{"paper_id":"2f09c98c-1fe8-4a6a-9172-e3f94263a26b","body_text":"Metabolic flux analysis in hiPSC-CMs reveals insights into cardiac dysfunction in propionic acidemia | 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 Research Article Metabolic flux analysis in hiPSC-CMs reveals insights into cardiac dysfunction in propionic acidemia Eva Richard, Hannah Marchuk, Mar Álvarez, Wentao He, Xiaoxin Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5874705/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Apr, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 4 You are reading this latest preprint version Abstract Propionic acidemia is an inborn error of metabolism caused by mutations in either the PCCA or PCCB genes. Patients with propionic acidemia experience a range of complications, including life-threatening cardiac dysfunctions. However, the pathological mechanisms underlying propionic acidemia-associated cardiac diseases remain largely unknown. To gain insights into the metabolic alterations in propionic acidemia, we studied human induced pluripotent stem cell-derived cardiomyocytes generated from a patient with propionic acidemia with two pathogenic PCCA mutations ( p.Cys616_Val633del and p.Gly477Glufs9* ) and from a healthy individual. Using stable isotope-based metabolic flux analysis, we confirmed that the PCCA mutations lead to impaired propionyl-CoA carboxylase activity in human induced pluripotent stem cell-derived cardiomyocytes. In addition to being converted to propionylcarnitine, the accumulated propionyl-CoA can also be hydrolyzed to propionate and exported out of the cell, serving as a secondary “pressure valve” to regulate cellular propionyl-CoA levels. Interestingly, the deficiency of propionyl-CoA carboxylase was found to shift fuel metabolism from fatty acid oxidation to increased glucose metabolism human in induced pluripotent stem cell-derived cardiomyocytes from patients with propionic acidemia. This metabolic switch is less energy-efficient and may contribute to the development of chronic cardiac dysfunction in patients with propionic acidemia. propionic acidemia human induced pluripotent stem cell-derived cardiomyocytes metabolic flux glucose metabolism fatty acid metabolism cardiac diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Propionic acidemia (PA) is an inborn error of metabolism inherited in an autosomal recessive manner [ 18 , 36 , 37 , 59 ]. Mutations in either the PCCA or PCCB gene result in the malfunction of propionyl-CoA carboxylase (PCC), a critical mitochondrial enzyme. The global incidence of PA varies, ranging from 1 in 50,000 to 1 in 100,000 live births. The adoption of tandem mass spectrometry for neonatal screening has significantly increased the detection rate of PA cases [ 49 ]. PCC is a crucial mitochondrial enzyme, though it remains relatively understudied [ 56 ]. Whole-body PCC knockout in mouse models has been shown to be neonatal lethal [ 10 , 21 , 25 , 40 ]. Patients with PA, often present symptoms in early infancy such as poor feeding, vomiting, and low muscle tone [ 2 , 32 ]. As the disease progresses, patients may develop a variety of complications, including life-threatening cardiac dysfunctions [ 30 , 31 , 55 ]. Understanding the pathological mechanisms underlying PA-associated cardiac diseases is an urgent and critical need. PCC catalyzes the carboxylation of propionyl-CoA to methylmalonyl-CoA, which is subsequently converted to succinyl-CoA and enters the tricarboxylic acid (TCA) cycle as part of an anaplerotic process [ 8 , 27 , 28 , 39 , 41 ]. Malfunctioning PCC disrupts propionyl-CoA metabolism, leading to the accumulation of propionyl-CoA and its metabolites, including methylcitrate, 3-hydroxypropionate, and maleic acid [ 51 , 54 ]. These accumulated metabolites have been reported to inhibit the TCA cycle and mitochondrial energy production in in vitro enzyme assays using high concentrations of these metabolites [ 6 , 12 , 45 , 46 , 54 ]. Additionally, oxidative stress, disruption of potassium channels, and altered miRNA expression have been implicated in the cardiac dysfunction observed in patients with PA [ 7 , 11 , 17 , 19 , 20 , 44 , 47 , 49 ]. Propionyl-CoA, an intracellular metabolite, cannot cross the plasma membrane [ 52 ]. Instead, accumulated propionyl-CoA is converted to propionylcarnitine, which can be released into the blood and urine [ 9 , 14 ]. L-carnitine has been shown to effectively treat PA and is recommended as a supplement for patients [ 15 , 42 , 43 ]. The conversion of propionyl-CoA to propionylcarnitine is considered a critical metabolic pathway for the disposal of accumulated propionyl-CoA, with elevated propionylcarnitine serving as a biomarker for PA. However, PA is also associated with elevated levels of circulating propionate, which is partly due to reduced hepatic disposal [ 53 ]. The exact source of circulating propionate remains unclear. While it is known to originate from the microbiome, it is uncertain whether it might also arise from intracellular propionyl-CoA within the host. This could represent a secondary regulatory mechanism for managing intracellular propionyl-CoA, in addition to its conversion to propionylcarnitine. Previously, we reported that supraphysiological levels of propionate dramatically increase propionyl-CoA and deplete free Coenzyme A (CoA) in perfused rat hearts [ 51 ]. This CoA depletion causes a metabolic shift from the high energy-efficient fatty acid oxidation to low energy-efficient glucose metabolism [ 51 ]. However, in the Pcca −/− (A138T) mouse model of PA, CoA or carnitine depletion in the heart occurs only upon the acute administration of high doses of propionate (500 mg/kg) [ 24 ]. This highlights the need for a more representative PA model to investigate the pathological mechanisms underlying cardiac diseases associated with PA. Animal experiments often face limitations in their applicability to humans. In this study, we utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a control individual and a PA patient to investigate metabolic alterations and better understand the pathological mechanisms of PA. Using stable isotope-based metabolic flux analysis, we confirmed the metabolic phenotype of PCC deficiency in PA patient-derived hiPSC-CMs. Our experiments demonstrated that accumulated propionyl-CoA can be hydrolyzed to propionate, which is then exported from the cell as an additional \"pressure valve.\" Furthermore, PA hiPSC-CMs exhibited a metabolic switch from energy-efficient fatty acid oxidation to energy-inefficient glucose metabolism. This shift in fuel utilization may be a key pathological factor contributing to the cardiac dysfunction observed in PA patients. Methods Maintenance of hiPSC lines The hiPSC lines utilized in this study included: (i) a PCCA-deficient hiPSC line (PCCA23-FiPS4F6 or UAMi001-A), created by reprogramming fibroblasts from a patient with PCCA gene mutations (c.1899 + 4_1899 + 7delAGTA; p.(Cys616_Val633del) and c.1430-?_1643+?del; p.(Gly477Glufs*9)) via Sendai virus; and (ii) a normal control hiPSC line (FIPS Ctrl2-SV4F-1) acquired from the National Bank of Cell Lines at the Carlos III Health Institute (ISCIII, Madrid, Spain). These human iPSC lines were cultured on 60 mm dishes coated with Matrigel (hESC-qualified matrix, Corning, New York, NY, USA) and maintained in mTESR™ Plus medium (StemCell™ Technologies, Vancouver, BC, Canada), with media changes every other day. The hiPSCs were passaged every four days using ReleSR™ (StemCell™ Technologies) and 10 µM Rock inhibitor (StemCell™ Technologies) at a splitting ratio of 1:3 to 1:5. Differentiation of hiPSCs into cardiomyocytes hiPSCs cultured in mTESR™ Plus medium were dissociated into single cells using StemPro Accutase (Gibco, Waltham, MA, USA). A total of 1x10 6 cells in 1.5 ml of mTESR™ Plus medium enriched with 10 µM Rock inhibitor were plated on Matrigel-coated 12-well plates. The differentiation into cardiomyocytes was conducted using the STEMdiff™ Cardiomyocyte Differentiation and Maintenance Kits (StemCell™ Technologies) following the guidelines provided by the manufacturer. The characterization of cardiomyocytes was achieved by analyzing the expression of various cardiac-specific markers, including cardiac troponin T, α-smooth muscle actin, GATA4, and α-actinin 2 through immunocytochemistry as previously described [ 5 ]. Experimental conditions and metabolic treatments of hiPSC-derived cardiomyocytes Control and PA hiPSC-derived cardiomyocytes were subjected to four experimental conditions to assess the impact of various metabolites: Experiment #1: hiPSC-CMs were cultured in RPMI/B27 medium incubated for four days at 37°C in a 5% CO 2 atmosphere. Experiment #2 (Tracing experiment): hiPSC-CMs were cultured in RPMI/B27 medium with 1 mM [ 13 C 3 ]propionate (Sigma-Aldrich). Cells were incubated for two days at 37°C in a 5% CO 2 atmosphere. Experiment #3 (Tracing experiment): hiPSC-CMs were cultured in RPMI/B27 medium without glucose and supplemented with 11 mM [ 13 C 6 ]glucose (Sigma-Aldrich) and incubated for two days at 37°C in a 5% CO 2 atmosphere. Experiment #4 (Tracing experiment): hiPSC-CMs were cultured in RPMI/B27 with 0.4 mM [ 13 C 16 ]palmitate conjugated with BSA (Sigma-Aldrich) medium and incubated for two days at 37°C in a 5% CO 2 atmosphere. After the incubation period, both the medium and the cell pellets were collected and frozen at -80°C for subsequent analysis. Short-chain fatty acids analysis by LC-MS/MS An LC-MS/MS method was adapted to analyze short-chain fatty acids including propionate in media [ 53 ]. A 30-µl media sample was mixed with 30 µl internal standard (200 µM [2,2,2- 2 H 3 -1,2- 13 C 2 ]aceate (D5 acetate), 20 µM [2,2,3,3,3- 2 H 5 ]propionate (D5 propionate), 20 µM [2,2,3,3,4,4,4- 2 H 7 ]butyrate (D7 butyrate), 20 µM [2,2,3,3,4,4,5,5,5- 2 H 9 ]pentanoate (D9 pentanoate), and 20 µM [2,2,3,3,4,4,5,5,6,6,- 2 H 11 ]hexanoate (D11 hexanoate)). Acetonitrile (1ml) was added to precipitate protein. The supernatant was transferred to a new Eppendorf vial and completely dried by nitrogen gas after samples were vortexed and centrifugated at 10000 ×g for 20 minutes. The dried residue was resuspended in 50 µl HPLC water and 20 µl 3-Nitrophenylhydrazine hydrochloride (EDC, 120 mM) and 20 µl (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (3-NPH, 200 mM) for derivatization at 40°C for 30 minutes. The reaction mixture was centrifuged for 10 minutes at 10,000 × g and the supernatant was transferred to an LC-MS/MS vial for analysis. LC-MS/MS was run with a Sciex QTRAP 6500 + MS connected with a Sciex AD UHPLC. An Agilent C18 column (Pursuit XRs C18 150 × 2.0 mm, 5 µm) was employed for separation at room temperature with a flow rate of 0.4 ml/min. A gradient method was conducted with two mobile phases. Mobile phase A was 98% H 2 O and 2% acetonitrile containing 0.1% formic acid. Mobile phase B was 98% acetonitrile and 2% H 2 O containing 0.1% formic acid. The gradient started with 2% B for the first 0.5 minutes and increased to 90% at 8 minutes. B was maintained at 90% for 4.5 minutes and returned to its initial condition within 0.5 minutes. Finally, the column was re-equilibrated for 9 minutes with the initial condition before the next injection. The injection volume was 3 µl. MRM in negative mode was used for short-chain fatty acids assay. The MS/MS parameters were set at the following: curtain gas: 35 psi, source temperature: 600°C, Gas 1: 55 psi, Gas 2: 55 psi, CAD: 10, Ion spray voltage: -4500 V, EP: -10 V, and CXP: -14. Cell pellets metabolic profiling by GC-MS A previously established GC-MS method was adopted to measure the isotope labeling of organic acids and amino acids in the cultured pellets [ 22 – 24 , 51 , 53 , 57 ]. Briefly, approximately 1 million cells were spiked with 2 nmol of norvaline and 0.2 nmol [ 2 H 9 ]L-carnitine or mixed stable isotope labeled metabolites as internal standards and then subjected to sonication extraction with 1 ml methanol for 3 minutes. The samples were centrifuged for 20 minutes. The upper phase, approximately 500 µl in volume, was transferred to a fresh Eppendorf vial and subsequently evaporated using nitrogen gas. The resulting dried residues underwent sequential derivatization with methoxylamine hydrochloride and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (TBDMS). Specifically, 40 µl of methoxylamine hydrochloride (2% (w/v) in pyridine) was added to the dried residues, followed by incubation for 90 minutes at 40°C. Subsequently, 20 µL of TBDMS with 1% tert-butylchlorodimethylsilane was added, and the mixture was incubated for an additional 30 minutes at 80°C. The derivatized samples were then centrifuged for 10 minutes at 12,000 × g, and the supernatants were transferred to GC vials for further analysis. For GC/MS analysis, we employed an Agilent 7890B GC system with an Agilent 5977A Mass Spectrometer, following the methodology described in our previous work. Specifically, 1 µl of the derivatized sample was injected into the GC column. The GC temperature gradient began at 80°C for 2 minutes, increased at a rate of 7°C per minute to 280°C, and was maintained at 280°C until the 40-minute run time was completed. The ionization was conducted via electron impact (EI) at 70 eV, with Helium flow at 1 mL/min. Temperatures of the source, the MS quadrupole, the interface, and the inlet were maintained at 230°C, 150°C, 280°C, and 250°C, respectively. Mass spectra (m/z) in the range of 50 to 700 were recorded in mass scan mode. Acylcarnitines profile by LC-MS/MS A 100 µl medium sample or ~ 1 million cell pellets were used for acylcarnitine assay with the spiked internal standard (20 µl 0.01 mM D9 carnitine). The detailed LC-MS/MS method for the acylcarnitine profile was described in our previous work [ 23 , 24 , 51 , 53 ]. The pellet sample extracts (500 µl) from the previous sample preparation were completely dried using nitrogen gas. The medium samples were deproteinized by adding 500 ul methanol and 500 ul acetonitrile. After centrifugation at 12000 g for 15 minutes, the supernatants were completely dried using nitrogen gas. The dried residues were then methylated with a 3 M HCl methanol solution (100 µl) at 50°C for 25 minutes. After methylation, the samples were once again dried completely using nitrogen gas and then reconstituted in 20 µl of methanol and 60 µl of water. The derivatized samples were subsequently analyzed using an LC-QTRAP 6500 + -MS/MS (Sciex, Concord, Ontario). A gradient HPLC method with two mobile phases (mobile phase A was 98% water with 2% acetonitrile and 0.1% formic acid and mobile phase B was 98% acetonitrile with 2% H 2 O and 0.1% formic acid) was adopted to run with an Agilent Pursuit XRs 5 C18 column (150 × 2.0 mm). The gradient started with 0% B within first 2 minutes and then increased to 80% at 13 minutes. The column was washed out by 90% B for 4 minutes and equilibrated with initial condition (2% B) for 5 minutes before next injection. The flow rate was 0.4 ml/minute and the column oven was set at room temperature. The injection volume was 2 µl. The parameters for Sciex QTRAP 6500 + mass spectrometry were optimized as following: DP: 33 V, EP 10 V, CXP: 10 V, source temperature: 680°C, gas 1: 65, gas 2: 65, curtain gas: 35, CAD: 10, and ion spray voltage: 5500 V. The Q1 of all the methylated acylcarnitines was scanned from m/z 218 to m/z 444 with the same fragment (Q3) at m/z 99. L-carnitine had the ion transition of Q1 (m/z 176) and Q3 (m/z 85 or m/z 117). [ 2 H 9 ]L-carnitine has the shifted Q1 at m/z 179 or m/z 185 with the same Q3 at m/z 85 or m/z 117. Medium glucose assay by LC-Q-Exactive-MS Glucose in the medium was assayed according to our previous method. Briefly, a 10 µl of medium sample was added to a tube prior to folch extraction using the following solvents: 200 µl methanol, 200 µl distilled