Lower and higher dietary intake of branched-chain amino acids impact cardiac structure and functions in high-fat fed LDLR -/- mice

Lower and higher dietary intake of branched-chain amino acids impact cardiac structure and functions in high-fat fed LDLR -/- mice | 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 Lower and higher dietary intake of branched-chain amino acids impact cardiac structure and functions in high-fat fed LDLR -/- mice Qingxia Li, Xian Gao, Fen Chen, Huaxing Zhang, Tianyi Zhao, Shiyao Liu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6995118/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Elevation of dietary branched-chain amino acids (BCAAs) is regarded as a risk factor for heart failure. However, exposure to lower or higher levels of BCAAs on cardiac structure and function remained unclear. Mthods: The male C57BL/6J LDLR −/− mice were divided into 4 groups (n = 6) as feeding standard diet, high-fat diet, high-fat diet with low BCAAs, and high-fat diet with high BCAAs for 24 weeks. They underwent echocardiography after 12 and 24 weeks of dietary intervention. Then the mice were sacrificed and the heart organs were harvested for histological examination. Results The results showed that low or high nutritional BCAAs intake possessed different phenotypes on cardiac structure and function. Reduced BCAAs intake caused edema and a smaller inner diameter of the hearts in mice, while high BCAAs intake caused thinning and a larger inner diameter of the hearts. Pathological results showed that reducing the intake of BCAAs caused swelling of cardiomyocytes, loose fibers, and fragmentation of myocardial mitochondria. In contrast, higher intake of BCAAs increased the formation of lipid droplets and giant mitochondria. Conclusion Either decreased or increased dietary BCAAs intake induced negative effects on heart structure and function. The present research findings help the understanding of nutritional BCAAs intake at low or high levels related to pathological changes in the heart. branched-chain amino acids cardiac structure cardiac function heart failure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key summary points 1.Elevation of dietary branched-chain amino acids (BCAAs) is regarded as a risk factor for heart failure. However, exposure to lower or higher levels of BCAAs on cardiac structure and function remained unclear. 2.In the present study, we explored the influence of dietary BCAAs supplementation at low or high doses for different durations on cardiac structure and function in mice. 3.Either decreased or increased dietary BCAAs intake induced negative effects on heart structure and function. 4.Patients with cardiovascular disease should not blindly reduce dietary BCAAs intake. 1. Introduction Branched-chain amino acids (BCAAs), which encompass Leucine (Leu), Isoleucine (Ile), and Valine (Val), are essential amino acids [ 1 ] . BCAAs constitute about 35% of the free essential amino acids in human plasma [ 2 ] and play a crucial role in maintaining intestinal health, modulating immune function, providing energy, promoting nutrient metabolism, and facilitating protein synthesis [ 2 , 3 ] . The heart is one of the major organs of BCAAs metabolism. The correlation between BCAAs (including their metabolites) and cardiac diseases has attracted considerable research attention. A substantial body of evidence points towards a potential detrimental effect of increased BCAAs levels on cardiac function. In a previous study, the expression of BCAAs catabolic genes is notably diminished in both human and murine-failing hearts, indicating a significant downregulation of this metabolic pathway [ 4 ] . Reduced BCAAs levels in extracardiac organs have also been linked to lowered blood pressure and a reduced risk of heart failure [ 5 ] . In a knockout mouse model lacking the 2C-type serine-threonine protein phosphatase (PP2Cm), defective BCAAs catabolism is associated with glucose metabolic disruptions and an enhanced vulnerability of the heart to ischemia-reperfusion injury [ 6 ] . Also, correlations between serum BCAAs levels and the severity of cardiac dysfunction have been identified in rats with chronic heart failure [ 7 ] . An increase in cardiac BCAAs concentrations has also been reported in rats with heart failure post-myocardial infarction [ 8 ] . Similarly, elevated levels of BCAAs in the heart have been detected in patients with dilated cardiomyopathy [ 9 ] . In myocardial infarction model mice, dietary BCAAs supplementation has been implicated in exacerbating post-infarction contractile dysfunction and enlarging infarct size [ 8 ] . Contrarily, other research suggests a beneficial role for BCAAs in the heart, with short-term (8 weeks) administration demonstrating a reduction in infarct size and a cardioprotective effect in ischemia-reperfusion models [ 10 ] . Although the prevailing research