In HFrEF, glucose consumption by the TCA cycle decreases as glycolysis becomes uncoupled from OXPHOS\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Pyruvate is a key metabolite that links glycolysis to mitochondrial oxidative metabolism. The potential for using this metabolite to image physiological cardiac metabolism and disease-related changes has been demonstrated by hyperpolarized (HP) [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate magnetic resonance imaging (MRI) in both animals and human subjects\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Despite the encouraging developments in HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate cardiac MRI, the short half-life of the hyperpolarized species (T\u003csub\u003e1\u003c/sub\u003e relaxation time\u0026thinsp;=\u0026thinsp;1 min) and the requirement for supra-physiological masses of pyruvate may ultimately limit some applications of this technology for cardiac imaging\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this context, it is notable that \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC-labeled pyruvate positron emission tomography (PET) has not been widely pursued as a complementary or alternative imaging strategy. The physical properties of carbon-11 (t\u003csub\u003e1/2\u003c/sub\u003e = 20.4 min), which decays almost entirely by positron emission (β\u003csup\u003e+\u003c/sup\u003e = 99.8%, E\u003csub\u003eavg\u003c/sub\u003e = 385.7 keV), render \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC-labeled pyruvate an intriguing candidate for imaging changes in cardiac pyruvate metabolism. Two isotopologues of \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC-labeled pyruvate, [1-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate and [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate, are available in sufficient activities and purities for PET imaging\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Pyruvate requires monocarboxylate transporters (MCTs) for transport into cells and the mitochondrial pyruvate carriers 1 and 2 (MPC1/2) for transport across the mitochondrial membrane\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Since the discovery of MPC1/2 as essential transporters for pyruvate across the mitochondrial membrane, their role in cardiac metabolic changes associated with heart failure has been increasingly recognized\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Inhibition of pyruvate transport by DOX in rat cardiomyocytes was previously reported\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, suggesting that MPC1/2 might contribute to DOX-induced metabolic disruption in cardiomyocytes. In this study, we hypothesize that DOX exposure compromises murine cardiac pyruvate metabolism by disrupting its transport and reducing expression of MCT and MPC1/2. Most notably, we hypothesize that DOX-induced changes in cardiac MPC1/2 expression can be detected and quantified through dynamic cardiac PET imaging using [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate. We demonstrate that cardiac [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate flux is inversely related to MPC expression and use stable isotope tracing and HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate MRI to confirm that increased flux corresponds to decreased utilization of pyruvate by the TCA cycle.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMPC1/2 expression decreases in the murine heart after exposure to doxorubicin\u003c/h2\u003e\u003cp\u003eTo assess the initial effect of DOX exposure on cardiac metabolism, we analyzed cardiac tissue samples collected from mice 4 weeks after the first administration of DOX. At this point, we previously observed cardiac atrophy in the mice exposed to DOX\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, a finding that we confirmed in this study (Supplemental Fig.\u0026nbsp;1a). We performed bulk RNA sequencing analysis on these tissues and identified 1618 differentially expressed genes (DEGs) between the DOX and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Log2 fold change (FC) \u0026gt;|1|). The DEGs were used for Gene Ontology (GO) and KEGG pathway enrichment using the DAVID and STRING databases. Among the most significantly downregulated pathways were those involving pyruvate metabolism and processes supporting mitochondrial function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b, and Supplementary Tables\u0026nbsp;1, 2). These findings are consistent with prior observations of mitochondrial oxidative stress induced by DOX\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. From the EnhancedVolcano plot of the DEGs, we identified 9 significantly downregulated genes associated with pyruvate transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Two of these genes, Mpc1 and Mpc2, are highly expressed in the hearts of mice and humans (Supplemental Fig.\u0026nbsp;1b, c, and Supplementary Table\u0026nbsp;3) and encode subunits of the mitochondrial pyruvate carrier (MPC), which assembles as a heterodimer to facilitate pyruvate import into the mitochondrial matrix\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Given prior reports of MPC expression deficits in other cardiac pathologies\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and the interaction between MPC1/2 and the pyruvate dehydrogenase complex, which catalyzes the irreversible conversion of pyruvate to acetyl-CoA (Supplemental Fig.\u0026nbsp;1d and Supplementary Table\u0026nbsp;4), we evaluated the protein expression levels of MPC1 and MPC2 in the heart tissue samples. There was a significant reduction in MPC1 and MPC2 expression in the tissue taken from the mice exposed to DOX compared to the age-matched controls (p\u0026thinsp;=\u0026thinsp;0.0248 for MPC1; p\u0026thinsp;=\u0026thinsp;0.0028 for MPC2; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). In parallel, we observed a significant reduction in MCT1 transcripts (Slc16a1) and expression in these tissues (p\u0026thinsp;=\u0026thinsp;0.0300; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Supplemental Fig.\u0026nbsp;1e). By contrast, expression of MCT4 (Slc16a3) was not significantly different in the mice exposed to DOX compared to controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we evaluated myocardial carbohydrate metabolism and tricarboxylic acid (TCA) cycle flux by stable isotope tracing in mice using [3-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate at the 4-week time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). We chose this time point for the experiment because prior studies in healthy mouse hearts indicated that incorporation of multiple isotopic labels into TCA cycle intermediates, corresponding to multiple iterations of the TCA cycle, was possible when samples were collected 10 min post-injection (p.i.; Supplemental Fig.\u0026nbsp;1f). These experiments revealed no significant changes in the total cardiac lactate, alanine, citrate, and glutamate pools in hearts collected from the animals exposed to DOX at 10 minutes p.i. compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-j), although the total metabolite pool size was significantly lower (p\u0026thinsp;=\u0026thinsp;0.0079) in the mice exposed to DOX (Supplemental Fig.\u0026nbsp;1g). Fractional enrichment of the M\u0026thinsp;+\u0026thinsp;1 peak in lactate was slightly, but not statistically significantly, increased in the hearts from the DOX animals (n\u0026thinsp;=\u0026thinsp;5) compared to the controls (0.19 \u003cem\u003evs\u003c/em\u003e. 0.12; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), although we observed considerable variability within this group. We additionally observed reduced expression of lactate dehydrogenase (LDH) A and B, while there was no change in alanine aminotransferase (ALT) at the 4-week time point in mice exposed to DOX (Supplemental Fig.\u0026nbsp;1e). In agreement with the lactate data, M\u0026thinsp;+\u0026thinsp;1 fractional enrichment of alanine increased in the hearts exposed to DOX, although the variability within this group ensured that the difference to the control hearts was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). By contrast, M\u0026thinsp;+\u0026thinsp;1 fractional enrichment of citrate (p\u0026thinsp;=\u0026thinsp;0.0230) and glutamate (p\u0026thinsp;=\u0026thinsp;0.0052) was significantly lower in the hearts extracted from the mice exposed to DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei and j). Similarly, M\u0026thinsp;+\u0026thinsp;1 fractional enrichment of other TCA cycle intermediates, including aconitate, fumarate, malate, oxoglutarate, and succinate, was significantly lower in the DOX hearts (Supplementary Fig.\u0026nbsp;1h). The pool size of these TCA cycle metabolites was generally comparable between the two groups, with only deficits in cardiac fumarate and malate in the DOX group reaching statistical significance. Given the reduced MPC1 and MPC2 expression in DOX-treated hearts and the corresponding decrease in M\u0026thinsp;+\u0026thinsp;1 enrichment of TCA cycle metabolites, our findings suggest that DOX exposure impairs pyruvate entry into mitochondria and promotes its conversion to lactate and alanine in the cytosol.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMPC1 and MPC2 are key mediators of pyruvate metabolism in human cardiomyocytes exposed to DOX\u003c/h3\u003e\n\u003cp\u003eTo test our hypothesis that DOX impairs mitochondrial pyruvate oxidation by targeting MPC1/2, we investigated whether DOX contributes to alterations in MPC1/2-mediated pyruvate metabolism in human cardiomyocytes. Consistent with previously established conditions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, human cardiomyocytes (HCM) were treated with 0.1 \u0026micro;M DOX for two consecutive 48-hour periods (Supplemental Fig.