H 2 O, and 200 µl chloroform. The sample mixture was vortexed and centrifuged for 20 minutes at 10,000 × g at 4°C. The upper phase (~ 350 µl) was dried completely by nitrogen gas at 37°C. The dried residue was resuspended in 60 µl distilled water, vortexed, and placed in an autosampler vial for LC-MS analysis. LC-Q-Exactive + -Orbitrap-MS was used for the final quantitation in this work. The Vanquish Binary Pump was used to deliver the mobile phase (98% H 2 O and 2% methanol containing 0.01% formic acid) at a flow rate of 0.3 ml/min in isocratic elution mode. The column was a Microsorb-MV C18 column (100 × 4.6 mm, 3 µm) with a C18 guard column and was kept at 40°C in the column oven compartment. The autosampler was maintained at 5°C, and the injection volume was 1 µl. The total running time is 10 minutes. The parameters for Q-Exactive + -MS equipped with a HESI probe: heat temperature: 425°C; sheath gas: 30, auxiliary gas, 13; sweep gas, 3; spray voltage, 3.5 kV for positive mode; the capillary temperature was set at 320°C, and S-lens was 45. A full scan range was set at 60 to 900 (m/z). The resolution was set at 70,000 (at m/z 200). The maximum injection time (max IT) was 200 ms. Automated gain control (AGC) was targeted at 3 × 10 6 ions. Statistics All cell experiments were conducted using two differentiations with a total of n = 4 biological replicates. Measured mass isotopologues distributions expressed as mol percent were corrected for natural enrichment [ 16 , 50 ]. M0, M1, M2, ..., Mn denote the isotopologues of molecules containing n heavy atoms. Statistical differences were analyzed using Prism software. A Student's t-test was used for comparisons between two groups, while a two-way ANOVA followed by a post hoc Tukey test was conducted for comparisons among more than two groups. Results Metabolic profile of human induced pluripotent stem cell-derived cardiomyocytes from a control Individual and a PCCA-deficient patient Metabolic profiling was performed on control and PCCA-deficient hiPSC-CMs using both cell pellets and cultured media samples. PCC deficiency significantly increased propionylcarnitine (C3 AC) in the cultured media (Fig. 1 A) and led to the accumulation of cellular methylcitrate (Supplemental Fig. 1A). Interestingly, medium-chain acylcarnitines were found to be lower in the cultured media from the PCCA-deficient group (Figs. 1 B- 1 D). This decrease in acylcarnitines may indicate reduced fatty acid oxidation or a redistribution of acylcarnitines toward increased propionylcarnitine synthesis. In line with this, there was a trend toward a reduction in cellular L-carnitine (p = 0.076, Supplemental Fig. 1B). The accumulation of propionylcarnitine, along with the reduction of free carnitine, could alter fuel metabolism in the cardiomyocytes, which depend on proper fuel metabolism for mechanical contraction. PCC deficiency disrupts propionate and propionyl-CoA metabolism in hiPSC-CMs One of the reliable biomarkers of PA is elevated circulating propionate, which can reach millimolar levels in PA patients [ 26 , 53 ]. However, this biomarker is not often reported due to challenges with analytical methodology. Our previous work suggests that impaired hepatic disposal of propionate leads to increased circulating levels, which may elevate the risk of cardiac disease [ 53 ]. In the present study, we aimed to investigate the impact of PCC deficiency on propionate metabolism in cardiomyocytes derived from both control and PCCA-deficient hiPSC. After culturing the cells for 48 hours with 1 mM [ 13 C 3 ]propionate, both pre-culture and post-culture media were collected for propionate quantification (Fig. 2 A). PCCA-deficient hiPSC-CMs released 9.6 times more unlabeled propionate compared to control hiPSC-CMs (Fig. 2 B). The released unlabeled propionate is likely generated endogenously, probably from hydrolysis of propionyl-CoA, which is elevated in PA patient-derived hiPSC-CMs. After 48 hours of culturing, [ 13 C 3 ]propionate was consumed by 25% in the control hiPSC-CMs (Fig. 2 C). In contrast, [ 13 C 3 ]propionate levels in the medium remained almost unchanged in PA hiPSC-CMs (Fig. 2 C). This strongly suggests that the PCC deficiency not only affects propionyl-CoA metabolism but also disrupts propionate metabolism, consistent with our previous findings in the livers of Pcca −/− (A138T) mice [ 53 ]. [ 13 C 3 ]Propionate, used as a tracer, allows for tracking both propionate and propionyl-CoA metabolism [ 24 ]. TCA cycle intermediates are downstream metabolites of propionyl-CoA, and stable isotope labeling of these intermediates reflects propionyl-CoA metabolism (Fig. 3 A). The stable isotopomer labeling and average carbon labeling of malate and citrate are shown in Figs. 3 B- 3 E. The low labeling of malate and citrate in PA hiPSC-CMs confirms that the PCCA mutation impairs PCC activity and demonstrates that this disrupts propionyl-CoA anaplerosis into the TCA cycle. The dramatic reduction in labeling of TCA cycle intermediates is further corroborated by other metabolite labeling data (Supplemental Fig. 2). PCC deficiency enhances glucose metabolism in hiPSC-CMs Approximately 25% or more of patients with PA develop cardiac diseases, although the pathological mechanisms remain largely unknown [ 34 ]. Fuel metabolism is crucial for cardiac function, as it sustains energy production required for mechanical contraction. To better understand the metabolic alterations in PA, we investigated the major fuel metabolism pathways in PA hiPSC-CMs. We replaced unlabeled glucose (11 mM) with 11 mM [ 13 C 6 ]glucose in the medium to trace glucose metabolism. First, we measured the consumption of [ 13 C 6 ]glucose by quantifying the levels of [ 13 C 6 ]glucose in both pre- and post-culture media using LC-Q Exactive + -MS (Fig. 4 A). After 48 hours of incubation, [ 13 C 6 ]glucose levels were much lower in the medium of PA hiPSC-CMs compared to control hiPSC-CMs (Fig. 4 B), indicating that PA hiPSC-CMs metabolize [ 13 C 6 ]glucose at a higher rate than control cells. Using 13 C labeled tracer, we traced the downstream metabolites of glucose to assess glucose metabolic rate through stable isotopomer analysis (Fig. 5 A). The stable isotope labeling of glycolytic intermediates—such as 3-phosphoglycerate (3PG), phosphoenolpyruvate (PEP), pyruvate, and lactate—was measured (Figs. 5 B- 5 E). The stable isotope labeling of these intermediates was significantly higher in PA hiPSC-CMs, reaching 80%, compared to 60% in control hiPSC-CMs. This confirms that glucose metabolism is elevated in PA-derived cardiomyocytes. Further downstream in the glucose metabolic pathway, TCA cycle intermediates were labeled by [ 13 C 6 ]glucose (Fig. 6 A). Figures 6 B- 6 E show the stable isotopomer labeling and average carbon labeling of malate and citrate in control and PA hiPSC-CMs. Consistent with the increased glucose metabolism in PA hiPSC-CMs, the stable isotope labeling of malate was significantly higher in PA than in control cells. Additionally, the shift to a higher isotopomer of citrate in PA hiPSC-CMs demonstrates increased metabolic flux of glucose into the TCA cycle (Fig. 6 D). The lower M2 citrate in PA hiPSC-CMs also supports the finding of reduced unlabeled oxaloacetate/malate (Fig. 6 F), as M2 citrate is primarily derived from M0 oxaloacetate and M2 acetyl-CoA (M2 citrate = M0 oxaloacetate × M2 oxaloacetate). This pattern was also observed in other detected TCA cycle intermediates (Supplemental Fig. 3). Interestingly, M3 malate was the second most abundant isotopomer after M2 (Fig. 6 B). The minor amount of M3 malate could be generated from multiple turns of the TCA cycle after M2 acetyl-CoA enters the cycle (Supplement Fig. 4 ). M2 malate and M3 malate are primarily derived from M2 acetyl-CoA via pyruvate dehydrogenase and M3 pyruvate via pyruvate carboxylase, respectively. The increased glucose metabolism in PA hiPSC-CMs is likely driven by enhanced metabolic flux through both pyruvate dehydrogenase and pyruvate carboxylase. Increased secretion of labeled acetate and unlabeled propionate in PA hiPSC-CMs Acyl-CoAs are intracellular metabolites that cannot cross the plasma membrane [ 52 ]. To prevent the accumulation of acyl-CoAs, which can be toxic due to their detergent-like effect, they are converted into their counterparts, acylcarnitines. This metabolic conversion serves as a protective mechanism. In the context of PA, carnitine supplementation can facilitate the conversion of accumulated propionyl-CoA to propionylcarnitine, helping to remove excess propionyl-CoA from cells. However, it remains unclear whether acyl-CoAs could also be hydrolyzed into free fatty acids, offering an additional means for the intracellular metabolites to escape cells and enter the bloodstream (Fig. 7 A). In this study, we measured short-chain fatty acids in the cultured media and detected M2 acetate, derived from labeled M2 acetyl-CoA, in the media from [ 13 C 6 ]glucose (Fig. 7 B). This confirmed the activity of acyl-CoA hydrolase, which plays a secondary role in regulating cellular acyl-CoA levels. The increased secretion of labeled acetate (M2 acetate) from [ 13 C 6 ]glucose in PA hiPSC-CMs also suggests enhanced glucose metabolism in these cells. As anticipated, accumulated propionyl-CoA led to the increased release of propionate into the medium (Fig. 7 C) via acyl-CoA hydrolase, consistent with results from the [¹³C₃]propionate experiment (Fig. 2 B). In contrast, the release of butyrate and hexanoate decreased, consistent with the reduction of medium-chain acylcarnitines (Figs. 7 D, 7 E, 1 B- 1 E). This decrease in butyrate, hexanoate, and their corresponding acylcarnitines further supports the shift from fatty acid metabolism to glucose metabolism in PA hiPSC-CMs. Reduced fatty acid oxidation in PA hiPSC-CMs The increased lipid droplets have been observed in the heart from patients with PA [ 33 ]. A recent study using stable isotope techniques also demonstrated altered lipid metabolism in PA patients [ 48 ]. Given the increased glucose metabolism, we sought to investigate fatty acid metabolism in PA hiPSC-CMs using [ 13 C 16 ]palmitate. After 48 hours of culturing, we measured the labeling of metabolites derived from [ 13 C 16 ]palmitate (Fig. 8 A). In the cultured medium samples, acylcarnitine intermediates from M16 palmitate were significantly lower in PA hiPSC-CMs (Figs. 8 B- 8 E and Supplemental Figs. 5A-5C). Notably, M2 acetate labeling was also significantly lower in the cultured media of PA hiPSC-CMs (Fig. 8 F). As observed in the [ 13 C 6 ]glucose tracing experiment, M2 acetate is derived from the hydrolysis of M2 acetyl-CoA, which is produced during the metabolism of both [ 13 C 6 ]glucose and [ 13 C 16 ]palmitate. However, in contrast to the [ 13 C 6 ]glucose experiment (Fig. 7 B), M2 acetate labeling in PA hiPSC-CMs was significantly lower than in control cells when [ 13 C 16 ]palmitate was used as a tracer (Fig. 8 F). Together, these findings suggest that PCC deficiency induces a fuel switch from fatty acid metabolism to increased glucose utilization. Free carnitine plays a crucial role in transporting long-chain fatty acids into the mitochondria for complete β-oxidation. We observed a reduction, or a trend toward reduced, cellular L-carnitine in all experiments (Supplemental. Figure 1 B and Supplemental Fig. 6). The L-carnitine deficiency in PA hiPSC-CMs impedes fatty acid oxidation, which in turn promotes increased glucose metabolism. Discussion PA is a rare metabolic disorder caused by mutations in PCCA or PCCB genes, resulting in impaired propionyl-CoA metabolism. PA is typically diagnosed early in life, often within days of birth. If left unmanaged, PA can lead to severe complications, including cardiac dysfunction. The underlying mechanisms driving these cardiac complications are not yet fully understood. Chronic metabolic alterations associated with PA are believed to contribute to these complications. Energy metabolism is crucial for the heart to perform mechanical work. Fatty acids are the heart's preferred and most efficient fuel for ATP production. In failing hearts, however, fuel metabolism switches from fatty acids to increased glucose and ketone metabolism [ 13 ]. This shift may be particularly pronounced in PA patients due to several factors: (1) the depletion of L-carnitine and free CoA, caused by the accumulation of propionylcarnitine and propionyl-CoA, which impairs fatty acid oxidation, (2) the accumulation of lipid droplets in the heart, indicating disrupted fatty acid metabolism [ 4 , 5 ], and (3) the inhibition of fatty acid oxidation during exercise in PA patients [ 48 ]. In this study, we observed that PA hiPSC-CMs exhibit a significant increase in glucose utilization and a decrease in fatty acid oxidation using stable isotope-based metabolic flux analysis. This chronic metabolic switch could impair cardiac energy metabolism and lead to progressive cardiac dysfunction, as the heart preferentially utilizes fatty acids for energy. Interestingly, while resting fatty acid oxidation in PA patients may not differ significantly from healthy individuals, PA patients are unable to fully utilize fatty acids during exercise when the whole-body switches to fatty acid oxidation [ 48 ]. The accumulation of propionylcarnitine in PA leads to decreased intracellular L-carnitine levels, particularly in tissues like the heart. One PA patient with fatal cardiomyopathy had low carnitine levels in the heart despite supplementation [ 38 ]. Reduced intracellular L-carnitine (Supplemental Fig. 1B and Supplemental Fig. 6) limits the transport of long-chain fatty acids into mitochondria for complete β-oxidation. Chronic reductions in fatty acid oxidation could trigger cardiac disease, as seen in patients with long-chain fatty acid β-oxidation disorders (LCFAOD), who frequently develop cardiomyopathy [ 29 ]. Propionate in plasma and urine is another reliable biomarker for PA. Short-chain fatty acids (SCFAs), typically produced by the microbiome, are usually at low levels in the portal vein of germ-free mice [ 23 , 53 ]. The liver plays a key role in metabolizing and disposing of propionate, maintaining low levels ( ~ < 1 µM) [ 53 ]. However, it remains unclear whether SCFAs could be produced endogenously, given two key observations: (1) acetate, the most abundant SCFA in the blood, correlates well with acetyl-CoA levels, which are among the highest of all acyl-CoAs, and (2) propionate arises in PA due to propionyl-CoA accumulation. This raises the question of whether acyl-CoA hydrolysis might be another source of SCFAs. Acyl-CoA hydrolysis, a process that releases free fatty acids that can be transported out of cells, is a potential regulatory mechanism that controls intracellular acyl-CoA levels and prevents their accumulation. Although the role of acyl-CoA hydrolases is understudied, they could play an essential role in regulating CoA for other metabolic events in the cell [ 1 , 3 ]. In peroxisomes, acyl-CoAs are hydrolyzed into free fatty acids, which are then transported to mitochondria for complete β-oxidation [ 58 ]. We confirmed the acyl-CoA hydrolysis activity using stable isotope tracing in this study. Labeled acetate and unlabeled propionate, hydrolyzed from labeled acetyl-CoA and unlabeled propionyl-CoA, occur in mitochondria, where pyruvate oxidation to acetyl-CoA and propionyl-CoA metabolism predominantly take place. Free acetate from pyruvate metabolism could also be directly catalyzed by pyruvate dehydrogenase or 2-ketoglutarate dehydrogenase according to Liu et al.’s work [ 35 ]. However, the release of propionate, butyrate and hexanoate confirms the acyl-CoA hydrolase activity. The physiological implications of propionyl-CoA hydrolysis into propionate in PA warrant further investigation. This hydrolysis could represent a second regulatory mechanism to control intracellular propionyl-CoA levels, serving as a \"pressure valve\" to mitigate the toxicity of accumulated propionyl-CoA in mitochondria. This study is the first to utilize cardiomyocytes derived from patients with PA for metabolic discovery, emphasizing its strong clinical relevance. However, the authors acknowledge the following limitations: This is an in vitro experiment, which does not fully capture the complexity of metabolic regulation present in vivo . The in vitro conditions may not accurately reflect the physiological environment in vivo . These limitations highlight the need for future in vivo studies, which face the following challenges: (1) Metabolic data from the heart in vivo is often confounded by secondary metabolites originating from other organs. (2) Experimental findings using heart tissue from animal models may not directly translate to human patients. In summary, using stable isotope tracing and hiPSC-CMs derived from PA patients, we provide the direct evidence that PA induces a fuel switch from fatty acids to glucose in cardiomyocytes derived from patients with PA. This shift may underlie the cardiac complications observed in PA. Additionally, we identify a second metabolic regulatory mechanism—acyl-CoA hydrolysis—that helps control propionyl-CoA levels in PA. This pathway may offer a potential therapeutic target for treating PA. Declarations Author Contributions Material preparation, data collection and analysis were performed by Eva Richard, Hannah Marchuk, Mar Álvarez and Wentao He. Eva Richard, Xiaoxin Chen, Lourdes R. Desvia and Guof-Fang Zhang contributed to the study conception and design. The first draft of the manuscript was written by Guo-Fang Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by Propionic Acidemia Foundation awards: to G.F.Z. and PAF113 to E. R., NIH R01 AA030026 and NIH R21TR005163 to X.C and G.F.Z, the Fundación Ramón Areces (XX National Call 2020) to E.R. and the MICIU/AEI/10.13039/501100011033/ and ERDF A way of making Europe (PID2022-137238OB-100) to L.R.D. and E.R. Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces and is a Severo Ochoa Center of Excellence (MICIN, Award CEX2021-001154-S). Availability of data and material Data will be made available from authors on request. Conflict of interest. The authors declare that there are no conflicts of interest. Ethical approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Autonomous University of Madrid (project identification code CEI-134-2830; date of approval: 3 November 2023); and by the authorization of “Dirección General de Investigación Sanitaria y Documentación”, Community of Madrid (data of approval: 19 February 2024). Written informed consent for research has been obtained from the patient’s family. Consent for publication . All authors read and approved the final version of this manuscript. References Agius L, Wright PD, Alberti KG (1987) Carnitine acyltransferases and acyl-CoA hydrolases in human and rat liver. Clin Sci (Lond) 73:3-10 doi:10.1042/cs0730003 Al-Hamed MH, Imtiaz F, Al-Hassnan Z, Al-Owain M, Al-Zaidan H, Alamoudi MS, Faqeih E, Alfadhel M, Al-Asmari A, Saleh MM, Almutairi F, Moghrabi N, AlSayed M (2019) Spectrum of mutations underlying Propionic acidemia and further insight into a genotype-phenotype correlation for the common mutation in Saudi Arabia. Mol Genet Metab Rep 18:22-29 doi:10.1016/j.ymgmr.2018.12.004 Alexson SE, Nedergaard J (1988) A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria. J Biol Chem 263:13564-13571 Alonso-Barroso E, Perez B, Desviat LR, Richard E (2021) Cardiomyocytes Derived from Induced Pluripotent Stem Cells as a Disease Model for Propionic Acidemia. Int J Mol Sci 22 doi:10.3390/ijms22031161 Alvarez M, Ruiz-Sala P, Perez B, Desviat LR, Richard E (2023) Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy. Int J Mol Sci 24 doi:10.3390/ijms24032182 Amaral AU, Cecatto C, Castilho RF, Wajner M (2016) 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria. J Neurochem 137:62-75 doi:10.1111/jnc.13544 Bodi I, Grunert SC, Becker N, Stoelzle-Feix S, Spiekerkoetter U, Zehender M, Bugger H, Bode C, Odening KE (2016) Mechanisms of acquired long QT syndrome in patients with propionic academia. Heart Rhythm 13:1335-1345 doi:10.1016/j.hrthm.2016.02.003 Brunengraber H, Roe CR (2006) Anaplerotic molecules: current and future. J Inherit Metab Dis 29:327-331 doi:10.1007/s10545-006-0320-1 Chalmers RA, Roe CR, Stacey TE, Hoppel CL (1984) Urinary excretion of l-carnitine and acylcarnitines by patients with disorders of organic acid metabolism: evidence for secondary insufficiency of l-carnitine. Pediatr Res 18:1325-1328 doi:10.1203/00006450-198412000-00021 Chandler RJ, Chandrasekaran S, Carrillo-Carrasco N, Senac JS, Hofherr SE, Barry MA, Venditti CP (2011) Adeno-associated virus serotype 8 gene transfer rescues a neonatal lethal murine model of propionic acidemia. Hum Gene Ther 22:477-481 doi:10.1089/hum.2010.164 Chapman KA, Ostrovsky J, Rao M, Dingley SD, Polyak E, Yudkoff M, Xiao R, Bennett MJ, Falk MJ (2018) Propionyl-CoA carboxylase pcca-1 and pccb-1 gene deletions in Caenorhabditis elegans globally impair mitochondrial energy metabolism. J Inherit Metab Dis 41:157-168 doi:10.1007/s10545-017-0111-x Cheema-Dhadli S, Leznoff CC, Halperin ML (1975) Effect of 2-methylcitrate on citrate metabolism: implications for the management of patients with propionic acidemia and methylmalonic aciduria. Pediatr Res 9:905-908 doi:10.1203/00006450-197512000-00008 Davila-Roman VG, Vedala G, Herrero P, de las Fuentes L, Rogers JG, Kelly DP, Gropler RJ (2002) Altered myocardial fatty acid and glucose metabolism in idiopathic dilated cardiomyopathy. J Am Coll Cardiol 40:271-277 doi:10.1016/s0735-1097(02)01967-8 Di Donato S, Rimoldi M, Garavaglia B, Uziel G (1984) Propionylcarnitine excretion in propionic and methylmalonic acidurias: a cause of carnitine deficiency. Clin Chim Acta 139:13-21 doi:10.1016/0009-8981(84)90187-6 Duran M, Ketting D, Beckeringh TE, Leupold D, Wadman SK (1986) Direct identification of propionylcarnitine in propionic acidaemia: biochemical and clinical results of oral carnitine supplementation. J Inherit Metab Dis 9:202-207 doi:10.1007/BF01799460 Fernandez CA, Des Rosiers C, Previs SF, David F, Brunengraber H (1996) Correction of 13C mass isotopomer distributions for natural stable isotope abundance. J Mass Spectrom 31:255-262 doi:10.1002/(SICI)1096-9888(199603)31:3<255::AID-JMS290>3.0.CO;2-3 Fulgencio-Covian A, Alonso-Barroso E, Guenzel AJ, Rivera-Barahona A, Ugarte M, Perez B, Barry MA, Perez-Cerda C, Richard E, Desviat LR (2020) Pathogenic implications of dysregulated miRNAs in propionic acidemia related cardiomyopathy. Transl Res 218:43-56 doi:10.1016/j.trsl.2019.12.004 Galarreta Aima CI, Shchelochkov OA, Jerves Serrano T, Venditti CP (1993) Propionic Acidemia. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A (eds) GeneReviews((R)). Seattle (WA) Gallego-Villar L, Perez-Cerda C, Perez B, Abia D, Ugarte M, Richard E, Desviat LR (2013) Functional characterization of novel genotypes and cellular oxidative stress studies in propionic acidemia. J Inherit Metab Dis 36:731-740 doi:10.1007/s10545-012-9545-3 Gallego-Villar L, Rivera-Barahona A, Cuevas-Martin C, Guenzel A, Perez B, Barry MA, Murphy MP, Logan A, Gonzalez-Quintana A, Martin MA, Medina S, Gil-Izquierdo A, Cuezva JM, Richard E, Desviat LR (2016) In vivo evidence of mitochondrial dysfunction and altered redox homeostasis in a genetic mouse model of propionic acidemia: Implications for the pathophysiology of this disorder. Free Radic Biol Med 96:1-12 doi:10.1016/j.freeradbiomed.2016.04.007 Guenzel AJ, Hofherr SE, Hillestad M, Barry M, Weaver E, Venezia S, Kraus JP, Matern D, Barry MA (2013) Generation of a hypomorphic model of propionic acidemia amenable to gene therapy testing. Mol Ther 21:1316-1323 doi:10.1038/mt.2013.68 He W, Berthiaume JM, Previs S, Kasumov T, Zhang GF (2023) Ischemia promotes acyl-CoAs dephosphorylation and propionyl-CoA accumulation. Metabolomics 19:12 doi:10.1007/s11306-023-01975-2 He W, Marchuk H, Koeberl D, Kasumov T, Chen X, Zhang GF (2024) Fasting alleviates metabolic alterations in mice with propionyl-CoA carboxylase deficiency due to Pcca mutation. Commun Biol 7:659 doi:10.1038/s42003-024-06362-8 He W, Wang Y, Xie EJ, Barry MA, Zhang GF (2021) Metabolic perturbations mediated by propionyl-CoA accumulation in organs of mouse model of propionic acidemia. Mol Genet Metab 134:257-266 doi:10.1016/j.ymgme.2021.09.009 Hofherr SE, Senac JS, Chen CY, Palmer DJ, Ng P, Barry MA (2009) Short-term rescue of neonatal lethality in a mouse model of propionic acidemia by gene therapy. Hum Gene Ther 20:169-180 doi:10.1089/hum.2008.158 Hommes FA, Kuipers JR, Elema JD, Jansen JF, Jonxis JH (1968) Propionicacidemia, a new inborn error of metabolism. Pediatr Res 2:519-524 doi:10.1203/00006450-196811000-00010 Kasumov T, Cendrowski AV, David F, Jobbins KA, Anderson VE, Brunengraber H (2007) Mass isotopomer study of anaplerosis from propionate in the perfused rat heart. Arch Biochem Biophys 463:110-117 doi:10.1016/j.abb.2007.02.022 Kinman RP, Kasumov T, Jobbins KA, Thomas KR, Adams JE, Brunengraber LN, Kutz G, Brewer WU, Roe CR, Brunengraber H (2006) Parenteral and enteral metabolism of anaplerotic triheptanoin in normal rats. Am J Physiol Endocrinol Metab 291:E860-866 doi:10.1152/ajpendo.00366.2005 Knottnerus SJG, Bleeker JC, Ferdinandusse S, Houtkooper RH, Langeveld M, Nederveen AJ, Strijkers GJ, Visser G, Wanders RJA, Wijburg FA, Boekholdt SM, Bakermans AJ (2020) Subclinical effects of long-chain fatty acid beta-oxidation deficiency on the adult heart: A case-control magnetic resonance study. J Inherit Metab Dis 43:969-980 doi:10.1002/jimd.12266 Kolker S, Garcia-Cazorla A, Valayannopoulos V, Lund AM, Burlina AB, Sykut-Cegielska J, Wijburg FA, Teles EL, Zeman J, Dionisi-Vici C, Baric I, Karall D, Augoustides-Savvopoulou P, Aksglaede L, Arnoux JB, Avram P, Baumgartner MR, Blasco-Alonso J, Chabrol B, Chakrapani A, Chapman K, EC IS, Couce ML, de Meirleir L, Dobbelaere D, Dvorakova V, Furlan F, Gleich F, Gradowska W, Grunewald S, Jalan A, Haberle J, Haege G, Lachmann R, Laemmle A, Langereis E, de Lonlay P, Martinelli D, Matsumoto S, Muhlhausen C, de Baulny HO, Ortez C, Pena-Quintana L, Ramadza DP, Rodrigues E, Scholl-Burgi S, Sokal E, Staufner C, Summar ML, Thompson N, Vara R, Pinera IV, Walter JH, Williams M, Burgard P (2015) The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 1: the initial presentation. J Inherit Metab Dis 38:1041-1057 doi:10.1007/s10545-015-9839-3 Kolker S, Valayannopoulos V, Burlina AB, Sykut-Cegielska J, Wijburg FA, Teles EL, Zeman J, Dionisi-Vici C, Baric I, Karall D, Arnoux JB, Avram P, Baumgartner MR, Blasco-Alonso J, Boy SP, Rasmussen MB, Burgard P, Chabrol B, Chakrapani A, Chapman K, Cortes ISE, Couce ML, de Meirleir L, Dobbelaere D, Furlan F, Gleich F, Gonzalez MJ, Gradowska W, Grunewald S, Honzik T, Horster F, Ioannou H, Jalan A, Haberle J, Haege G, Langereis E, de Lonlay P, Martinelli D, Matsumoto S, Muhlhausen C, Murphy E, de Baulny HO, Ortez C, Pedron CC, Pintos-Morell G, Pena-Quintana L, Ramadza DP, Rodrigues E, Scholl-Burgi S, Sokal E, Summar ML, Thompson N, Vara R, Pinera IV, Walter JH, Williams M, Lund AM, Garcia-Cazorla A (2015) The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 2: the evolving clinical phenotype. J Inherit Metab Dis 38:1059-1074 doi:10.1007/s10545-015-9840-x Kor D, Seker-Yilmaz B, Bulut FD, Kilavuz S, Oktem M, Ceylaner S, Yildizdas D, Onenli-Mungan N (2019) Clinical features of 27 Turkish Propionic acidemia patients with 12 novel mutations. Turk J Pediatr 61:330-336 doi:10.24953/turkjped.2019.03.003 Kott-Blumenkranz R, Pappas CT, Bensch KG (1981) A study of the ultrastructure of the organs and of cultured fibroblasts incubated with isoleucine from a patient with propionic acidemia. Hum Pathol 12:1141-1148 doi:10.1016/s0046-8177(81)80336-x Kovacevic A, Garbade SF, Horster F, Hoffmann GF, Gorenflo M, Mereles D, Kolker S, Staufner C (2022) Detection of early cardiac disease manifestation in propionic acidemia - Results of a monocentric cross-sectional study. Mol Genet Metab 137:349-358 doi:10.1016/j.ymgme.2022.10.007 Liu X, Cooper DE, Cluntun AA, Warmoes MO, Zhao S, Reid MA, Liu J, Lund PJ, Lopes M, Garcia BA, Wellen KE, Kirsch DG, Locasale JW (2018) Acetate Production from Glucose and Coupling to Mitochondrial Metabolism in Mammals. Cell 175:502-513 e513 doi:10.1016/j.cell.2018.08.040 Maines E, Moretti M, Vitturi N, Gugelmo G, Fasan I, Lenzini L, Piccoli G, Gragnaniello V, Maiorana A, Soffiati M, Burlina A, Franceschi R (2023) Understanding the Pathogenesis of Cardiac Complications in Patients with Propionic Acidemia and Exploring Therapeutic Alternatives for Those Who Are Not Eligible or Are Waiting for Liver Transplantation. Metabolites 13 doi:10.3390/metabo13040563 Marchuk H, Wang Y, Ladd ZA, Chen X, Zhang GF (2023) Pathophysiological mechanisms of complications associated with propionic acidemia. Pharmacol Ther 249:108501 doi:10.1016/j.pharmthera.2023.108501 Mardach R, Verity MA, Cederbaum SD (2005) Clinical, pathological, and biochemical studies in a patient with propionic acidemia and fatal cardiomyopathy. Mol Genet Metab 85:286-290 doi:10.1016/j.ymgme.2005.04.004 Martini WZ, Stanley WC, Huang H, Rosiers CD, Hoppel CL, Brunengraber H (2003) Quantitative assessment of anaplerosis from propionate in pig heart in vivo. Am J Physiol Endocrinol Metab 284:E351-356 doi:10.1152/ajpendo.00354.2002 Miyazaki T, Ohura T, Kobayashi M, Shigematsu Y, Yamaguchi S, Suzuki Y, Hata I, Aoki Y, Yang X, Minjares C, Haruta I, Uto H, Ito Y, Muller U (2001) Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene. J Biol Chem 276:35995-35999 doi:10.1074/jbc.M105467200 Reszko AE, Kasumov T, Pierce BA, David F, Hoppel CL, Stanley WC, Des Rosiers C, Brunengraber H (2003) Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver. J Biol Chem 278:34959-34965 doi:10.1074/jbc.M302013200 Ribas GS, Manfredini V, de Marco MG, Vieira RB, Wayhs CY, Vanzin CS, Biancini GB, Wajner M, Vargas CR (2010) Prevention by L-carnitine of DNA damage induced by propionic and L-methylmalonic acids in human peripheral leukocytes in vitro. Mutat Res 702:123-128 doi:10.1016/j.mrgentox.2010.07.008 Ribas GS, Manfredini V, de Mari JF, Wayhs CY, Vanzin CS, Biancini GB, Sitta A, Deon M, Wajner M, Vargas CR (2010) Reduction of lipid and protein damage in patients with disorders of propionate metabolism under treatment: a possible protective role of L-carnitine supplementation. Int J Dev Neurosci 28:127-132 doi:10.1016/j.ijdevneu.2010.01.002 Rivera-Barahona A, Fulgencio-Covian A, Perez-Cerda C, Ramos R, Barry MA, Ugarte M, Perez B, Richard E, Desviat LR (2017) Dysregulated miRNAs and their pathogenic implications for the neurometabolic disease propionic acidemia. Sci Rep 7:5727 doi:10.1038/s41598-017-06420-8 Roginski AC, Cecatto C, Wajner SM, Camera FD, Castilho RF, Wajner M, Amaral AU (2019) Experimental evidence that maleic acid markedly compromises glutamate oxidation through inhibition of glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase activities in kidney of developing rats. Mol Cell Biochem 458:99-112 doi:10.1007/s11010-019-03534-7 Roginski AC, Zemniacak AB, Marschner RA, Wajner SM, Ribeiro RT, Wajner M, Amaral AU (2022) Disruption of mitochondrial functions involving mitochondrial permeability transition pore opening caused by maleic acid in rat kidney. J Bioenerg Biomembr 54:203-213 doi:10.1007/s10863-022-09945-4 Salmi H, Leonard JV, Lapatto R (2012) Patients with organic acidaemias have an altered thiol status. Acta Paediatr 101:e505-508 doi:10.1111/j.1651-2227.2012.02799.x Storgaard JH, Madsen KL, Lokken N, Vissing J, van Hall G, Lund AM, Orngreen MC (2020) Impaired lipolysis in propionic acidemia: A new metabolic myopathy? JIMD Rep 53:16-21 doi:10.1002/jmd2.12113 Tajima G, Kagawa