indicates that elevated intake of BCAAs could adversely affect the heart, the cardiac implications of reduced BCAAs levels remain equivocal. As essential amino acids, the impact of short-term fluctuations in BCAAs intake on the heart may differ from long-term effects. Current research in this area is sparse, highlighting the need to study the effects of increasing or decreasing BCAAs on cardiac structure and function and to explore whether the duration of intervention influences outcomes. Atherosclerosis, a cardinal pathogenic factor in cardiovascular diseases (CVDs), exerts a profound impact on global health, necessitating vigilant study. The etiology of atherosclerosis encompasses inflammatory processes and lipid metabolism anomalies [ 11 ] . Initial insights have been gleaned into the nexus between BCAAs and oxidative stress, inflammatory pathways, coronary artery calcification, and carotid intima-media thickness [ 12 ] . However, a comprehensive understanding of their role in atherosclerotic cardiovascular pathology, especially concerning the heart's structure and function, is lacking. Consequently, the present investigation seeks to delineate the effects of BCAAs dietary modulation on cardiac structure and function in atherosclerotic murine models induced by a high-fat diet over both short-term (12 weeks) and long-term (24 weeks). Furthermore, this study initially explored the investigation of the underlying mechanisms of BCAAs’ action in the atherosclerotic heart. 2. Materials and Methods 2.1 Animal care and diets. The animals used in this study were 24 male C57BL/6J LDLR −/− mice at 8 weeks of age (Changzhou Cavens Laboratory Animal Co., Ltd.). After a one-week acclimation period, they were randomly divided into four groups using the random number method: control group (C) with a standard diet, model group with high-fat diet (HF), low BCAAs group (LB) with high-fat and half dose of BCAAs diet, high BCAAs group (HB) with high-fat and 2 fold of BCAAs diet. The standard diet contained 0.87g leucine, 0.48g isoleucine, and 0.57g valine per 100g. Two mice were housed per cage. The mice were allowed to eat ad libitum and food intake was recorded twice a week. All mice had free access to water. The environmental conditions were maintained at a temperature of (20 ± 5)°C and a relative humidity of (50 ± 10)%, with a 12-hour light-dark cycle. The feed was purchased from Beijing Keao Xieli Feed Co., Ltd., and the feed formulation is detailed in Supplementary Material Table S1 . The animal study protocol was approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University ( Approval No. IACUC-Hebmu-2024045). We confirm that the study is reported by ARRIVE guidelines. Clinical trial number: not applicable. 2.2 Small Animal Ultrasound Imaging Echocardiography was performed on the hearts of the mice at 12 and 24 weeks of intervention. After a 4-hour fasting period, the ventral thoracic region of the mice was depilated using a hair removal cream. Subsequently, inhalation anesthesia was administered using an EZ-SA800 Single Animal System from E-Z Systems Inc., America, with isoflurane (Ruiwo De) maintained at a gas pressure of 0.1 Mpa. Once fully anesthetized, the mice were prepared with conductive gel for ultrasonographic imaging. They were immobilized on a heating platform ventral side up to maintain the body temperature at 37°C ± 0.5°C. The Vevo @2100 Imaging System by FUJIFILM VisualSonics Inc., Canada, was utilized, selecting a short-axis view at the level of the papillary muscle with an 11.4 MHz high-frequency cardiac ultrasound transducer. For each animal, three cardiac cycles were measured, and the average values were recorded. The measured parameters included the left ventricular (LV) anterior wall end-diastolic thickness (LVAWD), LV anterior wall end-systolic thickness (LVAWS), the LV end-diastolic dimension (LVEDD), the LV end-systolic dimension (LVESD), Other parameters such as the LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), ejection fraction (EF), fractional shortening (FS), stroke volume (SV), cardiac output (CO) and LV Mass were derived automatically by Vevo 2100 software (Version-3.2.0) from the following formulas: (1) LVEDV = [7.0 / (2.4 + LVEDD)] × LVEDD 3 ; (2) LVESV = [7.0 / (2.4 + LVESD)] × LVEDD 3 ; (3) EF (%) = 100 × [(LVEDV – LVESV)/LVEDV]; (4) FS (%) = 100 × [(LVEDD – LVESD) / LVEDD]; (5) SV = LVEDV – LVESV; (6) CO = SV × HR; (7) LV Mass = 1.053 × ((LVIDD + LVPWD + LVAWD) 3 –LVIDD 3 ); (8) Corrected LV Mass = LV Mass × 0.8. 