\u0026nbsp;2a). There was no difference in cell viability between groups (Supplemental Fig.\u0026nbsp;2b). We incubated control and DOX-treated HCM with [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate for up to 8 minutes and observed significantly decreased time-dependent retention of signal in the DOX-treated HCMs (p\u0026thinsp;=\u0026thinsp;0.0137; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The difference in uptake in the DOX-treated HCMs was then compared to HCM treated with UK5099, a selective pharmacological inhibitor of MPC,\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e for 72 hours before the addition of [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate, as shown in Supplemental Fig.\u0026nbsp;2a. These conditions were previously demonstrated to downregulate MPC1/2\u003csup\u003e33\u003c/sup\u003e. We confirmed downregulation of MPC1/2 in the UK5099- and DOX-treated cells and also determined by Western blotting that these treatments decreased expression of MCT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These experiments revealed that [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate uptake after 8 minutes was reduced by 2.3-fold in UK5099-treated HCM compared to the untreated control cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for CTRL versus UK5099 or DOX; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). By contrast, [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate uptake was comparable between the UK5099-treated HCM and the DOX-treated HCM. These results indicate that DOX-induced changes in pyruvate metabolism are comparable to those induced by UK5099. Treatment with DOX or UK5099 did not affect cell viability but did result in a lower pH (6.8 vs. 7.5) in the media (Supplemental Fig.\u0026nbsp;2c, d). We attributed this acidification to increased lactate production resulting from impaired mitochondrial import of pyruvate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo disentangle the contributions of MCT1 and MPC1/2 to reduced pyruvate metabolism in DOX-treated cardiomyocytes, we used MPC1- and MPC2-specific siRNA to prepare HCM with selective inhibition of MPC1/2 (Supplemental Fig.\u0026nbsp;2a, e). These cells demonstrated a 25% reduction in [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate uptake compared to the scrambled control cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), although there were no changes in the expression of MCT1, MCT4, LDHA, LDHB, ALT, or the PDH complex (Supplemental Fig.\u0026nbsp;2f). A comparison these results with the changes induced by DOX or UK5099 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) suggests that deficits in DOX-induced pyruvate flux in cardiomyocytes are attributable to both impaired transport of pyruvate across the cell membrane (loss of MCT1 expression) and impaired transport across the mitochondrial membrane (loss of MPC1/2 expression).\u003c/p\u003e\u003cp\u003eOne limitation of the uptake assay is that it is not possible to determine the chemical form of the \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC label after its addition to the well. There are three fates of the \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC label: it can be retained in the cell (as [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate, a TCA cycle intermediate, or another metabolite), released into the media (as [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]lactate or [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate), or released as [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee. We hypothesized that one major difference between HCM consuming [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate in the TCA cycle and HCM deficient in MPC would be the production of [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e, which could be formed after multiple iterations of the TCA cycle (Supplemental Fig.\u0026nbsp;2g). Consequently, we performed a [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e capture assay\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e following incubation of HCM with [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate for 8 min. This experiment confirmed that lower activities of [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e were evolved in the HCM with reduced MPC1/2 levels (p\u0026thinsp;=\u0026thinsp;0.0002 for CTRL versus MPC1/2 siRNA; p\u0026thinsp;=\u0026thinsp;0.0004 for CTRL versus UK5099; p\u0026thinsp;=\u0026thinsp;0.0137 for CTRL versus DOX; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The total \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC counts, representing the sum of the [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e captured, the cell-associated activity, and the remaining activity in the media, did not change (Supplemental Fig.\u0026nbsp;2h). Taken together, our findings indicate that the DOX-induced reduction in pyruvate metabolism in cardiomyocytes reflects impaired pyruvate transport and is partially mediated by MPC1/2.\u003c/p\u003e\n\u003ch3\u003e[3-C]Pyruvate PET detects DOX-induced changes in cardiac pyruvate flux at 4 weeks\u003c/h3\u003e\n\u003cp\u003eGiven the potential significance of cardiac MPC as a target of DOX, we aimed to develop a method for assessing MPC levels in vivo. We hypothesized that measurements of the cardiac flux of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate by positron emission tomography (PET) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) would highlight deficiencies in pyruvate transport and thereby permit MPC expression to be inferred. The rationale for this hypothesis was the difference in [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e production between cells that utilize pyruvate in the TCA cycle and cells that do not. Based on our in vitro studies, we anticipated rapid clearance of [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e from cardiac tissue and a correspondingly rapid activity flux in hearts without impaired transport. To test our hypothesis, we acquired 30 minute dynamic PET images upon intravenous administration of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate to mice exposed to DOX (n\u0026thinsp;=\u0026thinsp;10) and age-matched controls (n\u0026thinsp;=\u0026thinsp;10) at the 4-week time point (Supplemental Fig.\u0026nbsp;3a). Summed sagittal-axis PET images over the first 10 minutes post-injection showed a markedly enhanced [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate signal in the DOX group at 4 weeks compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Consistent with PET images and [3-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate metabolomics, the time-activity curves (TACs) drawn using regions-of-interest (ROIs) over the whole heart from the 10-minute acquisitions\u0026mdash;normalized to peak uptake\u0026mdash;highlighted a slower clearance of activity from the hearts of mice in the DOX group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Normalization to peak uptake allows us to account for differences in injected activity and heart size. This translated to a significantly increased transit time of 320\u0026thinsp;\u0026plusmn;\u0026thinsp;52 s in the animals exposed to DOX (n\u0026thinsp;=\u0026thinsp;9) compared to 272\u0026thinsp;\u0026plusmn;\u0026thinsp;36 s in the control group (n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;=\u0026thinsp;0.0355). Three mice, 1 from the DOX group and 2 from the control group, were excluded from the analysis because the cardiac transit time in these animals was greater than 2 standard deviations from the mean. The transit time is proportional to the clearance rate from cardiac tissue and supports the conclusion that [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e is not generated as quickly in the hearts exposed to DOX.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs a further test of our hypothesis, we performed magnetic resonance spectroscopy (MRS) of hyperpolarized (HP) [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate MRI in the two groups at 4 weeks. In contrast to PET, MRS of HP pyruvate allows the metabolism of pyruvate to be directly observed in the mouse heart\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Although the HP tracer bore the isotopic label at the C-1 position rather than the C-3 position, the metabolism of these two isotopologues would be expected to be identical through PDH-catalyzed entry into the TCA cycle. We observed a significant increase in the cardiac lactate/pyruvate ratio in the mice exposed to DOX (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This is consistent with enhanced lactate production resulting from decreased utilization of pyruvate by the TCA cycle. Indeed, in a separate experiment using \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR, we measured a higher lactate pool in cardiac tissues from mice in the DOX group compared to the control group (Supplementary Fig.\u0026nbsp;3b). In addition, the bicarbonate/lactate ratio was significantly lower for the mice from the DOX group (p\u0026thinsp;=\u0026thinsp;0.0260; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). The alanine/lactate ratio did not change between groups (Supplementary Fig.\u0026nbsp;3c). Collectively, these studies support the interpretation that slower cardiac pyruvate flux corresponds to lower pyruvate oxidation and higher conversion to lactate, and that flux measurements can be used to infer MPC expression in the heart.