R, Sakura F, Nakamura-Utsunomiya A, Hara K, Yuasa M, Hasegawa Y, Sasai H, Okada S (2021) Current Perspectives on Neonatal Screening for Propionic Acidemia in Japan: An Unexpectedly High Incidence of Patients with Mild Disease Caused by a Common PCCB Variant. Int J Neonatal Screen 7 doi:10.3390/ijns7030035 Tomcik K, Ibarra RA, Sadhukhan S, Han Y, Tochtrop GP, Zhang GF (2011) Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications. Anal Biochem 410:110-117 doi:10.1016/j.ab.2010.11.030 Wang Y, Christopher BA, Wilson KA, Muoio D, McGarrah RW, Brunengraber H, Zhang GF (2018) Propionate-induced changes in cardiac metabolism, notably CoA trapping, are not altered by l-carnitine. Am J Physiol Endocrinol Metab 315:E622-E633 doi:10.1152/ajpendo.00081.2018 Wang Y, Yang H, Geerts C, Furtos A, Waters P, Cyr D, Wang S, Mitchell GA (2023) The multiple facets of acetyl-CoA metabolism: Energetics, biosynthesis, regulation, acylation and inborn errors. Mol Genet Metab 138:106966 doi:10.1016/j.ymgme.2022.106966 Wang Y, Zhu S, He W, Marchuk H, Richard E, Desviat LR, Young SP, Koeberl D, Kasumov T, Chen X, Zhang GF (2024) The attenuated hepatic clearance of propionate increases cardiac oxidative stress in propionic acidemia. Basic Res Cardiol doi:10.1007/s00395-024-01066-w Wilson KA, Han Y, Zhang M, Hess JP, Chapman KA, Cline GW, Tochtrop GP, Brunengraber H, Zhang GF (2017) Inter-relations between 3-hydroxypropionate and propionate metabolism in rat liver: relevance to disorders of propionyl-CoA metabolism. Am J Physiol Endocrinol Metab 313:E413-E428 doi:10.1152/ajpendo.00105.2017 Wolf B, Hsia YE, Sweetman L, Gravel R, Harris DJ, Nyhan WL (1981) Propionic acidemia: a clinical update. J Pediatr 99:835-846 doi:10.1016/s0022-3476(81)80004-2 Wongkittichote P, Ah Mew N, Chapman KA (2017) Propionyl-CoA carboxylase - A review. Mol Genet Metab 122:145-152 doi:10.1016/j.ymgme.2017.10.002 Zhang GF, Jensen MV, Gray SM, El K, Wang Y, Lu D, Becker TC, Campbell JE, Newgard CB (2021) Reductive TCA cycle metabolism fuels glutamine- and glucose-stimulated insulin secretion. Cell Metab 33:804-817 e805 doi:10.1016/j.cmet.2020.11.020 Zhang Y, Agarwal KC, Beylot M, Soloviev MV, David F, Reider MW, Anderson VE, Tserng KY, Brunengraber H (1994) Nonhomogeneous labeling of liver extra-mitochondrial acetyl-CoA. Implications for the probing of lipogenic acetyl-CoA via drug acetylation and for the production of acetate by the liver. J Biol Chem 269:11025-11029 Zhang Y, Peng C, Wang L, Chen S, Wang J, Tian Z, Wang C, Chen X, Zhu S, Zhang GF, Wang Y (2023) Prevalence of propionic acidemia in China. Orphanet J Rare Dis 18:281 doi:10.1186/s13023-023-02898-w Supplementary Files 20250110Supplementalfigures.pptx Cite Share Download PDF Status: Published Journal Publication published 02 Apr, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Reviewers agreed at journal 24 Jan, 2025 Reviewers invited by journal 24 Jan, 2025 Editor assigned by journal 23 Jan, 2025 First submitted to journal 21 Jan, 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5874705\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":406832815,\"identity\":\"37de2f06-0e49-4b87-80ad-28022d2fd3ab\",\"order_by\":0,\"name\":\"Eva Richard\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad Autónoma de Madrid: Universidad Autonoma de Madrid\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Eva\",\"middleName\":\"\",\"lastName\":\"Richard\",\"suffix\":\"\"},{\"id\":406832816,\"identity\":\"d460c2e7-6b8e-430a-8e50-0f023ffa4e43\",\"order_by\":1,\"name\":\"Hannah Marchuk\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Duke University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hannah\",\"middleName\":\"\",\"lastName\":\"Marchuk\",\"suffix\":\"\"},{\"id\":406832817,\"identity\":\"f74e1851-e8d7-44f6-a7d1-a200e787f178\",\"order_by\":2,\"name\":\"Mar Álvarez\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad Autónoma de Madrid: Universidad Autonoma de Madrid\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mar\",\"middleName\":\"\",\"lastName\":\"Álvarez\",\"suffix\":\"\"},{\"id\":406832818,\"identity\":\"257ca306-4670-41a6-b8db-a254f1c7f257\",\"order_by\":3,\"name\":\"Wentao He\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Duke University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wentao\",\"middleName\":\"\",\"lastName\":\"He\",\"suffix\":\"\"},{\"id\":406832819,\"identity\":\"228319c8-7bf8-48f7-91d8-74757c189e62\",\"order_by\":4,\"name\":\"Xiaoxin Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Coriell Institute for Medical Research\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaoxin\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":406832820,\"identity\":\"74f4e8c0-9a05-441a-8682-5e8c146b6e4b\",\"order_by\":5,\"name\":\"Lourdes R. Desviat\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad Autónoma de Madrid: Universidad Autonoma de Madrid\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lourdes\",\"middleName\":\"R.\",\"lastName\":\"Desviat\",\"suffix\":\"\"},{\"id\":406832821,\"identity\":\"0f1d0d1d-c984-498a-8988-be4034212d7e\",\"order_by\":6,\"name\":\"Guo-Fang Zhang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYJCCD2BSAkRUMENoHvw6GGcgtJwhWQtjGxFa5COSDzb83FHLID+7+dnDr/OsE2e2NzA+eNuGW4vhjbTExt4zxxkY5xwzN5bdlp44m+cAs+FcfFpm5Jg/4G07xsAskWAmLbntcOI8iQQ2aV78Wgwb/wK1sEmkf5OWnAPUIv+A/Tc+LfISOYbNvG01DDwSOWaSHxsOJ86WYGBjxqfFgOdZYrNs2wEeCYmcMmmGY+nGM3sSmyXnnMNjS3vywca3bXVy8jPSt0n+qLGWnXH88MEPb8rw2HIATB0GRwQzJDoYG3CrB9kCka6DqP2BV+0oGAWjYBSMVAAAEdtSFSy7U50AAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0003-3484-5864\",\"institution\":\"Duke University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Guo-Fang\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-01-21 16:17:47\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5874705/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5874705/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s00018-025-05661-5\",\"type\":\"published\",\"date\":\"2025-04-02T15:57:11+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":74947814,\"identity\":\"b15e835b-69ec-4c6f-9bbd-5aebf9710466\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 15:52:10\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":173001,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelease of acylcarnitines from hiPSC-CMs into the culture medium\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea-d\\u003c/strong\\u003e) Levels of propionylcarnitine (C3 AC), butyrylcarnitine (C4 AC), hexanoylcarnitine (C6 AC), and octanoylcarnitine (C8 AC) in the culture medium after a 4-day incubation with RPMI/B27 medium. Data are presented as mean ± SE, with N=4 per group. ** and **** indicate p-values \\u0026lt; 0.01 and \\u0026lt; 0.001, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/9c686d7050f29a91d8dd62c0.png\"},{\"id\":74947811,\"identity\":\"fe4ad2f8-9a7f-4fe4-9ce6-223cae647877\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 15:52:10\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":368869,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of PCC deficiency on [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate consumption\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of the experimental design and procedure. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Levels of unlabeled propionate (M0 propionate) in the pre-culture medium and post-culture medium of hiPSC-CMs derived from healthy controls (Control) and PA patients (PA). (\\u003cstrong\\u003ec\\u003c/strong\\u003e) Residual [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate (M3 propionate) in the culture medium after 2 days of incubation with 1 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate in RPMI/B27 medium, comparing hiPSC-CMs from Control and PA groups. Data are presented as mean ± SE, with N=4 per group. **** indicates p-values \\u0026lt; 0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/7e208d74cf5a7f4317456833.png\"},{\"id\":74949236,\"identity\":\"1eba2771-bf5c-4cad-8b08-0152d88ab8cf\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 16:00:09\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":281283,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCC deficiency reduces [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate metabolism\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Simplified schematic of the [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate tracing pathway during the first round of TCA cycle entry. (\\u003cstrong\\u003eb-c\\u003c/strong\\u003e) Stable isotopomer labeling and average carbon (C) labeling of malate. (\\u003cstrong\\u003ed-e\\u003c/strong\\u003e) Stable isotopomer labeling and average carbon (C) labeling of citrate. hiPSC-CMs from control individual and PA patient were incubated with 1 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate for 48 hours. Data are presented as mean ± SE, with N=4 per group. ** and **** represent p-values \\u0026lt; 0.01 and \\u0026lt; 0.001, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/5e884a732222aa5a168c7c99.png\"},{\"id\":74947815,\"identity\":\"27151b4c-871f-45d6-bc2d-6b435d24765a\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 15:52:10\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":299051,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCC deficiency increases [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose consumption\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of the experimental design and procedure for measuring [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose consumption from pre-culture and post-culture media. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]Glucose concentrations in the culture media after 48 hours of incubation with hiPSC-CMs derived from a healthy control individual and a PA patient, using 11 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose. Data are presented as mean ± SE, with N=4 per group. ** indicates p-values \\u0026lt; 0.01.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/dec93adfde39175d89795773.png\"},{\"id\":74947813,\"identity\":\"16b84b7f-39b5-4cba-97f7-880fd886fd4c\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 15:52:10\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":247074,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCC deficiency enhances [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose glycolysis\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose metabolism through glycolysis, showing the labeling pathway. G6P, F6P, DHAP, GADP, 1,3PG, 2PG, PYR, and LAC represent glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 2-phosphoglycerate, pyruvate, and lactate, respectively. (\\u003cstrong\\u003eb-e\\u003c/strong\\u003e) M3 isotopomer labeling of glycolytic intermediates, including phosphoenolpyruvate (PEP), 3-phosphoglycerate (3PG), pyruvate, and lactate, in hiPSC-CMs from the control individual and the PA patient after 48 hours of culture with 11 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose. Data are presented as mean ± SE, with N=4 per group. **, ***, and **** indicate p-values \\u0026lt; 0.01, \\u0026lt; 0.005, and \\u0026lt; 0.001, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/a672859d628216f1dca00ebf.png\"},{\"id\":74947824,\"identity\":\"e6d14593-c7c9-4d83-8123-7668086e1593\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 15:52:11\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":334460,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCC deficiency enhances [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose metabolism through the TCA cycle\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose metabolism through the first round of the TCA cycle, showing [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]pyruvate entry via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). Key intermediates include acetyl-CoA, citrate, malate and \\u0026nbsp;oxaloacetate (OAA). (\\u003cstrong\\u003eb-e\\u003c/strong\\u003e) Stable isotopomer labeling and average carbon (C) labeling of malate and citrate derived from [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose in hiPSC-CMs from the control individual and the PA patient after 48 hours of culture. (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Percentage of unlabeled malate in the same samples. Data are presented as mean ± SE, with N=4 per group. ** indicates p-values \\u0026lt; 0.01.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/5a6d51776a123b6aea7babff.png\"},{\"id\":74949238,\"identity\":\"83787e7c-194e-49f5-9e82-3cc384a490fe\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 16:00:10\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":226653,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eShort-chain fatty acids are hydrolyzed from acyl-CoAs in hiPSC-CMs\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of acyl-CoA hydrolase-mediated hydrolysis of acyl-CoAs into free fatty acids. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Concentration of M2 acetate in the cultured media from control and PA hiPSC-CMs after 48 hours of incubation with [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose. (\\u003cstrong\\u003ec-e\\u003c/strong\\u003e) Concentrations of unlabeled propionate, butyrate, and hexanoate in the pre- and post-cultured media from control and PA hiPSC-CMs after 48 hours of incubation with [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose. Data are presented as mean ± SE, with N=4 per group. ** and *** denote p-values \\u0026lt; 0.01 and \\u0026lt; 0.005, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/00c53a3415de18fd1f4b6218.png\"},{\"id\":74949241,\"identity\":\"08220701-8fdf-4295-bf23-d210156f57ab\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 16:00:10\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":294525,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCC deficiency reduces [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate metabolism\\u003cstrong\\u003e. \\u003c/strong\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic representation of [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate metabolism. (\\u003cstrong\\u003eb-f\\u003c/strong\\u003e) Stable isotopomer labeling of M16 palmitoylcarnitine (M16 C16 AC), M14 myristoylcarnitine (M14 C14 AC), M12 lauroylcarnitine (M12 C12 AC), M10 caproylcarnitine (M10 C10 AC), and M2 acetate in the cultured media from control and PA hiPSC-CMs after 48 hours of incubation with 0.4 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate. Data are presented as mean ± SE, with N=4 per group. *, **, and *** indicate p-values \\u0026lt; 0.05, \\u0026lt; 0.01, and \\u0026lt; 0.005, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/542e6c316895cf75fb85b129.png\"},{\"id\":80082350,\"identity\":\"18b7bd92-f72d-4980-a47a-5c498e2a8069\",\"added_by\":\"auto\",\"created_at\":\"2025-04-07 16:08:30\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2956775,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/b0d00cf7-c364-4e21-8139-22dda64cfa34.pdf\"},{\"id\":74947805,\"identity\":\"aadf672f-2866-44dd-a152-836a0a0a3045\",\"added_by\":\"auto\",\"created_at\":\"2025-01-28 15:52:10\",\"extension\":\"pptx\",\"order_by\":12,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1046560,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"20250110Supplementalfigures.pptx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5874705/v1/fd3207970802bc91a6c33305.pptx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Metabolic flux analysis in hiPSC-CMs reveals insights into cardiac dysfunction in propionic acidemia\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePropionic acidemia (PA) is an inborn error of metabolism inherited in an autosomal recessive manner [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e]. Mutations in either the \\u003cem\\u003ePCCA\\u003c/em\\u003e or \\u003cem\\u003ePCCB\\u003c/em\\u003e gene result in the malfunction of propionyl-CoA carboxylase (PCC), a critical mitochondrial enzyme. The global incidence of PA varies, ranging from 1 in 50,000 to 1 in 100,000 live births. The adoption of tandem mass spectrometry for neonatal screening has significantly increased the detection rate of PA cases [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePCC is a crucial mitochondrial enzyme, though it remains relatively understudied [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. Whole-body PCC knockout in mouse models has been shown to be neonatal lethal [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Patients with PA, often present symptoms in early infancy such as poor feeding, vomiting, and low muscle tone [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. As the disease progresses, patients may develop a variety of complications, including life-threatening cardiac dysfunctions [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. Understanding the pathological mechanisms underlying PA-associated cardiac diseases is an urgent and critical need.\\u003c/p\\u003e \\u003cp\\u003ePCC catalyzes the carboxylation of propionyl-CoA to methylmalonyl-CoA, which is subsequently converted to succinyl-CoA and enters the tricarboxylic acid (TCA) cycle as part of an anaplerotic process [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. Malfunctioning PCC disrupts propionyl-CoA metabolism, leading to the accumulation of propionyl-CoA and its metabolites, including methylcitrate, 3-hydroxypropionate, and maleic acid [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. These accumulated metabolites have been reported to inhibit the TCA cycle and mitochondrial energy production in \\u003cem\\u003ein vitro\\u003c/em\\u003e enzyme assays using high concentrations of these metabolites [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. Additionally, oxidative stress, disruption of potassium channels, and altered miRNA expression have been implicated in the cardiac dysfunction observed in patients with PA [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePropionyl-CoA, an intracellular metabolite, cannot cross the plasma membrane [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. Instead, accumulated propionyl-CoA is converted to propionylcarnitine, which can be released into the blood and urine [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. L-carnitine has been shown to effectively treat PA and is recommended as a supplement for patients [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. The conversion of propionyl-CoA to propionylcarnitine is considered a critical metabolic pathway for the disposal of accumulated propionyl-CoA, with elevated propionylcarnitine serving as a biomarker for PA. However, PA is also associated with elevated levels of circulating propionate, which is partly due to reduced hepatic disposal [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. The exact source of circulating propionate remains unclear. While it is known to originate from the microbiome, it is uncertain whether it might also arise from intracellular propionyl-CoA within the host. This could represent a secondary regulatory mechanism for managing intracellular propionyl-CoA, in addition to its conversion to propionylcarnitine.\\u003c/p\\u003e \\u003cp\\u003ePreviously, we reported that supraphysiological levels of propionate dramatically increase propionyl-CoA and deplete free Coenzyme A (CoA) in perfused rat hearts [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. This CoA depletion causes a metabolic shift from the high energy-efficient fatty acid oxidation to low energy-efficient glucose metabolism [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. However, in the \\u003cem\\u003ePcca\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e(A138T) mouse model of PA, CoA or carnitine depletion in the heart occurs only upon the acute administration of high doses of propionate (500 mg/kg) [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. This highlights the need for a more representative PA model to investigate the pathological mechanisms underlying cardiac diseases associated with PA.\\u003c/p\\u003e \\u003cp\\u003eAnimal experiments often face limitations in their applicability to humans. In this study, we utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a control individual and a PA patient to investigate metabolic alterations and better understand the pathological mechanisms of PA. Using stable isotope-based metabolic flux analysis, we confirmed the metabolic phenotype of PCC deficiency in PA patient-derived hiPSC-CMs. Our experiments demonstrated that accumulated propionyl-CoA can be hydrolyzed to propionate, which is then exported from the cell as an additional \\\"pressure valve.\\\" Furthermore, PA hiPSC-CMs exhibited a metabolic switch from energy-efficient fatty acid oxidation to energy-inefficient glucose metabolism. This shift in fuel utilization may be a key pathological factor contributing to the cardiac dysfunction observed in PA patients.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMaintenance of hiPSC lines\\u003c/h2\\u003e \\u003cp\\u003eThe hiPSC lines utilized in this study included: (i) a PCCA-deficient hiPSC line (PCCA23-FiPS4F6 or UAMi001-A), created by reprogramming fibroblasts from a patient with \\u003cem\\u003ePCCA\\u003c/em\\u003e gene mutations (c.1899\\u0026thinsp;+\\u0026thinsp;4_1899\\u0026thinsp;+\\u0026thinsp;7delAGTA; p.(Cys616_Val633del) and c.1430-?_1643+?del; p.(Gly477Glufs*9)) via Sendai virus; and (ii) a normal control hiPSC line (FIPS Ctrl2-SV4F-1) acquired from the National Bank of Cell Lines at the Carlos III Health Institute (ISCIII, Madrid, Spain).\\u003c/p\\u003e \\u003cp\\u003eThese human iPSC lines were cultured on 60 mm dishes coated with Matrigel (hESC-qualified matrix, Corning, New York, NY, USA) and maintained in mTESR\\u0026trade; Plus medium (StemCell\\u0026trade; Technologies, Vancouver, BC, Canada), with media changes every other day. The hiPSCs were passaged every four days using ReleSR\\u0026trade; (StemCell\\u0026trade; Technologies) and 10 \\u0026micro;M Rock inhibitor (StemCell\\u0026trade; Technologies) at a splitting ratio of 1:3 to 1:5.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eDifferentiation of hiPSCs into cardiomyocytes\\u003c/h3\\u003e\\n\\u003cp\\u003ehiPSCs cultured in mTESR\\u0026trade; Plus medium were dissociated into single cells using StemPro Accutase (Gibco, Waltham, MA, USA). A total of 1x10\\u003csup\\u003e6\\u003c/sup\\u003e cells in 1.5 ml of mTESR\\u0026trade; Plus medium enriched with 10 \\u0026micro;M Rock inhibitor were plated on Matrigel-coated 12-well plates. The differentiation into cardiomyocytes was conducted using the STEMdiff\\u0026trade; Cardiomyocyte Differentiation and Maintenance Kits (StemCell\\u0026trade; Technologies) following the guidelines provided by the manufacturer. The characterization of cardiomyocytes was achieved by analyzing the expression of various cardiac-specific markers, including cardiac troponin T, α-smooth muscle actin, GATA4, and α-actinin 2 through immunocytochemistry as previously described [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e].\\u003c/p\\u003e\\n\\u003ch3\\u003eExperimental conditions and metabolic treatments of hiPSC-derived cardiomyocytes\\u003c/h3\\u003e\\n\\u003cp\\u003eControl and PA hiPSC-derived cardiomyocytes were subjected to four experimental conditions to assess the impact of various metabolites: Experiment #1: hiPSC-CMs were cultured in RPMI/B27 medium incubated for four days at 37\\u0026deg;C in a 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere. Experiment #2 (Tracing experiment): hiPSC-CMs were cultured in RPMI/B27 medium with 1 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate (Sigma-Aldrich). Cells were incubated for two days at 37\\u0026deg;C in a 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere. Experiment #3 (Tracing experiment): hiPSC-CMs were cultured in RPMI/B27 medium without glucose and supplemented with 11 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose (Sigma-Aldrich) and incubated for two days at 37\\u0026deg;C in a 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere. Experiment #4 (Tracing experiment): hiPSC-CMs were cultured in RPMI/B27 with 0.4 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate conjugated with BSA (Sigma-Aldrich) medium and incubated for two days at 37\\u0026deg;C in a 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere. After the incubation period, both the medium and the cell pellets were collected and frozen at -80\\u0026deg;C for subsequent analysis.\\u003c/p\\u003e\\n\\u003ch3\\u003eShort-chain fatty acids analysis by LC-MS/MS\\u003c/h3\\u003e\\n\\u003cp\\u003eAn LC-MS/MS method was adapted to analyze short-chain fatty acids including propionate in media [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. A 30-\\u0026micro;l media sample was mixed with 30 \\u0026micro;l internal standard (200 \\u0026micro;M [2,2,2-\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e3\\u003c/sub\\u003e-1,2-\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e2\\u003c/sub\\u003e]aceate (D5 acetate), 20 \\u0026micro;M [2,2,3,3,3-\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e5\\u003c/sub\\u003e]propionate (D5 propionate), 20 \\u0026micro;M [2,2,3,3,4,4,4-\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e7\\u003c/sub\\u003e]butyrate (D7 butyrate), 20 \\u0026micro;M [2,2,3,3,4,4,5,5,5-\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e9\\u003c/sub\\u003e]pentanoate (D9 pentanoate), and 20 \\u0026micro;M [2,2,3,3,4,4,5,5,6,6,-\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e11\\u003c/sub\\u003e]hexanoate (D11 hexanoate)). Acetonitrile (1ml) was added to precipitate protein. The supernatant was transferred to a new Eppendorf vial and completely dried by nitrogen gas after samples were vortexed and centrifugated at 10000 \\u0026times;g for 20 minutes. The dried residue was resuspended in 50 \\u0026micro;l HPLC water and 20 \\u0026micro;l 3-Nitrophenylhydrazine hydrochloride (EDC, 120 mM) and 20 \\u0026micro;l (N-(3-Dimethylaminopropyl)-N\\u0026prime;-ethylcarbodiimide (3-NPH, 200 mM) for derivatization at 40\\u0026deg;C for 30 minutes. The reaction mixture was centrifuged for 10 minutes at 10,000 \\u0026times; g and the supernatant was transferred to an LC-MS/MS vial for analysis. LC-MS/MS was run with a Sciex QTRAP 6500\\u003csup\\u003e+\\u003c/sup\\u003e MS connected with a Sciex AD UHPLC. An Agilent C18 column (Pursuit XRs C18 150 \\u0026times; 2.0 mm, 5 \\u0026micro;m) was employed for separation at room temperature with a flow rate of 0.4 ml/min. A gradient method was conducted with two mobile phases. Mobile phase A was 98% H\\u003csub\\u003e2\\u003c/sub\\u003eO and 2% acetonitrile containing 0.1% formic acid. Mobile phase B was 98% acetonitrile and 2% H\\u003csub\\u003e2\\u003c/sub\\u003eO containing 0.1% formic acid. The gradient started with 2% B for the first 0.5 minutes and increased to 90% at 8 minutes. B was maintained at 90% for 4.5 minutes and returned to its initial condition within 0.5 minutes. Finally, the column was re-equilibrated for 9 minutes with the initial condition before the next injection. The injection volume was 3 \\u0026micro;l. MRM in negative mode was used for short-chain fatty acids assay. The MS/MS parameters were set at the following: curtain gas: 35 psi, source temperature: 600\\u0026deg;C, Gas 1: 55 psi, Gas 2: 55 psi, CAD: 10, Ion spray voltage: -4500 V, EP: -10 V, and CXP: -14.\\u003c/p\\u003e\\n\\u003ch3\\u003eCell pellets metabolic profiling by GC-MS\\u003c/h3\\u003e\\n\\u003cp\\u003eA previously established GC-MS method was adopted to measure the isotope labeling of organic acids and amino acids in the cultured pellets [\\u003cspan additionalcitationids=\\\"CR23\\\" citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. Briefly, approximately 1\\u0026nbsp;million cells were spiked with 2 nmol of norvaline and 0.2 nmol [\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e9\\u003c/sub\\u003e]L-carnitine or mixed stable isotope labeled metabolites as internal standards and then subjected to sonication extraction with 1 ml methanol for 3 minutes. The samples were centrifuged for 20 minutes. The upper phase, approximately 500 \\u0026micro;l in volume, was transferred to a fresh Eppendorf vial and subsequently evaporated using nitrogen gas. The resulting dried residues underwent sequential derivatization with methoxylamine hydrochloride and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (TBDMS). Specifically, 40 \\u0026micro;l of methoxylamine hydrochloride (2% (w/v) in pyridine) was added to the dried residues, followed by incubation for 90 minutes at 40\\u0026deg;C. Subsequently, 20 \\u0026micro;L of TBDMS with 1% tert-butylchlorodimethylsilane was added, and the mixture was incubated for an additional 30 minutes at 80\\u0026deg;C. The derivatized samples were then centrifuged for 10 minutes at 12,000 \\u0026times; g, and the supernatants were transferred to GC vials for further analysis. For GC/MS analysis, we employed an Agilent 7890B GC system with an Agilent 5977A Mass Spectrometer, following the methodology described in our previous work. Specifically, 1 \\u0026micro;l of the derivatized sample was injected into the GC column. The GC temperature gradient began at 80\\u0026deg;C for 2 minutes, increased at a rate of 7\\u0026deg;C per minute to 280\\u0026deg;C, and was maintained at 280\\u0026deg;C until the 40-minute run time was completed. The ionization was conducted via electron impact (EI) at 70 eV, with Helium flow at 1 mL/min. Temperatures of the source, the MS quadrupole, the interface, and the inlet were maintained at 230\\u0026deg;C, 150\\u0026deg;C, 280\\u0026deg;C, and 250\\u0026deg;C, respectively. Mass spectra (m/z) in the range of 50 to 700 were recorded in mass scan mode.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAcylcarnitines profile by LC-MS/MS\\u003c/h2\\u003e \\u003cp\\u003eA 100 \\u0026micro;l medium sample or ~\\u0026thinsp;1\\u0026nbsp;million cell pellets were used for acylcarnitine assay with the spiked internal standard (20 \\u0026micro;l 0.01 mM D9 carnitine). The detailed LC-MS/MS method for the acylcarnitine profile was described in our previous work [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. The pellet sample extracts (500 \\u0026micro;l) from the previous sample preparation were completely dried using nitrogen gas. The medium samples were deproteinized by adding 500 ul methanol and 500 ul acetonitrile. After centrifugation at 12000 g for 15 minutes, the supernatants were completely dried using nitrogen gas. The dried residues were then methylated with a 3 M HCl methanol solution (100 \\u0026micro;l) at 50\\u0026deg;C for 25 minutes. After methylation, the samples were once again dried completely using nitrogen gas and then reconstituted in 20 \\u0026micro;l of methanol and 60 \\u0026micro;l of water. The derivatized samples were subsequently analyzed using an LC-QTRAP 6500\\u003csup\\u003e+\\u003c/sup\\u003e-MS/MS (Sciex, Concord, Ontario). A gradient HPLC method with two mobile phases (mobile phase A was 98% water with 2% acetonitrile and 0.1% formic acid and mobile phase B was 98% acetonitrile with 2% H\\u003csub\\u003e2\\u003c/sub\\u003eO and 0.1% formic acid) was adopted to run with an Agilent Pursuit XRs 5 C18 column (150 \\u0026times; 2.0 mm). The gradient started with 0% B within first 2 minutes and then increased to 80% at 13 minutes. The column was washed out by 90% B for 4 minutes and equilibrated with initial condition (2% B) for 5 minutes before next injection. The flow rate was 0.4 ml/minute and the column oven was set at room temperature. The injection volume was 2 \\u0026micro;l. The parameters for Sciex QTRAP 6500\\u0026thinsp;+\\u0026thinsp;mass spectrometry were optimized as following: DP: 33 V, EP 10 V, CXP: 10 V, source temperature: 680\\u0026deg;C, gas 1: 65, gas 2: 65, curtain gas: 35, CAD: 10, and ion spray voltage: 5500 V. The Q1 of all the methylated acylcarnitines was scanned from m/z 218 to m/z 444 with the same fragment (Q3) at m/z 99. L-carnitine had the ion transition of Q1 (m/z 176) and Q3 (m/z 85 or m/z 117). [\\u003csup\\u003e2\\u003c/sup\\u003eH\\u003csub\\u003e9\\u003c/sub\\u003e]L-carnitine has the shifted Q1 at m/z 179 or m/z 185 with the same Q3 at m/z 85 or m/z 117.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eMedium glucose assay by LC-Q-Exactive-MS\\u003c/h3\\u003e\\n\\u003cp\\u003eGlucose in the medium was assayed according to our previous method. Briefly, a 10 \\u0026micro;l of medium sample was added to a tube prior to folch extraction using the following solvents: 200 \\u0026micro;l methanol, 200 \\u0026micro;l distilled H\\u003csub\\u003e2\\u003c/sub\\u003eO, and 200 \\u0026micro;l chloroform. The sample mixture was vortexed and centrifuged for 20 minutes at 10,000 \\u0026times; g at 4\\u0026deg;C. The upper phase (~\\u0026thinsp;350 \\u0026micro;l) was dried completely by nitrogen gas at 37\\u0026deg;C. The dried residue was resuspended in 60 \\u0026micro;l distilled water, vortexed, and placed in an autosampler vial for LC-MS analysis.\\u003c/p\\u003e \\u003cp\\u003eLC-Q-Exactive\\u003csup\\u003e+\\u003c/sup\\u003e-Orbitrap-MS was used for the final quantitation in this work. The Vanquish Binary Pump was used to deliver the mobile phase (98% H\\u003csub\\u003e2\\u003c/sub\\u003eO and 2% methanol containing 0.01% formic acid) at a flow rate of 0.3 ml/min in isocratic elution mode. The column was a Microsorb-MV C18 column (100 \\u0026times; 4.6 mm, 3 \\u0026micro;m) with a C18 guard column and was kept at 40\\u0026deg;C in the column oven compartment. The autosampler was maintained at 5\\u0026deg;C, and the injection volume was 1 \\u0026micro;l. The total running time is 10 minutes. The parameters for Q-Exactive\\u003csup\\u003e+\\u003c/sup\\u003e-MS equipped with a HESI probe: heat temperature: 425\\u0026deg;C; sheath gas: 30, auxiliary gas, 13; sweep gas, 3; spray voltage, 3.5 kV for positive mode; the capillary temperature was set at 320\\u0026deg;C, and S-lens was 45. A full scan range was set at 60 to 900 (m/z). The resolution was set at 70,000 (at m/z 200). The maximum injection time (max IT) was 200 ms. Automated gain control (AGC) was targeted at 3 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e ions.\\u003c/p\\u003e\\n\\u003ch3\\u003eStatistics\\u003c/h3\\u003e\\n\\u003cp\\u003eAll cell experiments were conducted using two differentiations with a total of n\\u0026thinsp;=\\u0026thinsp;4 biological replicates. Measured mass isotopologues distributions expressed as mol percent were corrected for natural enrichment [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. M0, M1, M2, ..., Mn denote the isotopologues of molecules containing n heavy atoms. Statistical differences were analyzed using Prism software. A Student's t-test was used for comparisons between two groups, while a two-way ANOVA followed by a post hoc Tukey test was conducted for comparisons among more than two groups.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eMetabolic profile of human induced pluripotent stem cell-derived cardiomyocytes from a control Individual and a PCCA-deficient patient\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eMetabolic profiling was performed on control and PCCA-deficient hiPSC-CMs using both cell pellets and cultured media samples. PCC deficiency significantly increased propionylcarnitine (C3 AC) in the cultured media (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA) and led to the accumulation of cellular methylcitrate (Supplemental Fig.\\u0026nbsp;1A). Interestingly, medium-chain acylcarnitines were found to be lower in the cultured media from the PCCA-deficient group (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB-\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). This decrease in acylcarnitines may indicate reduced fatty acid oxidation or a redistribution of acylcarnitines toward increased propionylcarnitine synthesis. In line with this, there was a trend toward a reduction in cellular L-carnitine (p\\u0026thinsp;=\\u0026thinsp;0.076, Supplemental Fig.\\u0026nbsp;1B). The accumulation of propionylcarnitine, along with the reduction of free carnitine, could alter fuel metabolism in the cardiomyocytes, which depend on proper fuel metabolism for mechanical contraction.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePCC deficiency disrupts propionate and propionyl-CoA metabolism in hiPSC-CMs\\u003c/h2\\u003e \\u003cp\\u003eOne of the reliable biomarkers of PA is elevated circulating propionate, which can reach millimolar levels in PA patients [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. However, this biomarker is not often reported due to challenges with analytical methodology. Our previous work suggests that impaired hepatic disposal of propionate leads to increased circulating levels, which may elevate the risk of cardiac disease [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. In the present study, we aimed to investigate the impact of PCC deficiency on propionate metabolism in cardiomyocytes derived from both control and PCCA-deficient hiPSC. After culturing the cells for 48 hours with 1 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate, both pre-culture and post-culture media were collected for propionate quantification (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). PCCA-deficient hiPSC-CMs released 9.6 times more unlabeled propionate compared to control hiPSC-CMs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The released unlabeled propionate is likely generated endogenously, probably from hydrolysis of propionyl-CoA, which is elevated in PA patient-derived hiPSC-CMs. After 48 hours of culturing, [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate was consumed by 25% in the control hiPSC-CMs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). In contrast, [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]propionate levels in the medium remained almost unchanged in PA hiPSC-CMs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). This strongly suggests that the PCC deficiency not only affects propionyl-CoA metabolism but also disrupts propionate metabolism, consistent with our previous findings in the livers of \\u003cem\\u003ePcca\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e(A138T) mice [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e[\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e3\\u003c/sub\\u003e]Propionate, used as a tracer, allows for tracking both propionate and propionyl-CoA metabolism [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. TCA cycle intermediates are downstream metabolites of propionyl-CoA, and stable isotope labeling of these intermediates reflects propionyl-CoA metabolism (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). The stable isotopomer labeling and average carbon labeling of malate and citrate are shown in Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB-\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE. The low labeling of malate and citrate in PA hiPSC-CMs confirms that the PCCA mutation impairs PCC activity and demonstrates that this disrupts propionyl-CoA anaplerosis into the TCA cycle. The dramatic reduction in labeling of TCA cycle intermediates is further corroborated by other metabolite labeling data (Supplemental Fig.\\u0026nbsp;2).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePCC deficiency enhances glucose metabolism in hiPSC-CMs\\u003c/h2\\u003e \\u003cp\\u003eApproximately 25% or more of patients with PA develop cardiac diseases, although the pathological mechanisms remain largely unknown [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Fuel metabolism is crucial for cardiac function, as it sustains energy production required for mechanical contraction. To better understand the metabolic alterations in PA, we investigated the major fuel metabolism pathways in PA hiPSC-CMs. We replaced unlabeled glucose (11 mM) with 11 mM [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose in the medium to trace glucose metabolism. First, we measured the consumption of [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose by quantifying the levels of [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose in both pre- and post-culture media using LC-Q Exactive\\u003csup\\u003e+\\u003c/sup\\u003e-MS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). After 48 hours of incubation, [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose levels were much lower in the medium of PA hiPSC-CMs compared to control hiPSC-CMs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB), indicating that PA hiPSC-CMs metabolize [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose at a higher rate than control cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eUsing \\u003csup\\u003e13\\u003c/sup\\u003eC labeled tracer, we traced the downstream metabolites of glucose to assess glucose metabolic rate through stable isotopomer analysis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). The stable isotope labeling of glycolytic intermediates\\u0026mdash;such as 3-phosphoglycerate (3PG), phosphoenolpyruvate (PEP), pyruvate, and lactate\\u0026mdash;was measured (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB-\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). The stable isotope labeling of these intermediates was significantly higher in PA hiPSC-CMs, reaching 80%, compared to 60% in control hiPSC-CMs. This confirms that glucose metabolism is elevated in PA-derived cardiomyocytes.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFurther downstream in the glucose metabolic pathway, TCA cycle intermediates were labeled by [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). Figures\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB-\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE show the stable isotopomer labeling and average carbon labeling of malate and citrate in control and PA hiPSC-CMs. Consistent with the increased glucose metabolism in PA hiPSC-CMs, the stable isotope labeling of malate was significantly higher in PA than in control cells. Additionally, the shift to a higher isotopomer of citrate in PA hiPSC-CMs demonstrates increased metabolic flux of glucose into the TCA cycle (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD). The lower M2 citrate in PA hiPSC-CMs also supports the finding of reduced unlabeled oxaloacetate/malate (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF), as M2 citrate is primarily derived from M0 oxaloacetate and M2 acetyl-CoA (M2 citrate\\u0026thinsp;=\\u0026thinsp;M0 oxaloacetate \\u0026times; M2 oxaloacetate). This pattern was also observed in other detected TCA cycle intermediates (Supplemental Fig.\\u0026nbsp;3).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eInterestingly, M3 malate was the second most abundant isotopomer after M2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). The minor amount of M3 malate could be generated from multiple turns of the TCA cycle after M2 acetyl-CoA enters the cycle (Supplement Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). M2 malate and M3 malate are primarily derived from M2 acetyl-CoA via pyruvate dehydrogenase and M3 pyruvate via pyruvate carboxylase, respectively. The increased glucose metabolism in PA hiPSC-CMs is likely driven by enhanced metabolic flux through both pyruvate dehydrogenase and pyruvate carboxylase.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIncreased secretion of labeled acetate and unlabeled propionate in PA hiPSC-CMs\\u003c/h2\\u003e \\u003cp\\u003eAcyl-CoAs are intracellular metabolites that cannot cross the plasma membrane [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. To prevent the accumulation of acyl-CoAs, which can be toxic due to their detergent-like effect, they are converted into their counterparts, acylcarnitines. This metabolic conversion serves as a protective mechanism. In the context of PA, carnitine supplementation can facilitate the conversion of accumulated propionyl-CoA to propionylcarnitine, helping to remove excess propionyl-CoA from cells. However, it remains unclear whether acyl-CoAs could also be hydrolyzed into free fatty acids, offering an additional means for the intracellular metabolites to escape cells and enter the bloodstream (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA). In this study, we measured short-chain fatty acids in the cultured media and detected M2 acetate, derived from labeled M2 acetyl-CoA, in the media from [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB). This confirmed the activity of acyl-CoA hydrolase, which plays a secondary role in regulating cellular acyl-CoA levels. The increased secretion of labeled acetate (M2 acetate) from [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose in PA hiPSC-CMs also suggests enhanced glucose metabolism in these cells. As anticipated, accumulated propionyl-CoA led to the increased release of propionate into the medium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eC) via acyl-CoA hydrolase, consistent with results from the [\\u0026sup1;\\u0026sup3;C₃]propionate experiment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). In contrast, the release of butyrate and hexanoate decreased, consistent with the reduction of medium-chain acylcarnitines (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eD, \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eE, \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB-\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE). This decrease in butyrate, hexanoate, and their corresponding acylcarnitines further supports the shift from fatty acid metabolism to glucose metabolism in PA hiPSC-CMs.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eReduced fatty acid oxidation in PA hiPSC-CMs\\u003c/h2\\u003e \\u003cp\\u003eThe increased lipid droplets have been observed in the heart from patients with PA [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. A recent study using stable isotope techniques also demonstrated altered lipid metabolism in PA patients [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. Given the increased glucose metabolism, we sought to investigate fatty acid metabolism in PA hiPSC-CMs using [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate. After 48 hours of culturing, we measured the labeling of metabolites derived from [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA). In the cultured medium samples, acylcarnitine intermediates from M16 palmitate were significantly lower in PA hiPSC-CMs (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eB-\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eE and Supplemental Figs.\\u0026nbsp;5A-5C). Notably, M2 acetate labeling was also significantly lower in the cultured media of PA hiPSC-CMs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eF). As observed in the [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose tracing experiment, M2 acetate is derived from the hydrolysis of M2 acetyl-CoA, which is produced during the metabolism of both [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose and [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate. However, in contrast to the [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e]glucose experiment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB), M2 acetate labeling in PA hiPSC-CMs was significantly lower than in control cells when [\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e16\\u003c/sub\\u003e]palmitate was used as a tracer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eF). Together, these findings suggest that PCC deficiency induces a fuel switch from fatty acid metabolism to increased glucose utilization.