\u003c/p\u003e\n\u003ch3\u003eRecovery of MPC1/2 expression is coincident with accelerated cardiac growth\u003c/h3\u003e\n\u003cp\u003eExposure to DOX is known to induce cardiac atrophy followed by a period of accelerated growth\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, we sought to investigate whether MPC1/2 expression changes during the growth phase. For this purpose, we collected heart tissue from mice 16 weeks after the first administration of DOX. The heart-weight-to-tibia-length (HW/TL) ratio was 5.94 for these mice, compared to 7.55 for the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). At 4 weeks, the ratios were 7.69 and 5.13, respectively. As such, the HW/TL ratio in DOX-treated tissues at 16 weeks demonstrated a 1.2-fold increase relative to those at 4 weeks of DOX exposure (p\u0026thinsp;=\u0026thinsp;0.0023; Supplementary Fig.\u0026nbsp;4a). We correspondingly observed increased expression of markers of cardiomyocyte growth and cardiomyopathy, such as p53\u003csup\u003e37\u003c/sup\u003e and p21\u003csup\u003e38\u003c/sup\u003e, as shown in Supplementary Fig.\u0026nbsp;4b. Bulk RNA sequencing analysis conducted at 16 weeks (false discovery rate; FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for KEGG pathways reinforced the growth profile of the heart, as shown in Supplementary Fig.\u0026nbsp;4c and Supplemental Table\u0026nbsp;5. Additionally, the EnhancedVolcano plot of the DEGs revealed neither Mpc1 (Log2FC = -0.00012) and Mpc2 (Log2FC = -0.02665) nor Mct1 (Log2FC\u0026thinsp;=\u0026thinsp;0.00508) and Mct4 (Log2FC = -0.04813) to be significantly different between the mice exposed to DOX and the control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Similarly, and in contrast to the expression profile in the samples collected at 4 weeks, there were neither significant differences in MPC1 and MPC2 protein expression nor MCT1 and MCT4 expression between the mice in the DOX and control groups at 16 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d, Supplemental Fig.\u0026nbsp;4d). Collectively, these observations suggest that recovery of pyruvate transport, including MCT1 and MPC1/2 expression, after DOX exposure is associated with accelerated cardiac growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCardiac [3-C]pyruvate flux reflects longitudinal changes in MPC1/2 expression\u003c/h3\u003e\n\u003cp\u003eHaving observed the recovery of MCT1 and MPC1/2 expression at 16 weeks, we sought to determine how these changes affected metabolism. We conducted stable isotope tracing metabolomics at 16 weeks using the same methodology as at 4 weeks. In contrast to the 4-week time point, we detected significantly increased M\u0026thinsp;+\u0026thinsp;1 fractional enhancement of lactate (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), alanine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and glutamate (p\u0026thinsp;=\u0026thinsp;0.0002) in the animals exposed to DOX, while fractional enhancement of citrate was comparable between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). The total pool of lactate was comparable to the control samples, but the alanine pool was significantly greater (p\u0026thinsp;=\u0026thinsp;0.0295), and the glutamate and citrate pools were significantly lower (p\u0026thinsp;=\u0026thinsp;0.0157 and p\u0026thinsp;=\u0026thinsp;0.0086, respectively) in the hearts exposed to DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). Notably, we observed substantially increased incorporation of the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC label into metabolites associated with the TCA cycle, with M\u0026thinsp;+\u0026thinsp;1 fractional enhancement of key metabolites for TCA cycle, including aconitate, fumarate, malate, oxoglutarate, and succinate, comparable or significantly greater than the corresponding controls (Supplemental Fig.\u0026nbsp;5a). However, the total pool size of these metabolites \u0026ndash; and the total of all metabolites detected \u0026ndash; was significantly lower (p\u0026thinsp;=\u0026thinsp;0.0035) in the mice exposed to DOX than the controls (Supplemental Fig.\u0026nbsp;5b), evidencing metabolic reprogramming even 16 weeks after the initial DOX exposure. Expression of other key components of pyruvate metabolism, such as LDHA, LDHB, and ALT, as well as MCT1 and MCT4, was comparable between the groups, as shown in Supplemental Fig.\u0026nbsp;4d, suggesting recovery of pyruvate transport. A similar trend was evident in genes encoding key proteins involved in β-oxidation, whose expression was significantly increased at 16 weeks compared to 4 weeks (Supplemental Fig.\u0026nbsp;5c), albeit without recovering to the expression levels in the control hearts. These findings highlight the restoration of pyruvate flux through mitochondria at 16 weeks.\u003c/p\u003e\u003cp\u003eWhen these mice were imaged by [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate PET, we observed no significant difference in cardiac flux between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In contrast to the 4-week time point, [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate flux in the DOX-treated mice (n\u0026thinsp;=\u0026thinsp;7) was more rapid than in the control animals (n\u0026thinsp;=\u0026thinsp;4) during the first 10 minutes (p\u0026thinsp;=\u0026thinsp;0.0426; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). This was reflected in a transit time of 315\u0026thinsp;\u0026plusmn;\u0026thinsp;42 s for the DOX mice and 323\u0026thinsp;\u0026plusmn;\u0026thinsp;27 s for the control mice. Notably, the accelerated cardiac flux observed in the DOX group is consistent with restored pyruvate utilization via the TCA cycle. However, as in week 4, the cardiac time-activity curves for [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate trended towards convergence after 30 minutes (Supplemental Fig.\u0026nbsp;5d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next performed cardiac segmentation using the Carimas software package\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and performed compartmental modeling using the image-derived left ventricular input function. Using Akaike\u0026rsquo;s information criterion, we identified the two-tissue, four-parameter compartmental model as the best fit for 93% of the images (n\u0026thinsp;=\u0026thinsp;27/29), as shown in Supplemental Fig.\u0026nbsp;6. The two remaining curves were best modeled by a one-tissue compartment model. No compartment model fit the curves obtained from any of the three outliers that we excluded in our analysis at 4, highlighting the discrepancy between these animals and the bulk of our subjects. The two-tissue, four-parameter model is in agreement with that derived for the utilization of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]lactate by the porcine heart\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In this model, k4 represents the oxidation of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate to [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e. We compared the k4 for each of the four groups, as shown in Supplemental Table\u0026nbsp;6 and found a trend towards lower k4 values in the DOX mice at 4 weeks compared to the other groups, although these differences were not statistically significant. We excluded 2 additional mice (n\u0026thinsp;=\u0026thinsp;1 for DOX 4W and control 4W) due to k4 values that were two standard deviations from the mean. These mice received lower activities of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate upon injection, which likely resulted in increased noise in the early time points of the TAC. There were no statistically significant differences between groups for kinetic constants VA, K1, k2, or k3 (Supplemental Table\u0026nbsp;7).\u003c/p\u003e\u003cp\u003eOverall, our findings support the hypothesis that exposure to DOX causes rapid changes in cardiac pyruvate metabolism by modulating pyruvate transport in cardiomyocytes. The deficits in MCT1 and MPC1/2 expression contribute to decreased utilization of pyruvate by the TCA cycle. After the initial decrease, MPC1/2 expression (and MCT1 expression) is restored in concert with pyruvate oxidation and accelerated cardiac growth. Significantly, this dynamic process can be imaged in vivo using PET-based measurements of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate cardiac flux.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHealthy cardiac tissue generates the bulk of its energy from oxidizable substrates via processes that take place in mitochondria, which comprise a large part of the cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, during the onset and progression of doxorubicin-induced cardiotoxicity, cardiac tissue undergoes significant molecular and metabolic remodeling necessitated by the emergence of mitochondrial dysfunction. Impaired mitochondrial function may ultimately lead to cardiomyocyte apoptosis and cell death\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In this context, we identified pyruvate to potentially be a key metabolite in a rodent model of cardiotoxicity. Pyruvate, the end-product of glycolysis, plays a pivotal role in mitochondrial ATP production after its entry into the TCA cycle via PDH or its conversion to oxaloacetate via pyruvate carboxylase. One of the major regulators of this process is the MPC, which is exclusively responsible for mitochondrial pyruvate import\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and reported by some groups to be a rate-limiting step in pyruvate oxidation in the heart\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. As MPC can regulate both pyruvate oxidation and anaplerosis, it may contribute more significantly to cardiac metabolic reprogramming than PDH\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To this end, reduced expression or genetic knockout of MPC1/2 was recently shown to promote hypertrophic cardiomyopathy in murine models of heart failure\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e while MPC1/2 abundance promoted survival of porcine cardiac tissue following ischemia-reperfusion\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Similarly, reduced MPC1/2 expression has been observed in failing hypertrophic human hearts\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and patients that failed to respond to left ventricular assist device implantation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite this emerging evidence of the importance of MPC in hypertrophic cardiomyopathy, we are not aware of any studies that have investigated its expression in hearts at risk of cardiotoxicity. Although doxorubicin-induced cardiotoxicity shares some characteristics, such as reduced left ventricular ejection fraction (LVEF), of these cardiomyopathies, in murine models it is associated with an initial phase of cardiac atrophy rather than hypertrophy\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In this context, it is significant that we observed decreased MPC expression and activity when atrophy, as assessed by HW/TL ratio, was more pronounced, while expression and activity recovered during a phase of accelerated cardiac growth. The stable isotope tracing and PET imaging experiments support decreased flux through MPC in the atrophy phase and increased flux through MPC when expression is higher, although the possibility that pyruvate uptake through MCT1 is rate-limiting cannot be discounted. Post-translational modification of MPC1 and MPC2 has been reported in other animal models\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, but the agreement between MPC expression and flux through MPC in our model suggests that MPC activity is not modulated by post-translational modification in our model.\u003c/p\u003e\u003cp\u003eOne plausible explanation for the increased MPC expression that we observed in the cardiac tissue samples collected from the DOX group at 16 weeks is that MPC promotes fibrosis. Inhibition of MPC reduced fibrosis in models of nonalcoholic steatohepatitis\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and corneal fibrosis\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This outcome was proposed to reflect the diversion of glutamine into the TCA cycle to compensate for the shortfall of mitochondrial acetyl-CoA derived from pyruvate\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Glutamine stimulates collagen biosynthesis in activated fibroblasts through its conversion to glutamate and proline\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, but these pathways are suppressed when glutamine is consumed by the TCA cycle. Our prior work with this model demonstrated increased cardiac fibrosis in mice exposed to DOX after 16 weeks compared to 4 weeks or control populations\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The relationship between MPC and cardiac fibrosis requires further investigation.\u003c/p\u003e\u003cp\u003eSimilarly, stable isotope tracing analyses in cultured cardiomyocytes revealed that synthesis of aspartate from glucose supports hypertrophy, likely due to the contribution of the amine group of aspartate to nucleotide biosynthesis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Aspartate is formed by transamination of oxaloacetate produced by the TCA cycle. In the heart samples collected at 16 weeks, M\u0026thinsp;+\u0026thinsp;1 fractional enhancement of aspartate is 43% higher in the hearts exposed to DOX (39.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3% vs. 27.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5% abundance, respectively). Our studies do not report on the metabolic fate of aspartate but do suggest another mechanism by which the recovery of MPC expression in the hearts exposed to DOX between 4 weeks and 16 weeks is associated with accelerated cardiac growth.\u003c/p\u003e\u003cp\u003eIn contrast to [1-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate, which rapidly produces [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e during PDH-catalyzed conversion to acetyl-CoA, [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate can only produce [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e by undergoing multiple rounds of the TCA cycle, as shown in Supplemental Fig.\u0026nbsp;2f. The rapid decarboxylation of [1-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate to [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e, as would be expected in the healthy heart, results in a lack of tissue signal retention\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. By contrast, administration of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate results in longer retention. We elected to use [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate for our studies because it is readily synthesized from [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]methyl iodide and a commercially available derivative of glycine\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, and the additional retention would enable flux measurements over a longer time interval. This rationale was previously used to support the use of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]lactate to measure myocardial lactate kinetics in porcine hearts\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]Lactate was found to track the fate of lactate in the heart, where it is either oxidized or backdiffused, and its kinetics were best described using a 2-tissue, 4-compartment model. Given the interconvertibility of pyruvate and lactate and the fact that both enter the mitochondria as pyruvate, we also fitted our data to a 2-tissue, 4-compartment model. Graphical analyses confirmed this model to be superior to other compartmental or simple exponential models, suggesting that [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate PET imaging can similarly be used to estimate myocardial pyruvate oxidation rates. However, this hypothesis will need to be tested by blood sampling in larger animals.\u003c/p\u003e\u003cp\u003eWe interpreted the slower cardiac flux of radioactivity in the DOX group at 4 weeks evident in the PET imaging to reflect differences in the rate of formation of [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e. Cardiomyocytes efficiently eliminate CO\u003csub\u003e2\u003c/sub\u003e through high CO\u003csub\u003e2\u003c/sub\u003e permeability and carbonic anhydrase activity\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate uptake studies in cultured human cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), confirm that evolution of [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e is related to MPC expression and consistent with our hypothesis. To reinforce this interpretation, we measured the cardiac bicarbonate-to-lactate (Bic/Lac) ratios in mice exposed to DOX and control animals at the 4-week time point by HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate MRI. This substrate bears the isotopic label on a different carbon atom to our PET probe, but the rapid release of [\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e upon entry of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate into the TCA cycle allowed use the formation of this metabolite as a means to distinguish between mitochondrial pyruvate oxidation and its metabolism by other pathways in a time course that is compatible with the hyperpolarization of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate (T1\u0026thinsp;=\u0026thinsp;90 s in D\u003csub\u003e2\u003c/sub\u003eO\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e). Hyperpolarized [2-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate MRI has previously been used to measure labeling of downstream mitochondrial metabolites in rat hearts\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and could represent a valuable follow up study for this work. Bicarbonate is formed when CO\u003csub\u003e2\u003c/sub\u003e is hydrated by reaction with carbonic anhydrase. Bic/Lac ratios were typically lower in the mice exposed to DOX, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee. Intriguingly, lower Bic/Lac ratios were associated with lower LVEF in human patients\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, highlighting a possible relationship between pyruvate metabolism through MPC and cardiac function. The statistically significant increase in Lac/Pyr ratio in the hearts exposed to DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) agrees with decreased flux of pyruvate through the TCA cycle and points toward a glycolytic phenotype in these hearts.