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFree carnitine plays a crucial role in transporting long-chain fatty acids into the mitochondria for complete β-oxidation. We observed a reduction, or a trend toward reduced, cellular L-carnitine in all experiments (Supplemental. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB and Supplemental Fig.\\u0026nbsp;6). The L-carnitine deficiency in PA hiPSC-CMs impedes fatty acid oxidation, which in turn promotes increased glucose metabolism.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003ePA is a rare metabolic disorder caused by mutations in \\u003cem\\u003ePCCA\\u003c/em\\u003e or \\u003cem\\u003ePCCB\\u003c/em\\u003e genes, resulting in impaired propionyl-CoA metabolism. PA is typically diagnosed early in life, often within days of birth. If left unmanaged, PA can lead to severe complications, including cardiac dysfunction. The underlying mechanisms driving these cardiac complications are not yet fully understood. Chronic metabolic alterations associated with PA are believed to contribute to these complications.\\u003c/p\\u003e \\u003cp\\u003eEnergy metabolism is crucial for the heart to perform mechanical work. Fatty acids are the heart's preferred and most efficient fuel for ATP production. In failing hearts, however, fuel metabolism switches from fatty acids to increased glucose and ketone metabolism [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. This shift may be particularly pronounced in PA patients due to several factors: (1) the depletion of L-carnitine and free CoA, caused by the accumulation of propionylcarnitine and propionyl-CoA, which impairs fatty acid oxidation, (2) the accumulation of lipid droplets in the heart, indicating disrupted fatty acid metabolism [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e], and (3) the inhibition of fatty acid oxidation during exercise in PA patients [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. In this study, we observed that PA hiPSC-CMs exhibit a significant increase in glucose utilization and a decrease in fatty acid oxidation using stable isotope-based metabolic flux analysis. This chronic metabolic switch could impair cardiac energy metabolism and lead to progressive cardiac dysfunction, as the heart preferentially utilizes fatty acids for energy. Interestingly, while resting fatty acid oxidation in PA patients may not differ significantly from healthy individuals, PA patients are unable to fully utilize fatty acids during exercise when the whole-body switches to fatty acid oxidation [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe accumulation of propionylcarnitine in PA leads to decreased intracellular L-carnitine levels, particularly in tissues like the heart. One PA patient with fatal cardiomyopathy had low carnitine levels in the heart despite supplementation [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Reduced intracellular L-carnitine (Supplemental Fig.\\u0026nbsp;1B and Supplemental Fig.\\u0026nbsp;6) limits the transport of long-chain fatty acids into mitochondria for complete β-oxidation. Chronic reductions in fatty acid oxidation could trigger cardiac disease, as seen in patients with long-chain fatty acid β-oxidation disorders (LCFAOD), who frequently develop cardiomyopathy [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePropionate in plasma and urine is another reliable biomarker for PA. Short-chain fatty acids (SCFAs), typically produced by the microbiome, are usually at low levels in the portal vein of germ-free mice [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. The liver plays a key role in metabolizing and disposing of propionate, maintaining low levels (\\u0026thinsp;~\\u0026thinsp;\\u0026lt;\\u0026thinsp;1 \\u0026micro;M) [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. However, it remains unclear whether SCFAs could be produced endogenously, given two key observations: (1) acetate, the most abundant SCFA in the blood, correlates well with acetyl-CoA levels, which are among the highest of all acyl-CoAs, and (2) propionate arises in PA due to propionyl-CoA accumulation. This raises the question of whether acyl-CoA hydrolysis might be another source of SCFAs.\\u003c/p\\u003e \\u003cp\\u003eAcyl-CoA hydrolysis, a process that releases free fatty acids that can be transported out of cells, is a potential regulatory mechanism that controls intracellular acyl-CoA levels and prevents their accumulation. Although the role of acyl-CoA hydrolases is understudied, they could play an essential role in regulating CoA for other metabolic events in the cell [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. In peroxisomes, acyl-CoAs are hydrolyzed into free fatty acids, which are then transported to mitochondria for complete β-oxidation [\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e]. We confirmed the acyl-CoA hydrolysis activity using stable isotope tracing in this study. Labeled acetate and unlabeled propionate, hydrolyzed from labeled acetyl-CoA and unlabeled propionyl-CoA, occur in mitochondria, where pyruvate oxidation to acetyl-CoA and propionyl-CoA metabolism predominantly take place. Free acetate from pyruvate metabolism could also be directly catalyzed by pyruvate dehydrogenase or 2-ketoglutarate dehydrogenase according to Liu et al.\\u0026rsquo;s work [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. However, the release of propionate, butyrate and hexanoate confirms the acyl-CoA hydrolase activity.\\u003c/p\\u003e \\u003cp\\u003eThe physiological implications of propionyl-CoA hydrolysis into propionate in PA warrant further investigation. This hydrolysis could represent a second regulatory mechanism to control intracellular propionyl-CoA levels, serving as a \\\"pressure valve\\\" to mitigate the toxicity of accumulated propionyl-CoA in mitochondria.\\u003c/p\\u003e \\u003cp\\u003eThis study is the first to utilize cardiomyocytes derived from patients with PA for metabolic discovery, emphasizing its strong clinical relevance. However, the authors acknowledge the following limitations: This is an \\u003cem\\u003ein vitro\\u003c/em\\u003e experiment, which does not fully capture the complexity of metabolic regulation present \\u003cem\\u003ein vivo\\u003c/em\\u003e. The \\u003cem\\u003ein vitro\\u003c/em\\u003e conditions may not accurately reflect the physiological environment \\u003cem\\u003ein vivo\\u003c/em\\u003e. These limitations highlight the need for future \\u003cem\\u003ein vivo\\u003c/em\\u003e studies, which face the following challenges: (1) Metabolic data from the heart \\u003cem\\u003ein vivo\\u003c/em\\u003e is often confounded by secondary metabolites originating from other organs. (2) Experimental findings using heart tissue from animal models may not directly translate to human patients.\\u003c/p\\u003e \\u003cp\\u003eIn summary, using stable isotope tracing and hiPSC-CMs derived from PA patients, we provide the direct evidence that PA induces a fuel switch from fatty acids to glucose in cardiomyocytes derived from patients with PA. This shift may underlie the cardiac complications observed in PA. Additionally, we identify a second metabolic regulatory mechanism\\u0026mdash;acyl-CoA hydrolysis\\u0026mdash;that helps control propionyl-CoA levels in PA. This pathway may offer a potential therapeutic target for treating PA.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMaterial preparation, data collection and analysis were performed by Eva Richard, Hannah Marchuk, Mar \\u0026Aacute;lvarez and Wentao He. Eva Richard, Xiaoxin Chen, Lourdes R. Desvia and Guof-Fang Zhang contributed to the study conception and design. The first draft of the manuscript was written by Guo-Fang Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by Propionic Acidemia Foundation awards: to G.F.Z. and PAF113 to E. R., NIH R01 AA030026 and NIH R21TR005163 to X.C and G.F.Z,\\u0026nbsp;the Fundaci\\u0026oacute;n Ram\\u0026oacute;n Areces (XX National Call 2020) to E.R. and the MICIU/AEI/10.13039/501100011033/ and ERDF A way of making Europe (PID2022-137238OB-100) to L.R.D. and E.R. Centro de Biolog\\u0026iacute;a Molecular Severo Ochoa receives an institutional grant from Fundaci\\u0026oacute;n Ram\\u0026oacute;n Areces and is a Severo Ochoa Center of Excellence (MICIN, Award CEX2021-001154-S).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and material\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eData will be made available from authors on request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest.\\u0026nbsp;\\u003c/strong\\u003eThe authors declare that there are no conflicts of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Autonomous University of Madrid (project identification code CEI-134-2830; date of approval: 3 November 2023); and by the authorization of \\u0026ldquo;Direcci\\u0026oacute;n General de Investigaci\\u0026oacute;n Sanitaria y Documentaci\\u0026oacute;n\\u0026rdquo;, Community of Madrid (data of approval: 19 February 2024). Written informed consent for research has been obtained from the patient\\u0026rsquo;s family.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e. All authors read and approved the final version of this manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAgius L, Wright PD, Alberti KG (1987) Carnitine acyltransferases and acyl-CoA hydrolases in human and rat liver. Clin Sci (Lond) 73:3-10 doi:10.1042/cs0730003\\u003c/li\\u003e\\n\\u003cli\\u003eAl-Hamed MH, Imtiaz F, Al-Hassnan Z, Al-Owain M, Al-Zaidan H, Alamoudi MS, Faqeih E, Alfadhel M, Al-Asmari A, Saleh MM, Almutairi F, Moghrabi N, AlSayed M (2019) Spectrum of mutations underlying Propionic acidemia and further insight into a genotype-phenotype correlation for the common mutation in Saudi Arabia. Mol Genet Metab Rep 18:22-29 doi:10.1016/j.ymgmr.2018.12.004\\u003c/li\\u003e\\n\\u003cli\\u003eAlexson SE, Nedergaard J (1988) A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria. J Biol Chem 263:13564-13571\\u003c/li\\u003e\\n\\u003cli\\u003eAlonso-Barroso E, Perez B, Desviat LR, Richard E (2021) Cardiomyocytes Derived from Induced Pluripotent Stem Cells as a Disease Model for Propionic Acidemia. Int J Mol Sci 22 doi:10.3390/ijms22031161\\u003c/li\\u003e\\n\\u003cli\\u003eAlvarez M, Ruiz-Sala P, Perez B, Desviat LR, Richard E (2023) Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy. Int J Mol Sci 24 doi:10.3390/ijms24032182\\u003c/li\\u003e\\n\\u003cli\\u003eAmaral AU, Cecatto C, Castilho RF, Wajner M (2016) 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria. J Neurochem 137:62-75 doi:10.1111/jnc.13544\\u003c/li\\u003e\\n\\u003cli\\u003eBodi I, Grunert SC, Becker N, Stoelzle-Feix S, Spiekerkoetter U, Zehender M, Bugger H, Bode C, Odening KE (2016) Mechanisms of acquired long QT syndrome in patients with propionic academia. Heart Rhythm 13:1335-1345 doi:10.1016/j.hrthm.2016.02.003\\u003c/li\\u003e\\n\\u003cli\\u003eBrunengraber H, Roe CR (2006) Anaplerotic molecules: current and future. J Inherit Metab Dis 29:327-331 doi:10.1007/s10545-006-0320-1\\u003c/li\\u003e\\n\\u003cli\\u003eChalmers RA, Roe CR, Stacey TE, Hoppel CL (1984) Urinary excretion of l-carnitine and acylcarnitines by patients with disorders of organic acid metabolism: evidence for secondary insufficiency of l-carnitine. Pediatr Res 18:1325-1328 doi:10.1203/00006450-198412000-00021\\u003c/li\\u003e\\n\\u003cli\\u003eChandler RJ, Chandrasekaran S, Carrillo-Carrasco N, Senac JS, Hofherr SE, Barry MA, Venditti CP (2011) Adeno-associated virus serotype 8 gene transfer rescues a neonatal lethal murine model of propionic acidemia. Hum Gene Ther 22:477-481 doi:10.1089/hum.2010.164\\u003c/li\\u003e\\n\\u003cli\\u003eChapman KA, Ostrovsky J, Rao M, Dingley SD, Polyak E, Yudkoff M, Xiao R, Bennett MJ, Falk MJ (2018) Propionyl-CoA carboxylase pcca-1 and pccb-1 gene deletions in Caenorhabditis elegans globally impair mitochondrial energy metabolism. J Inherit Metab Dis 41:157-168 doi:10.1007/s10545-017-0111-x\\u003c/li\\u003e\\n\\u003cli\\u003eCheema-Dhadli S, Leznoff CC, Halperin ML (1975) Effect of 2-methylcitrate on citrate metabolism: implications for the management of patients with propionic acidemia and methylmalonic aciduria. Pediatr Res 9:905-908 doi:10.1203/00006450-197512000-00008\\u003c/li\\u003e\\n\\u003cli\\u003eDavila-Roman VG, Vedala G, Herrero P, de las Fuentes L, Rogers JG, Kelly DP, Gropler RJ (2002) Altered myocardial fatty acid and glucose metabolism in idiopathic dilated cardiomyopathy. J Am Coll Cardiol 40:271-277 doi:10.1016/s0735-1097(02)01967-8\\u003c/li\\u003e\\n\\u003cli\\u003eDi Donato S, Rimoldi M, Garavaglia B, Uziel G (1984) Propionylcarnitine excretion in propionic and methylmalonic acidurias: a cause of carnitine deficiency. Clin Chim Acta 139:13-21 doi:10.1016/0009-8981(84)90187-6\\u003c/li\\u003e\\n\\u003cli\\u003eDuran M, Ketting D, Beckeringh TE, Leupold D, Wadman SK (1986) Direct identification of propionylcarnitine in propionic acidaemia: biochemical and clinical results of oral carnitine supplementation. J Inherit Metab Dis 9:202-207 doi:10.1007/BF01799460\\u003c/li\\u003e\\n\\u003cli\\u003eFernandez CA, Des Rosiers C, Previs SF, David F, Brunengraber H (1996) Correction of 13C mass isotopomer distributions for natural stable isotope abundance. J Mass Spectrom 31:255-262 doi:10.1002/(SICI)1096-9888(199603)31:3\\u0026lt;255::AID-JMS290\\u0026gt;3.0.CO;2-3\\u003c/li\\u003e\\n\\u003cli\\u003eFulgencio-Covian A, Alonso-Barroso E, Guenzel AJ, Rivera-Barahona A, Ugarte M, Perez B, Barry MA, Perez-Cerda C, Richard E, Desviat LR (2020) Pathogenic implications of dysregulated miRNAs in propionic acidemia related cardiomyopathy. Transl Res 218:43-56 doi:10.1016/j.trsl.2019.12.004\\u003c/li\\u003e\\n\\u003cli\\u003eGalarreta Aima CI, Shchelochkov OA, Jerves Serrano T, Venditti CP (1993) Propionic Acidemia. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A (eds) GeneReviews((R)). Seattle (WA)\\u003c/li\\u003e\\n\\u003cli\\u003eGallego-Villar L, Perez-Cerda C, Perez B, Abia D, Ugarte M, Richard E, Desviat LR (2013) Functional characterization of novel genotypes and cellular oxidative stress studies in propionic acidemia. J Inherit Metab Dis 36:731-740 doi:10.1007/s10545-012-9545-3\\u003c/li\\u003e\\n\\u003cli\\u003eGallego-Villar L, Rivera-Barahona A, Cuevas-Martin C, Guenzel A, Perez B, Barry MA, Murphy MP, Logan A, Gonzalez-Quintana A, Martin MA, Medina S, Gil-Izquierdo A, Cuezva JM, Richard E, Desviat LR (2016) In vivo evidence of mitochondrial dysfunction and altered redox homeostasis in a genetic mouse model of propionic acidemia: Implications for the pathophysiology of this disorder. Free Radic Biol Med 96:1-12 doi:10.1016/j.freeradbiomed.2016.04.007\\u003c/li\\u003e\\n\\u003cli\\u003eGuenzel AJ, Hofherr SE, Hillestad M, Barry M, Weaver E, Venezia S, Kraus JP, Matern D, Barry MA (2013) Generation of a hypomorphic model of propionic acidemia amenable to gene therapy testing. Mol Ther 21:1316-1323 doi:10.1038/mt.2013.68\\u003c/li\\u003e\\n\\u003cli\\u003eHe W, Berthiaume JM, Previs S, Kasumov T, Zhang GF (2023) Ischemia promotes acyl-CoAs dephosphorylation and propionyl-CoA accumulation. Metabolomics 19:12 doi:10.1007/s11306-023-01975-2\\u003c/li\\u003e\\n\\u003cli\\u003eHe W, Marchuk H, Koeberl D, Kasumov T, Chen X, Zhang GF (2024) Fasting alleviates metabolic alterations in mice with propionyl-CoA carboxylase deficiency due to Pcca mutation. Commun Biol 7:659 doi:10.1038/s42003-024-06362-8\\u003c/li\\u003e\\n\\u003cli\\u003eHe W, Wang Y, Xie EJ, Barry MA, Zhang GF (2021) Metabolic perturbations mediated by propionyl-CoA accumulation in organs of mouse model of propionic acidemia. Mol Genet Metab 134:257-266 doi:10.1016/j.ymgme.2021.09.009\\u003c/li\\u003e\\n\\u003cli\\u003eHofherr SE, Senac JS, Chen CY, Palmer DJ, Ng P, Barry MA (2009) Short-term rescue of neonatal lethality in a mouse model of propionic acidemia by gene therapy. Hum Gene Ther 20:169-180 doi:10.1089/hum.2008.158\\u003c/li\\u003e\\n\\u003cli\\u003eHommes FA, Kuipers JR, Elema JD, Jansen JF, Jonxis JH (1968) Propionicacidemia, a new inborn error of metabolism. Pediatr Res 2:519-524 doi:10.1203/00006450-196811000-00010\\u003c/li\\u003e\\n\\u003cli\\u003eKasumov T, Cendrowski AV, David F, Jobbins KA, Anderson VE, Brunengraber H (2007) Mass isotopomer study of anaplerosis from propionate in the perfused rat heart. Arch Biochem Biophys 463:110-117 doi:10.1016/j.abb.2007.02.022\\u003c/li\\u003e\\n\\u003cli\\u003eKinman RP, Kasumov T, Jobbins KA, Thomas KR, Adams JE, Brunengraber LN, Kutz G, Brewer WU, Roe CR, Brunengraber H (2006) Parenteral and enteral metabolism of anaplerotic triheptanoin in normal rats. Am J Physiol Endocrinol Metab 291:E860-866 doi:10.1152/ajpendo.00366.2005\\u003c/li\\u003e\\n\\u003cli\\u003eKnottnerus SJG, Bleeker JC, Ferdinandusse S, Houtkooper RH, Langeveld M, Nederveen AJ, Strijkers GJ, Visser G, Wanders RJA, Wijburg FA, Boekholdt SM, Bakermans AJ (2020) Subclinical effects of long-chain fatty acid beta-oxidation deficiency on the adult heart: A case-control magnetic resonance study. J Inherit Metab Dis 43:969-980 doi:10.1002/jimd.12266\\u003c/li\\u003e\\n\\u003cli\\u003eKolker S, Garcia-Cazorla A, Valayannopoulos V, Lund AM, Burlina AB, Sykut-Cegielska J, Wijburg FA, Teles EL, Zeman J, Dionisi-Vici C, Baric I, Karall D, Augoustides-Savvopoulou P, Aksglaede L, Arnoux JB, Avram P, Baumgartner MR, Blasco-Alonso J, Chabrol B, Chakrapani A, Chapman K, EC IS, Couce ML, de Meirleir L, Dobbelaere D, Dvorakova V, Furlan F, Gleich F, Gradowska W, Grunewald S, Jalan A, Haberle J, Haege G, Lachmann R, Laemmle A, Langereis E, de Lonlay P, Martinelli D, Matsumoto S, Muhlhausen C, de Baulny HO, Ortez C, Pena-Quintana L, Ramadza DP, Rodrigues E, Scholl-Burgi S, Sokal E, Staufner C, Summar ML, Thompson N, Vara R, Pinera IV, Walter JH, Williams M, Burgard P (2015) The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 1: the initial presentation. J Inherit Metab Dis 38:1041-1057 doi:10.1007/s10545-015-9839-3\\u003c/li\\u003e\\n\\u003cli\\u003eKolker S, Valayannopoulos V, Burlina AB, Sykut-Cegielska J, Wijburg FA, Teles EL, Zeman J, Dionisi-Vici C, Baric I, Karall D, Arnoux JB, Avram P, Baumgartner MR, Blasco-Alonso J, Boy SP, Rasmussen MB, Burgard P, Chabrol B, Chakrapani A, Chapman K, Cortes ISE, Couce ML, de Meirleir L, Dobbelaere D, Furlan F, Gleich F, Gonzalez MJ, Gradowska W, Grunewald S, Honzik T, Horster F, Ioannou H, Jalan A, Haberle J, Haege G, Langereis E, de Lonlay P, Martinelli D, Matsumoto S, Muhlhausen C, Murphy E, de Baulny HO, Ortez C, Pedron CC, Pintos-Morell G, Pena-Quintana L, Ramadza DP, Rodrigues E, Scholl-Burgi S, Sokal E, Summar ML, Thompson N, Vara R, Pinera IV, Walter JH, Williams M, Lund AM, Garcia-Cazorla A (2015) The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 2: the evolving clinical phenotype. J Inherit Metab Dis 38:1059-1074 doi:10.1007/s10545-015-9840-x\\u003c/li\\u003e\\n\\u003cli\\u003eKor D, Seker-Yilmaz B, Bulut FD, Kilavuz S, Oktem M, Ceylaner S, Yildizdas D, Onenli-Mungan N (2019) Clinical features of 27 Turkish Propionic acidemia patients with 12 novel mutations. Turk J Pediatr 61:330-336 doi:10.24953/turkjped.2019.03.003\\u003c/li\\u003e\\n\\u003cli\\u003eKott-Blumenkranz R, Pappas CT, Bensch KG (1981) A study of the ultrastructure of the organs and of cultured fibroblasts incubated with isoleucine from a patient with propionic acidemia. Hum Pathol 12:1141-1148 doi:10.1016/s0046-8177(81)80336-x\\u003c/li\\u003e\\n\\u003cli\\u003eKovacevic A, Garbade SF, Horster F, Hoffmann GF, Gorenflo M, Mereles D, Kolker S, Staufner C (2022) Detection of early cardiac disease manifestation in propionic acidemia - Results of a monocentric cross-sectional study. Mol Genet Metab 137:349-358 doi:10.1016/j.ymgme.2022.10.007\\u003c/li\\u003e\\n\\u003cli\\u003eLiu X, Cooper DE, Cluntun AA, Warmoes MO, Zhao S, Reid MA, Liu J, Lund PJ, Lopes M, Garcia BA, Wellen KE, Kirsch DG, Locasale JW (2018) Acetate Production from Glucose and Coupling to Mitochondrial Metabolism in Mammals. Cell 175:502-513 e513 doi:10.1016/j.cell.2018.08.040\\u003c/li\\u003e\\n\\u003cli\\u003eMaines E, Moretti M, Vitturi N, Gugelmo G, Fasan I, Lenzini L, Piccoli G, Gragnaniello V, Maiorana A, Soffiati M, Burlina A, Franceschi R (2023) Understanding the Pathogenesis of Cardiac Complications in Patients with Propionic Acidemia and Exploring Therapeutic Alternatives for Those Who Are Not Eligible or Are Waiting for Liver Transplantation. Metabolites 13 doi:10.3390/metabo13040563\\u003c/li\\u003e\\n\\u003cli\\u003eMarchuk H, Wang Y, Ladd ZA, Chen X, Zhang GF (2023) Pathophysiological mechanisms of complications associated with propionic acidemia. Pharmacol Ther 249:108501 doi:10.1016/j.pharmthera.2023.108501\\u003c/li\\u003e\\n\\u003cli\\u003eMardach R, Verity MA, Cederbaum SD (2005) Clinical, pathological, and biochemical studies in a patient with propionic acidemia and fatal cardiomyopathy. Mol Genet Metab 85:286-290 doi:10.1016/j.ymgme.2005.04.004\\u003c/li\\u003e\\n\\u003cli\\u003eMartini WZ, Stanley WC, Huang H, Rosiers CD, Hoppel CL, Brunengraber H (2003) Quantitative assessment of anaplerosis from propionate in pig heart in vivo. Am J Physiol Endocrinol Metab 284:E351-356 doi:10.1152/ajpendo.00354.2002\\u003c/li\\u003e\\n\\u003cli\\u003eMiyazaki T, Ohura T, Kobayashi M, Shigematsu Y, Yamaguchi S, Suzuki Y, Hata I, Aoki Y, Yang X, Minjares C, Haruta I, Uto H, Ito Y, Muller U (2001) Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene. J Biol Chem 276:35995-35999 doi:10.1074/jbc.M105467200\\u003c/li\\u003e\\n\\u003cli\\u003eReszko AE, Kasumov T, Pierce BA, David F, Hoppel CL, Stanley WC, Des Rosiers C, Brunengraber H (2003) Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver. J Biol Chem 278:34959-34965 doi:10.1074/jbc.M302013200\\u003c/li\\u003e\\n\\u003cli\\u003eRibas GS, Manfredini V, de Marco MG, Vieira RB, Wayhs CY, Vanzin CS, Biancini GB, Wajner M, Vargas CR (2010) Prevention by L-carnitine of DNA damage induced by propionic and L-methylmalonic acids in human peripheral leukocytes in vitro. Mutat Res 702:123-128 doi:10.1016/j.mrgentox.2010.07.008\\u003c/li\\u003e\\n\\u003cli\\u003eRibas GS, Manfredini V, de Mari JF, Wayhs CY, Vanzin CS, Biancini GB, Sitta A, Deon M, Wajner M, Vargas CR (2010) Reduction of lipid and protein damage in patients with disorders of propionate metabolism under treatment: a possible protective role of L-carnitine supplementation. Int J Dev Neurosci 28:127-132 doi:10.1016/j.ijdevneu.2010.01.002\\u003c/li\\u003e\\n\\u003cli\\u003eRivera-Barahona A, Fulgencio-Covian A, Perez-Cerda C, Ramos R, Barry MA, Ugarte M, Perez B, Richard E, Desviat LR (2017) Dysregulated miRNAs and their pathogenic implications for the neurometabolic disease propionic acidemia. Sci Rep 7:5727 doi:10.1038/s41598-017-06420-8\\u003c/li\\u003e\\n\\u003cli\\u003eRoginski AC, Cecatto C, Wajner SM, Camera FD, Castilho RF, Wajner M, Amaral AU (2019) Experimental evidence that maleic acid markedly compromises glutamate oxidation through inhibition of glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase activities in kidney of developing rats. Mol Cell Biochem 458:99-112 doi:10.1007/s11010-019-03534-7\\u003c/li\\u003e\\n\\u003cli\\u003eRoginski AC, Zemniacak AB, Marschner RA, Wajner SM, Ribeiro RT, Wajner M, Amaral AU (2022) Disruption of mitochondrial functions involving mitochondrial permeability transition pore opening caused by maleic acid in rat kidney. J Bioenerg Biomembr 54:203-213 doi:10.1007/s10863-022-09945-4\\u003c/li\\u003e\\n\\u003cli\\u003eSalmi H, Leonard JV, Lapatto R (2012) Patients with organic acidaemias have an altered thiol status. Acta Paediatr 101:e505-508 doi:10.1111/j.1651-2227.2012.02799.x\\u003c/li\\u003e\\n\\u003cli\\u003eStorgaard JH, Madsen KL, Lokken N, Vissing J, van Hall G, Lund AM, Orngreen MC (2020) Impaired lipolysis in propionic acidemia: A new metabolic myopathy? JIMD Rep 53:16-21 doi:10.1002/jmd2.12113\\u003c/li\\u003e\\n\\u003cli\\u003eTajima G, Kagawa R, Sakura F, Nakamura-Utsunomiya A, Hara K, Yuasa M, Hasegawa Y, Sasai H, Okada S (2021) Current Perspectives on Neonatal Screening for Propionic Acidemia in Japan: An Unexpectedly High Incidence of Patients with Mild Disease Caused by a Common PCCB Variant. Int J Neonatal Screen 7 doi:10.3390/ijns7030035\\u003c/li\\u003e\\n\\u003cli\\u003eTomcik K, Ibarra RA, Sadhukhan S, Han Y, Tochtrop GP, Zhang GF (2011) Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications. Anal Biochem 410:110-117 doi:10.1016/j.ab.2010.11.030\\u003c/li\\u003e\\n\\u003cli\\u003eWang Y, Christopher BA, Wilson KA, Muoio D, McGarrah RW, Brunengraber H, Zhang GF (2018) Propionate-induced changes in cardiac metabolism, notably CoA trapping, are not altered by l-carnitine. Am J Physiol Endocrinol Metab 315:E622-E633 doi:10.1152/ajpendo.00081.2018\\u003c/li\\u003e\\n\\u003cli\\u003eWang Y, Yang H, Geerts C, Furtos A, Waters P, Cyr D, Wang S, Mitchell GA (2023) The multiple facets of acetyl-CoA metabolism: Energetics, biosynthesis, regulation, acylation and inborn errors. Mol Genet Metab 138:106966 doi:10.1016/j.ymgme.2022.106966\\u003c/li\\u003e\\n\\u003cli\\u003eWang Y, Zhu S, He W, Marchuk H, Richard E, Desviat LR, Young SP, Koeberl D, Kasumov T, Chen X, Zhang GF (2024) The attenuated hepatic clearance of propionate increases cardiac oxidative stress in propionic acidemia. Basic Res Cardiol doi:10.1007/s00395-024-01066-w\\u003c/li\\u003e\\n\\u003cli\\u003eWilson KA, Han Y, Zhang M, Hess JP, Chapman KA, Cline GW, Tochtrop GP, Brunengraber H, Zhang GF (2017) Inter-relations between 3-hydroxypropionate and propionate metabolism in rat liver: relevance to disorders of propionyl-CoA metabolism. Am J Physiol Endocrinol Metab 313:E413-E428 doi:10.1152/ajpendo.00105.2017\\u003c/li\\u003e\\n\\u003cli\\u003eWolf B, Hsia YE, Sweetman L, Gravel R, Harris DJ, Nyhan WL (1981) Propionic acidemia: a clinical update. J Pediatr 99:835-846 doi:10.1016/s0022-3476(81)80004-2\\u003c/li\\u003e\\n\\u003cli\\u003eWongkittichote P, Ah Mew N, Chapman KA (2017) Propionyl-CoA carboxylase - A review. Mol Genet Metab 122:145-152 doi:10.1016/j.ymgme.2017.10.002\\u003c/li\\u003e\\n\\u003cli\\u003eZhang GF, Jensen MV, Gray SM, El K, Wang Y, Lu D, Becker TC, Campbell JE, Newgard CB (2021) Reductive TCA cycle metabolism fuels glutamine- and glucose-stimulated insulin secretion. Cell Metab 33:804-817 e805 doi:10.1016/j.cmet.2020.11.020\\u003c/li\\u003e\\n\\u003cli\\u003eZhang Y, Agarwal KC, Beylot M, Soloviev MV, David F, Reider MW, Anderson VE, Tserng KY, Brunengraber H (1994) Nonhomogeneous labeling of liver extra-mitochondrial acetyl-CoA. Implications for the probing of lipogenic acetyl-CoA via drug acetylation and for the production of acetate by the liver. J Biol Chem 269:11025-11029\\u003c/li\\u003e\\n\\u003cli\\u003eZhang Y, Peng C, Wang L, Chen S, Wang J, Tian Z, Wang C, Chen X, Zhu S, Zhang GF, Wang Y (2023) Prevalence of propionic acidemia in China. Orphanet J Rare Dis 18:281 doi:10.1186/s13023-023-02898-w\\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\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cellular-and-molecular-life-sciences\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"life\",\"sideBox\":\"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)\",\"snPcode\":\"18\",\"submissionUrl\":\"https://www.editorialmanager.com/life/default2.aspx\",\"title\":\"Cellular and Molecular Life Sciences\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Open\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"propionic acidemia, human induced pluripotent stem cell-derived cardiomyocytes, metabolic flux, glucose metabolism, fatty acid metabolism, cardiac diseases\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5874705/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5874705/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003ePropionic acidemia is an inborn error of metabolism caused by mutations in either the \\u003cem\\u003ePCCA\\u003c/em\\u003e or \\u003cem\\u003ePCCB\\u003c/em\\u003e genes. Patients with propionic acidemia experience a range of complications, including life-threatening cardiac dysfunctions. However, the pathological mechanisms underlying propionic acidemia-associated cardiac diseases remain largely unknown. To gain insights into the metabolic alterations in propionic acidemia, we studied human induced pluripotent stem cell-derived cardiomyocytes generated from a patient with propionic acidemia with two pathogenic PCCA mutations (\\u003cem\\u003ep.Cys616_Val633del\\u003c/em\\u003e and \\u003cem\\u003ep.Gly477Glufs9*\\u003c/em\\u003e) and from a healthy individual. Using stable isotope-based metabolic flux analysis, we confirmed that the \\u003cem\\u003ePCCA\\u003c/em\\u003e mutations lead to impaired propionyl-CoA carboxylase activity in human induced pluripotent stem cell-derived cardiomyocytes. In addition to being converted to propionylcarnitine, the accumulated propionyl-CoA can also be hydrolyzed to propionate and exported out of the cell, serving as a secondary \\u0026ldquo;pressure valve\\u0026rdquo; to regulate cellular propionyl-CoA levels. Interestingly, the deficiency of propionyl-CoA carboxylase was found to shift fuel metabolism from fatty acid oxidation to increased glucose metabolism human in induced pluripotent stem cell-derived cardiomyocytes from patients with propionic acidemia. This metabolic switch is less energy-efficient and may contribute to the development of chronic cardiac dysfunction in patients with propionic acidemia.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Metabolic flux analysis in hiPSC-CMs reveals insights into cardiac dysfunction in propionic acidemia\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-01-28 15:52:04\",\"doi\":\"10.21203/rs.3.rs-5874705/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2025-01-24T21:08:03+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-01-24T19:33:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-01-23T09:47:48+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Cellular and Molecular Life Sciences\",\"date\":\"2025-01-21T11:15:10+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cellular-and-molecular-life-sciences\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"life\",\"sideBox\":\"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)\",\"snPcode\":\"18\",\"submissionUrl\":\"https://www.editorialmanager.com/life/default2.aspx\",\"title\":\"Cellular and Molecular Life Sciences\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Open\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"5790c76a-a834-4879-94e6-47f3aeb71adc\",\"owner\":[],\"postedDate\":\"January 28th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-04-07T16:05:34+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5874705\",\"link\":\"https://doi.org/10.1007/s00018-025-05661-5\",\"journal\":{\"identity\":\"cellular-and-molecular-life-sciences\",\"isVorOnly\":false,\"title\":\"Cellular and Molecular Life Sciences\"},\"publishedOn\":\"2025-04-02 15:57:11\",\"publishedOnDateReadable\":\"April 2nd, 2025\"},\"versionCreatedAt\":\"2025-01-28 15:52:04\",\"video\":\"\",\"vorDoi\":\"10.1007/s00018-025-05661-5\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00018-025-05661-5\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5874705\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5874705\",\"identity\":\"rs-5874705\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}