\u003c/p\u003e\u003cp\u003eAlthough we did not specifically evaluate [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate PET as an imaging biomarker of cardiotoxicity, reports of cardiomyocyte metabolic reprogramming preceding changes in cardiac structure and function support its use for this application\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Cardiotoxicity is traditionally defined as a drop in LVEF of \u0026gt;\u0026thinsp;10% in symptomatic patients or \u0026gt;\u0026thinsp;5% in asymptomatic patients\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Such cardiac dysfunction is detected by echocardiography and may arise many years after cessation of DOX treatment\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. However, early detection of subacute or subclinical disease remains challenging\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, and new methods of doing so could greatly improve management of these conditions, especially when they convey information about specific biochemical pathways that are disrupted. In our previous work with this model\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we demonstrated that significant changes in fractional shortening were not evident until 10 weeks after the first exposure to DOX. This timeline was comparable to other murine models in which the mice received a similar cumulative dose\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In this light, it is potentially significant that we observed reduced cardiac [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate flux at 4 weeks. Future studies are required to determine whether [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate PET predicts changes in LVEF and therefore represents a biomarker of cardiotoxicity.\u003c/p\u003e\u003cp\u003eThe major limitation of our approach is that non-invasive measurement of radioactivity fluxes cannot necessarily isolate the effect of individual molecular targets. Our goal was to identify a method of non-invasively assessing MPC protein expression because decreased protein, but not gene expression, has been detected in cardiac tissue samples taken from patients with HFrEF\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. We present evidence that the decreased flux in the 4-week DOX group reflects decreased expression of MPC1 and MPC2, but we cannot definitively rule out the contribution of other enzymes and transporters to clearance patterns. Most significantly, expression of MCT1 mirrored that of MPC1/2 on a population level in our model, although individual differences in MCT1 expression might be responsible for the lower pool size and M\u0026thinsp;+\u0026thinsp;1 fractional enrichment of lactate observed in a subset of the mice (n\u0026thinsp;=\u0026thinsp;3) exposed to DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Conversion of HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate to [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]lactate and other downstream metabolites is rate-limited by MCT1-mediated transport across the plasma membrane in cancer cells\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, and the same kinetics likely apply to cardiomyocytes. One potential advantage of PET imaging in this context is that the tracer is administered in nanomolar concentrations, rather than the supra-physiological amounts required for HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate MR imaging and stable isotope tracing. As such, pyruvate transport through MCT1 may not influence radioactivity flux to the same extent and the PET time-activity curves might more closely reflect MPC expression levels. In addition, despite significantly lower levels of LDHB, the more prevalent isoform of LDH in the heart, we did not observe any impairment of pyruvate and lactate interconversion. This apparent contradiction can be explained by the observation that LDHB \u0026ndash; and many of the enzymes involved in pyruvate metabolism \u0026ndash; are subject to post-translational modification and substrate or product inhibition\u003csup\u003e\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. It is noteworthy that [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate flux was faster in the DOX-exposed hearts at 16 weeks compared to the controls and incorporation of the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC label into TCA cycle metabolites was similarly elevated even though neither MCT1 nor MPC1/2 expression was not significantly higher in the hearts exposed to DOX. This may indicate a compensatory increase in pyruvate oxidation following decreased fatty acid β-oxidation, a phenomenon previously reported for a rat model of sub-chronic DOX-induced cardiotoxicity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Indeed, although we observed increased expression of genes involved in β-oxidation in the hearts exposed to DOX at 16 weeks compared to 4 weeks, expression levels remained significantly lower than the age-matched controls (Supplemental Fig.\u0026nbsp;5b). This example highlights the challenge of relating flux measurements to changes in MPC protein expression levels alone, although development of a probe that selectively binds to MPC could further clarify this relationship. These efforts are ongoing in our laboratory.\u003c/p\u003e\u003cp\u003eCollectively, our findings support dynamic [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate PET as a method for imaging cardiac pyruvate metabolism in vivo that conveys information about MPC expression levels in this tissue. Exposure of cardiomyocytes to doxorubicin induces atrophy and loss of MPC1/2 and MCT1 expression in vitro and in a mouse model. At later time points, expression of these markers returns to baseline, where it appears to be related to rates of heart growth. Loss of MPC results in reduced oxidation of pyruvate in mitochondria and decreased flux of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate in the heart. Restoration of MPC expression levels are evident through increased [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate flux. Although further work is required, this study supports the potential of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate PET for detecting impaired pyruvate transport, which could serve as an early indicator of cardiotoxicity in cancer patients undergoing doxorubicin treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eEthics Statement\u003c/h2\u003e\u003cp\u003e All animal experiments carried out in this protocol were approved by the Institutional Animal Care and Use Committees (IACUC) of Weill Cornell Medicine and Memorial-Sloan Kettering Cancer Center (protocol 2019-0043).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eGeneral\u003c/h2\u003e\u003cp\u003eDoxorubicin (DOX) hydrochloride was purchased from Tocris Bioscience, USA and used without further purification. It was dissolved at a concentration of 0.75 mg/mL in sterile saline for injection (Hospira, USA) with the aid of sonication. The solution was stored in the dark at -20\u0026deg;C for up to 24 hours (h) before use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStable isotope tracing\u003c/h2\u003e\u003cp\u003eFour or 16 weeks after initial DOX exposure, DOX-treated mice (n\u0026thinsp;=\u0026thinsp;3 and n\u0026thinsp;=\u0026thinsp;5, respectively) and age-matched control mice (n\u0026thinsp;=\u0026thinsp;4 and n\u0026thinsp;=\u0026thinsp;5, respectively) received an intravenous bolus injection of 1 M sodium [3-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate (Millipore Sigma). The total injection volume was 100\u0026thinsp;\u0026plusmn;\u0026thinsp;5 \u0026micro;L. At 10 min post-injection, blood was removed by cardiac puncture and the mice were sacrificed by cervical dislocation. The heart was excised, dried, weighed, and flash frozen in liquid nitrogen. Metabolites were extracted using 80% methanol. The extracts were dried down and then re-dissolved in water. Targeted LC/MS analyses were performed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled to a Vanquish UPLC system (Thermo Scientific). The Q Exactive operated in polarity-switching mode. A Sequant ZIC-pHILIC column (2.1 mm i.d. \u0026times; 150 mm, particle size of 5 \u0026micro;m, Millipore Sigma) was used for the separation of metabolites. A 2.1 \u0026times; 20 mm guard column with the same packing material was used for the protection of the analytical column. The flow rate was set at 150 \u0026micro;L/min. The mobile phases consisted of 100% acetonitrile for mobile phase A and 0.1% NH\u003csub\u003e4\u003c/sub\u003eOH/20 mM CH\u003csub\u003e3\u003c/sub\u003eCOONH\u003csub\u003e4\u003c/sub\u003e in water for mobile phase B. The chromatographic gradient ran from 85\u0026ndash;30% A in 20 minutes, followed by a wash with 30% A and re-equilibration at 85% A. The raw data was processed using El-MAVEN (v0.12.0). Metabolites and their \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC isotopologues were identified on the basis of exact mass within 5 ppm and standard retention times. The fractional abundance of isotopically labeled metabolites was determined by determining the ratio of peak intensity of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled species to the peak intensity of the corresponding natural \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC metabolite. The fractional labeling of each metabolite was compared by two-tailed, unpaired t-test, with \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eRadiosynthesis\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003eGeneral\u003c/h2\u003e\u003cp\u003eA 5 M KOH solution was prepared by dissolving KOH, \u0026ge;\u0026thinsp;99.95% trace metals basis (Millipore Sigma) in a suitable volume of 18 mΩ H\u003csub\u003e2\u003c/sub\u003eO. Stock solutions of 5 mg/mL D-amino acid oxidase from porcine kidney (D-AAO; Millipore Sigma) and 5 mg/mL catalase from bovine liver (Millipore Sigma) were prepared in 18 mΩ H\u003csub\u003e2\u003c/sub\u003eO. A 1 M solution of HCl in dioxane was prepared by diluting 0.4 mL of a 4 M HCl/dioxane solution (Millipore Sigma) with 1.2 mL anhydrous dioxane (VWR). Stock solutions of 0.5 M and 0.05 M Tris-HCl, pH 8.5, was prepared by diluting 1 M Tris-HCl, pH 8.5 (VWR) with 18 mΩ H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eProduction of [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CH\u003csub\u003e3\u003c/sub\u003eI\u003c/h2\u003e\u003cp\u003e[\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e was produced by a [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eN(p,α)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC] transformation on a TR19 cyclotron (Advanced Cyclotron Systems, Inc.). The [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e was converted to [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CH\u003csub\u003e3\u003c/sub\u003eI using a TracerLab FX\u003csub\u003eC\u003c/sub\u003e Pro (GE Healthcare). Conversion of [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e to [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CH\u003csub\u003e3\u003c/sub\u003eI took approximately 14 min. [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CH\u003csub\u003e3\u003c/sub\u003eI was trapped on an ascarite column and distilled into the reaction vial in a stream of N\u003csub\u003e2\u003c/sub\u003e gas by heating the column to 250\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]Pyruvate\u003c/h2\u003e\u003cp\u003e[3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]Pyruvate was synthesized following published methods\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, with small modifications. Briefly, \u003cem\u003eN\u003c/em\u003e-(diphenylmethylene)-glycine \u003cem\u003etert\u003c/em\u003e-butyl ester (3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg, Millipore Sigma) was dissolved in 350 \u0026micro;L N,N-dimethylformamide (ThermoFisher) in a glass vial. Next, 10 \u0026micro;L 5 M KOH was added, and [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CH\u003csub\u003e3\u003c/sub\u003eI (approximately 6 GBq) was transferred to the vial. After the [\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]CH\u003csub\u003e3\u003c/sub\u003eI was trapped, the reaction was heated at 85\u0026deg;C for 5 min. The intermediate was diluted with 10 mL H\u003csub\u003e2\u003c/sub\u003eO and passed through a pre-conditioned Sep-Pak C18 plus short cartridge (Waters). The cartridge was washed with 5 mL H\u003csub\u003e2\u003c/sub\u003eO and eluted into a clean glass vial with 1.6 mL 1 M HCl in dioxane. Deprotection was effected by heating the reaction at 130\u0026deg;C for 5 min. The contents of the vial were taken up in 20 mL H\u003csub\u003e2\u003c/sub\u003eO and passed through a Bond Elut Jr SCX 1000 mg cartridge (Agilent Technologies) pre-conditioned with 10 mL H\u003csub\u003e2\u003c/sub\u003eO. The cartridge was washed successively with 5 mL H\u003csub\u003e2\u003c/sub\u003eO and 2 mL 0.5 M Tris-HCl, and D/L-[3-\u003csup\u003e11\u003c/sup\u003eC]alanine was eluted in 2 mL 0.05 M Tris-HCl, pH 8.5. The mixture was transferred to a ThermoMixer\u0026reg; C (Eppendorf) and 75 \u0026micro;L of the D-AAO solution and 5 \u0026micro;L of the catalase solution was added. The reaction was shaken for 6 min at 40\u0026deg;C before acidification to pH\u0026thinsp;\u0026lt;\u0026thinsp;2 with addition of 1 M HCl. The contents were passed through a pre-conditioned Bond Elut Jr SCX 1000 mg cartridge (Agilent Technologies). The filtrate was adjusted to pH 5\u0026ndash;6 by addition of 1 M NaOH. The radiochemical purity of the product was determined by analytical radioHPLC by injection onto a Chirex 3126 (D)-penicillamine, 4.6 x 150 mm column (Phenomenex). The isocratic mobile phase was 1 mM CuSO\u003csub\u003e4\u003c/sub\u003e set to a flow rate of 1 mL/min. The retention time, t\u003csub\u003eR\u003c/sub\u003e, of the final product was compared to the t\u003csub\u003eR\u003c/sub\u003e of sodium pyruvate (11.5 min).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSmall animal microPET/CT imaging experiments\u003c/h2\u003e\u003cp\u003ePrior to administration of [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate, the lateral tail vein of each mouse was cannulated for intravenous (i.v.) administration. A 27G \u0026times; \u0026frac12; inch, 8 cm catheter (SURFLO\u0026reg; Winged Infusion Set, USA) was utilized for the cannulation procedure. Following confirmation of proper catheter insertion using a small saline flush, the tubing was prefilled with sterile saline and capped. Mice were imaged at 4 weeks after initial DOX exposure (n\u0026thinsp;=\u0026thinsp;10 per group) or 16 weeks after initial DOX exposure (n\u0026thinsp;=\u0026thinsp;8 treated animals and n\u0026thinsp;=\u0026thinsp;4 controls).\u003c/p\u003e\u003cp\u003eCannulated mice were anesthetized under isoflurane (3.5% for induction, 1.5% for maintenance) and placed in pairs on the imaging bed. Prior to radiotracer administration, a CT acquisition was performed for anatomic co-registration and scatter and attenuation correction. The mice were administered 3.7\u0026ndash;11.1 MBq [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate in a total volume of 100\u0026ndash;150 \u0026micro;L. Imaging was performed using small animal microPET/CT (Siemens Inveon\u0026trade;, USA), and the 30-min acquisition began immediately upon tracer injection. The data were collected in list mode, histogrammed into 54 dynamic frames (12x5s, 12x10s, 8x15s, 10x30s, 10x60s, and 2x300s) and reconstructed using the OSEM-MAP algorithm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003emicroPET/CT image analysis\u003c/h2\u003e\u003cp\u003eCardiac segmentation was performed using Carimas\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The global time-activity curve (TAC) was plotted, and compartmental modeling was performed using the built-in software models. The input function was derived from the left ventricle. The TAC and compartmental modeling curves were plotted using GraphPad Prism. Using Akaike\u0026rsquo;s information criterion, we identified the two-tissue, four parameter compartmental model as the best fit for 93% of the images (n\u0026thinsp;=\u0026thinsp;27/29). The remaining curves were best modeled by a one tissue compartment model. Statistical comparison of each kinetic constant, VA, K1, k2, k3, and k4, was performed between groups by one-way ANOVA. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003cp\u003eTissue transit time (τ) in the heart was determined by dividing the area under the curve (AUC) of the TAC by its highest activity. The mean τ values and standard deviations for each of the four groups were determined. Three mice (n\u0026thinsp;=\u0026thinsp;1, DOX 4 weeks and n\u0026thinsp;=\u0026thinsp;2, control 4 weeks) were excluded from the comparison because their transit time was greater than 2 standard deviations below (DOX and control, n\u0026thinsp;=\u0026thinsp;1) or above (control, n\u0026thinsp;=\u0026thinsp;1). Statistical comparisons of transit times between DOX and control groups at each time point were performed by two-tailed, unpaired t-test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eHyperpolarization of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate\u003c/h2\u003e\u003cp\u003e35 \u0026micro;L neat [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvic acid doped with 15 mM AH111501 trityl radical was polarized in a SpinLab hyperpolarizer (GE Healthcare, USA) at 5 T and 0.8 K with 139.88 GHz microwave irradiation. After at least 45 min of solid-state polarization buildup, the frozen sample was dissolved with 10 mL superheated (~\u0026thinsp;400 K) D\u003csub\u003e2\u003c/sub\u003eO buffer containing 100 mM Tris and 1 mM EDTA for a final [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate concentration of 100 mM. The HP pyruvate solution was pH neutralized by a stoichiometric quantity of 10 N NaOH to the receiver flask. Dissolution polarization levels were estimated by measuring T1 decay on an aliquot of HP dissolution with a 1 T Spinsolve spectrometer (Magritek). The molar concentration of the pyruvate solution was measured by \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR at 11.7 T (Bruker; MSKCC NMR Core) with reference to a 100 mM [\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]urea standard.\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eC Magnetic resonance spectroscopy\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAnimal experiments were performed in a 3T Biospec MRI scanner (Bruker, USA) with a \u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e1\u003c/sup\u003eH volume quadrature coil (RAPID MR). A T1-weighted FLASH sequence was used for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH anatomic imaging of the heart. Pre-scans were performed while mice were under ~\u0026thinsp;1.5% isoflurane anesthesia. Immediately before initiating the hyperpolarized [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate dissolution process, the isoflurane flow was turned off and the mouse was allowed to wake up in the scanner. Mice (n\u0026thinsp;=\u0026thinsp;3 for the control group, n\u0026thinsp;=\u0026thinsp;2 for the DOX group) were restrained with three pieces of double-backed tape to prevent movement during the experiment and were not left awake under restraint for more than five min. The total time under isoflurane anesthesia was less than 30 min. For the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC spectroscopy experiment, the scanner was set to continuously acquire slice-selective spectra with a 60\u0026deg; flip angle, 2048 spectral points, and 1280 Hz (39.9 ppm) bandwidth. The HP pyruvate bolus was split into multiple injections to enhance the number of replicates measured per HP pyruvate dissolution\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. 100 mM HP pyruvate was injected via tail vein catheter four times separated by delays of at least 30 s to allow complete relaxation of the preceding HP pyruvate injection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eMR image analysis\u003c/h2\u003e\u003cp\u003eDynamic spectra were zero-filled, phased, apodized with 5 Hz exponential decay, and baseline-corrected in MNova software (Mestrelab Research). Pyruvate (171 ppm), lactate (183 ppm), alanine (176 ppm), and bicarbonate (161 ppm) signals were then quantified by integration. Treating each injection of HP pyruvate substrate as a distinct measurement, dynamic area under curve (AUC) values were calculated by summing the metabolite integral intensities over the time-course of the pyruvate bolus. Metabolite AUCs were normalized by calculating lactate-to-pyruvate ratios and bicarbonate- or alanine-to-lactate ratios. Mean values were statistically compared between groups by two-tailed, unpaired t-test, with \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eH NMR measurement of tissue lactate pool size\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFour weeks after initial exposure to DOX, mouse hearts (n\u0026thinsp;=\u0026thinsp;4 per group) were dissected and snap frozen in liquid nitrogen. At least 100 mg tissue was transferred to pre-filled bead mill tubes (Fisher Scientific, USA) containing 400 \u0026micro;L 4% (w/v) perchloric acid) and finely ground by a Fisherbrand\u0026trade; Bead Mill 24 Homogenizer (Fisher Scientific, USA). Homogenates were centrifuged for 15 minutes at 14000 rpm and 4\u0026deg;C, and the supernatant was transferred to a new tube containing 1 mL chloroform/tri-n-octylamine (78/22 v/v), followed by centrifugation for 15 minutes at 4000 rpm and 4\u0026deg;C. The aqueous phase was transferred to a new tube and lyophilized overnight. Dried samples were reconstituted in D\u003csub\u003e2\u003c/sub\u003eO solvent containing 1 mM sodium trimethylsilylpropanesulfonate standard and transferred to 5 mm NMR tubes. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra of metabolite extract samples were acquired in a 14.1 T spectrometer (Bruker; MSKCC NMR core). Spectra were processed and metabolite peaks were quantified with Chenomx NMR suite software (Chenomx, Canada).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of cardiac tissue for analysis\u003c/h2\u003e\u003cp\u003eThe mice were anesthetized by i.p. ketamine injection and perfused with phosphate-buffered saline (PBS) via the left ventricle at a constant pressure of 80 mmHg. To perform the molecular analysis, the whole hearts were homogenized by using liquid nitrogen and a mortar and pestle. The homogenized tissues were separated for RNA and protein extraction. The extracts were flash frozen in liquid nitrogen and stored at -78\u0026deg;C until further use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eRNA isolation\u003c/h2\u003e\u003cp\u003eFrozen heart tissue fractions were collected and soaked in Trizol (Invitrogen, USA), and RNeasy Fibrous tissue mini kit (Qiagen, USA) was used to isolate total RNA from heart tissues. Genomic DNA was removed by DNase I (Qiagen), and RNA was reverse transcribed using an iScript kit (Bio-Rad, USA). RNA extracts were validated prior to sequencing.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eBulk RNA-seq library construction and data analysis\u003c/h2\u003e\u003cp\u003eRNA libraries were sequenced with paired-end 50 bps on the NovaSeq 6000 Sequencer (Illumina, USA). The raw sequencing reads in BCL format were processed through bcl2fastq 2.20 (Illumina) for FASTQ conversion and demultiplexing. After trimming the adaptors with cutadapt (version 1.18; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cutadapt.readthedocs.io/en/v1.18/\u003c/span\u003e\u003cspan address=\"https://cutadapt.readthedocs.io/en/v1.18/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), RNA reads were aligned and mapped to the GRCm39 mouse reference genome by STAR (version 2.5.2; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/alexdobin/STAR\u003c/span\u003e\u003cspan address=\"https://github.com/alexdobin/STAR\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e68\u003c/sup\u003e, and transcriptome reconstruction was performed by Cufflinks (Version 2.1.1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cole-trapnell-lab.github.io/cufflinks/\u003c/span\u003e\u003cspan address=\"http://cole-trapnell-lab.github.io/cufflinks/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The abundance of transcripts was measured using Cufflinks, with fragments per kilobase of transcript per million mapped reads (FPKM) as the output\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Raw read counts per gene were extracted using HTSeq-count version 0.11.2 \u003csup\u003e71\u003c/sup\u003e. Gene expression profiles were constructed for differential expression, cluster, and principal component analyses with the DESeq2 package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioconductor.org/packages/release/bioc/html/DESeq2.html\u003c/span\u003e\u003cspan address=\"https://bioconductor.org/packages/release/bioc/html/DESeq2.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e72\u003c/sup\u003e. For differential expression analysis, pairwise comparisons were performed between two or more groups using parametric tests where read counts follow a negative binomial distribution with a gene-specific dispersion parameter. Corrected \u003cem\u003ep\u003c/em\u003e-values were calculated based on the Benjamini-Hochberg method to adjust for multiple testing. For the differentially expressed genes (DEGs) analysis in the 4-week groups, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 was used as the substantial signifier of statistical significance among a total 49,139 variables, and Log2FC (FC, fold change) \u0026gt;|1| was used to distinguish upregulated (Up) and downregulated (Down) DEGs, respectively. For the 16-week groups, those differentially expressed genes (DEGs) among a total 49,135 variables with a false discovery rate (FDR) of less than 0.05 were divided into up- and down-regulated groups for analysis. The EnhancedVolcano plot was generated using R studio for the overall distribution of DEGs.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eDAVID classification of DEGs\u003c/h2\u003e\u003cp\u003eThe Database for Annotation, Visualization, and Integrated Discovery (DAVID; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to classify differentially expressed genes (DEGs) based on their biological functions. For the 4-week group, 389 upregulated and 1,229 downregulated genes were analyzed, whereas 86 upregulated and 107 downregulated genes were assessed in the 16-week group. These DEGs were subjected to Gene Ontology (GO) enrichment analysis, focusing on biological processes (BP), as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. GO terms with a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eSTRING database analysis\u003c/h2\u003e\u003cp\u003eA protein-protein interaction (PPI) network was constructed to identify the associations between the target and related differentially expressed genes (DEGs) using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://string-db.org/\u003c/span\u003e\u003cspan address=\"http://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e73\u003c/sup\u003e. GO terms and PPI networks with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eFrozen heart tissue fractions were soaked in tissue protein extraction reagent (#78510, ThermoFisher, USA), supplemented with a protease inhibitor cocktail (#87786, ThermoFisher, USA) for protein extraction. Western blots were prepared and processed as previously reported\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The information on all primary and secondary antibodies can be found in Supplemental Table\u0026nbsp;8. The chemical luminescent signals were measured by the ChemiDoc imaging system (Bio-Rad, USA). Protein expression was quantified by drawing a region of interest (ROI) over the corresponding band using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eIn vitro [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate uptake in human cardiomyocytes\u003c/h2\u003e\u003cdiv id=\"Sec28\" class=\"Section4\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHuman cardiomyocytes (HCMs) isolated from adult left ventricles (PromoCell, Heidelberg, Germany) were cultured according to the manufacturer\u0026rsquo;s instructions. Briefly, once the cultures reached 80\u0026ndash;90% confluency, the cells were washed with PBS and refreshed with either control medium (DOX-free) or medium containing DOX or UK5099. Cells in the former group were incubated in media containing 0.1 \u0026micro;M DOX. The cells were incubated for 48 h, followed by a medium change with fresh medium. After an additional 48 h incubation, the medium was replaced again with 0.1 \u0026micro;M DOX-containing medium to complete the \"two-hit\" treatment\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Following a final incubation period of 48 h, the medium was removed and replaced with DOX-free control medium. All DOX-treated assays were performed on day 7. The second group of HCM was treated with 100 \u0026micro;M UK5099 for 72 h\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The control cells received an equal volume of dimethyl sulfoxide (DMSO).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eSilencing of MPC1 and MPC2\u003c/h2\u003e\u003cp\u003eMPC1 and MPC2 siRNA were used to downregulate MPC1/2-specific expression in HCMs. Pre-designed siRNA products targeting MPC1 (#4392420, ID s28488) and MPC2 (#4392420, ID s24657), along with negative control siRNA (#4390843, scramble) were purchased and 50 nM of MPC1 and MPC2 siRNA and 100 nM of scramble siRNA were mixed with Lipofectamine RNAiMAX (#13778100) or Lipofectamine Stem (STEM00008) and Opti-MEM (#31985070) according to established protocols\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. The siRNA mixture was applied to HCMs for 24 h, followed by replacement with fresh culture medium for an additional 24 h of incubation. MPC1 siRNA, sense; UGCUAUUCUUUGACAUUCAtt/ antisense; UGAAUGUCAAAGAAUAGCAac. MPC2 siRNA, sense; UCACUUGUAAUUAUUCCAAtt/ antisense; UUGGAAUAAUUACAAGUGAgt. All materials for siRNA work were purchased from ThermoFisher, USA.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e[3-C]Pyruvate uptake\u003c/h3\u003e\n\u003cp\u003eAll cells were seeded in 24-well plates. Each group of cells was washed with PBS and incubated with a Hanks' Balanced Salt Solution (HBSS, 21-023-CV, Corning, USA)-based labeling medium containing sodium [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate (0.1 mCi/ml, ARC0220, American Radiolabeled Chemicals, USA) diluted 1:2000 and 0.5% fatty acid-free bovine serum albumin (BSA, A7030, Sigma-Aldrich, USA) in each well for 0, 2, 4, and 8 min at 37\u0026deg;C. The cells were then washed twice with PBS and lysed with 1% sodium dodecyl sulfate (SDS). The radioactivity of the cell lysates was measured after dilution with scintillation buffer using a Liquid Scintillation Counter (Tri-carb 2910 TR, Perkin Elmer, USA). Cell uptake was corrected for activity added and normalized to the total protein concentration at the time of the assay (% of uptake/mg). Total protein concentrations were determined using the BCA protein assay kit (ThermoFisher, USA). Statistical analysis was performed using Pearson correlation coefficients from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9728), two-way ANOVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and unpaired t-test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e[\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e measurement\u003c/h2\u003e\u003cp\u003eAll cells were seeded in 35 mm culture plates. For the [\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e capture assay, a Whatman filter (Millipore Sigma, USA) was placed in the plate cap and moistened with 100 \u0026micro;L of 40% KOH, according to established procedures\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The cells were treated with sodium [3-\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC]pyruvate as described above and incubated at 37\u0026deg;C for 8 minutes. After incubation, the cells were washed twice with PBS and lysed using 1% SDS. The filter was suspended in scintillation buffer for counting on the Liquid Scintillation Counter. In parallel, cell lysates and the remaining supernatants were mixed with a scintillation buffer for radioactivity counting. Statistical analysis was performed using one-way ANOVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and Supplementary Fig.\u0026nbsp;2h).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eCell viability\u003c/h2\u003e\u003cp\u003eThe viability of the HCM treated with DOX or UK5099 was determined using the trypan blue method. Briefly, after cell harvesting by trypsinization and centrifugation, cell pellets were resuspended in PBS. An equal volume of trypan blue solution (ThermoFisher, USA) was added to the suspension and gently mixed. The mixture was incubated for 3 minutes at room temperature. Subsequently, 10 \u0026micro;L of the stained cell suspension was loaded onto a hemocytometer and examined under a light microscope. Viable cells (excluding dye) and non-viable cells (blue-stained) were counted manually, and the percentage of viable cells was calculated. Statistical analysis was performed using one-way ANOVA.\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003eCell counting\u003c/h2\u003e\u003cp\u003eHCMs were seeded in 96-well plates. Seven days following DOX treatment, both control and DOX-treated groups were washed with PBS and incubated with a CCK-8 solution for 2 h. Subsequent steps were performed following the manufacturer\u0026rsquo;s instructions (#K1018, APExBIO, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eExtracellular pH measurement\u003c/h2\u003e\u003cp\u003eAll cells were seeded in 24-well plates and treated with either DOX or UK5099 as described above. Control cells received an equal volume of DMSO. Each group of cells was washed with PBS and incubated in HBSS medium for 8 min at 37\u0026deg;C. The supernatant was collected, and the pH was measured using pH-Test 4.5\u0026ndash;10.0 indicator strips (VWR Chemicals, USA).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eStatistical analyses were performed as described using GraphPad Prism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eUnrelated to this work, K.R.K. is co-founder of Atish Technologies and serves on the Scientific Advisory Boards of NVision Imaging Technologies, Imaginostics and Mi2. He holds patents related to imaging and leveraging cellular metabolism. The other authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.M.K. secured funding for the project. J.M.K., J.W.B., and K.R.K. designed the experiments. C.H.L., T.R., S.D., A.W., G.F., and J.M.K performed the experiments and analyzed the data. S.N. and J.M.K. performed the kinetic modeling. C.H.L., T.R., and J.M.K. wrote the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to acknowledge the assistance of the Weill Cornell Medicine Proteomics and Metabolomics Core Facility for the stable isotope metabolomics experiments, the Weill Cornell Medicine Genomics Core Facility for the bulk RNAseq experiments, and the Citigroup Biomedical Imaging Center at Weill Cornell Medicine for the PET imaging experiments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files. DICOM files for PET images are available from the authors upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMartin, S. 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H.\u003cem\u003e et al.\u003c/em\u003e Cardiomyocyte-targeted siRNA delivery by prostaglandin E(2)-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis. \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 4439-4446 (2008). https://doi.org/10.1016/j.biomaterials.2008.07.047\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cardiotoxicity, cardiac pyruvate metabolism, doxorubicin, mitochondrial pyruvate carrier, monocarboxylate transporter, positron emission tomography","lastPublishedDoi":"10.21203/rs.3.rs-7465913/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7465913/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChanges in cardiac metabolism typically precede cardiac dysfunction and therefore represent an important target for diagnosis and treatment designed to prevent progression to heart failure, a leading cause of death. Profound changes in pyruvate metabolism, including reduced expression of the mitochondrial pyruvate carrier (MPC), are increasingly recognized as early maladaptive alterations in cardiomyopathies, but no methods currently exist to determine MPC expression in vivo. We exposed mice to doxorubicin (DOX), an anthracycline chemotherapeutic known to induce cardiotoxicity, and demonstrated that cardiac tissue levels of MPC decrease within 4 weeks of initial DOX exposure. Using a combination of stable isotope tracing metabolomics, hyperpolarized [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate magnetic resonance imaging (MRI), and [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate positron emission tomography (PET), we found that loss of MPC and monocarboxylate transporter 1 (MCT1) resulted in decreased utilization of pyruvate for mitochondrial oxidative metabolism and resulted in decreased cardiac carbon-11 flux. Significantly, cardiac [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]pyruvate flux was sensitive to MPC expression levels and was restored when expression rebounded 16 weeks after DOX exposure. [3-\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003eC]Pyruvate PET is therefore a promising approach to imaging cardiac pyruvate transport with potential application to the identification of maladaptive changes in MPC expression and monitoring response to therapy.\u003c/p\u003e","manuscriptTitle":"[3-11C]Pyruvate PET detects alterations in cardiac pyruvate metabolism induced by doxorubicin chemotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 15:14:14","doi":"10.21203/rs.3.rs-7465913/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-10T12:21:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T04:12:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T09:28:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90205172113208145068810282761959085947","date":"2025-09-07T14:31:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136235970774219397004072419904375681064","date":"2025-09-07T10:18:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-07T09:50:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T11:01:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-27T06:43:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Imaging","date":"2025-08-26T19:53:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"433f33b3-4487-4bcf-b37f-01ebc859176b","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54620978,"name":"Health sciences/Cardiology"},{"id":54620979,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-07T09:41:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 15:14:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7465913","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7465913","identity":"rs-7465913","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}