2.3 Animal Blood and Tissue Collection Following a 24-week BCAAs intervention and subsequent echocardiographic examination, animals were administered 2.5% sodium pentobarbital solution 50 mg/kg via intraperitoneal injection. Once deep anesthesia was achieved, the mice were euthanized by the cervical dislocation method and blood was obtained and centrifuged at 2500 g for 10 min at 4°C to obtain serum after coagulation. All the serum specimens were stored at -80℃. Then the heart was rapidly excised. Suitable tissue samples were then cut and either immersed in an electron microscopy preservative solution for subsequent scanning electron microscopy (SEM) analysis at Servicebio or fixed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining of the myocardial tissue. 2.4 Transmission electron microscopy of cardiac tissue The myocardial tissue was removed from the fixative, and then washed with 0.1 M PB (pH 7.4) 3 times, 15 min each. The next steps are briefly outlined as post-fix with 1% OsO4, dehydrate with a concentration gradient of ethanol, resin penetration, embedding with EMBed 812, and then polymerizing the resin blocks. The resin blocks were cut to 60-80nm thin on the ultramicrotome, and the tissues were fished out onto the 150-mesh cup rum grids with formvar film. After that, the tissue sections were stained with 2% uranium and 2.6% Lead citrate and observed under TEM. 2.5 Branched-Chain Amino Acids Targeted Quantitative Analysis Frozen serum was thawed at 4°C, then internal standard was added and AccQ Tag amino acid derived reagent was used to prepare samples to be tested. Chromatographic separation was performed using Waters ACQUITY UPLC I-CLASS ultra-performance liquid chromatography. The column model was Waters UPLC HSS T3 (1.7 µm, 2.1 mm×150 mm). The mobile phases were 0.1% formic acid (phase A) and acetonitrile (phase B), respectively. Mass spectrometry was then performed using a Waters XEVO TQ-S tandem quadrupole mass spectrometry system. The positive ion source voltage was 1.5kV and the cone voltage was 20V. The desolvation temperature was 600℃, and the desolvation gas flow rate was 1000 L/h. The gas flow rate of the cone hole was 10L/h. The peak area of the data was calculated by MassLynx quantitative software, the retention time allowed for an error of 15s, and the quantitative results were obtained by the standard curve method. 2.6 Statistical Analysis Data were analyzed using SPSS software version 27.0. Quantitative data conforming to a normal distribution were expressed as mean ± standard deviation (x ± s). The comparison of means between the two groups was performed using the independent samples t-test. For quantitative data not conforming to a normal distribution, the comparison between the two groups was conducted using the Mann-Whitney U test. P < 0.05 was considered to indicate a statistically significant difference. 3. Results 3.1 Increased Intake of BCAAs Leads to Increased Body Weight and Blood Glucose in LDLR −/− Mice In this study, dietary interventions were conducted on the mice as depicted in Fig. 1 A. Food intake data of mice are shown in Fig. 1 B. In terms of body weight, those fed a high-fat diet (HF, LB, and HB group) were heavier than those on a standard diet (C group). Specifically, the mice of the HB group were heavier than those of the HF and LB groups (Fig. 1 C). High-fat feeding induced an increase in blood glucose levels, and the HB group showed significantly higher fasting blood glucose levels compared to the other groups (Fig. 1 D). 3.2 Content of Branched-Chain Amino Acids in Serum The serum total BCAAs in HF, LB, and HB groups fed with a high-fat diet were about 40% higher than those in the control group fed with a standard diet (Fig. 1 E). However, there were no statistical differences in leucine, isoleucine, and valine among HF, LB, and HB groups (Fig. 1 E, F). 3.3 Divergent Impacts of BCAAs Intake at Varying Concentrations on Cardiac Structure in Mice. Following a 12-week intervention with high-fat diets with different doses of BCAAs, the results showed a higher average corrected left ventricular mass (LV Mass) of high-fat fed mice (HF, LB, and HB group) relative to the control group (Fig. 2 A). Prolonging of the intervention for 24 weeks elevated the corrected LV Mass in LB group than that of the HF group. No significant differences in the corrected LV Mass were observed among mice fed with high-fat diets when comparing short-term to long-term BCAAs intervention periods (Fig. 2 A). The difference between the control group at 12 and 24 weeks, may be due to the physiological ventricular hypertrophy that occurred in the mice due to the longer rearing time. We examined ventricular wall thickness at the end of systole in the heart. The LB group was significantly higher than the HB group for the left ventricular anterior wall thickness at end-systole (LVAW-s) at 24 weeks (Fig. 2 B). For the left ventricular posterior wall thickness at end-systole (LVPW-s) at 12 weeks (Fig. 2 C), the LB group was higher than HB group, too. When the intervention time was prolonged from 12 weeks to 24 weeks, the ventricular wall thickness at the end of the systole of the LB group increased most significantly. The ventricular anterior and posterior wall thickness at end-diastole ((LVAW-d and LVPW-d ) was also examined. For 12 weeks intervention, the LVPW-d of the HB group was thinner than the HF group (Fig. 2 E) and 15% lower than the LB group. While for 24 weeks intervention, both LVAW-d (Fig. 2 D) and LVPW-d (Fig. 2 E) the LB group were higher than the HB group, 14% and 13% respectively. At end-diastole, the ventricular wall thickness became thinner in the HB group and thicker in the LB group. However, no significant differences were observed between the groups under short-term and long-term intervention. After 12 weeks and 24 weeks of BCAAs intervention, the small animal ultrasound was detected, and the results of the two stages were compared. A: Corrected LV Mass: corrected left ventricular mass. B: LVAW-s: left ventricular anterior wall thickness at end-systole. C: LVPW-s: left ventricular posterior wall thickness at end-systole. D: LVAW-d: left ventricular anterior wall thickness at end-diastole. E: LVPW-d: left ventricular posterior wall thickness at end-diastole. The symbolic meaning of the analysis results is the same as in Fig. 1 . The increased ventricular wall thickness observed in the LB group and the decreased thickness in the HB group indicate that intake of BCAAs at varying concentrations can induce structural remodeling of the murine heart. The internal diameter and volume of the left ventricle reflect the cardiac structure and are crucial for evaluating the heart's pumping efficiency. After a 12-week intervention period, a comparative analysis between the HB group and the HF group demonstrated an enlargement in both the left ventricular systolic (LVSD) and diastolic (LVDD) internal diameters (Fig. 3 A, 3 B), 15% and 9% respectively. Correspondingly, there was a notable increase in the left ventricular end-systolic volume (LVESV) and end-diastolic volume (LVEDV) (Fig. 3 C, 3 D). These observations implied that an elevated intake of BCAAs over the short term can result in the dilation of the ventricular chamber and induce structural modifications within the heart. For long-term intervention, aside from the HB group's LVDD exceeding the LB group's, no significant differences in dimensions or volume were noted. These results indicated that BCAAs surplus may augment cardiac volume for short-term intervention. Moreover, no statistical variances in left ventricular dimensions and volume were detected within groups between the 12-week and 24-week interventions. This suggests a potential stabilization or reversal of structural adaptations over time, as the heart may achieve a new equilibrium through intrinsic mechanisms. In summary, the influences of elevated BCAAs intake on cardiac architecture were markedly more evident in the short term, as indicated by decreased ventricular wall thickness and increased internal diameter and volume. The effects of reducing BCAAs intake are predominantly reflected in the thickening of the murine ventricular wall. The results of the intra-group comparison showed that the prolongation of the intervention period mainly increased the systolic ventricular wall thickness. After 12 weeks and 24 weeks of BCAAs intervention, the small animal ultrasound was detected, and the results of the two stages were compared. A: LVSD: left ventricular systolic internal diameters. B: LVDD: left ventricular diastolic internal diameters. C: LVESV: left ventricular end-systolic volume. D: LVEDV: left ventricular end-diastolic volume. The symbolic meaning of the analysis results is the same as in Fig. 1 . 3.4 The Impact of Varying BCAAs Intake on Murine Cardiac Function We then detected the cardiac functions. For 12-week intervention, a tendency for decreased heart rate was noted across HF, LB, HB groups feeding with high-fat diets (Fig. 4 A). Interestingly, the trend reversed when the intervention was extended to 24 weeks (Fig. 4 A). The stroke volume of the mice exhibited no statistical differences in all groups under either short-term or long-term intervention (Fig. 4 B). Cardiac Output (CO), calculated as the product of heart rate and stroke volume, was consistent with the trend of heart rate (Fig. 4 C). Ejection Fraction (EF) and Fractional Shortening (FS) are pivotal indicators of cardiac systolic function. In our study, a 12-week short-term dietary intervention resulted in a decline in EF (15%) and FS (17%) within the HB group in contrast to the HF group. After a prolonged intervention, the trend was reversed, too. In contrast, a decreased intake of BCAAs does not appear to affect cardiac contractility, regardless of the intervention duration being short or long (Fig. 4 D, 4 E). Cardiac function was measured by echocardiography at 12 and 24 weeks after BCAAs intervention, and the results of the two stages were compared. The detection results presented in figures A-E are shown in the figure notes. The calculation of each index is described in the Materials and Methods section. The symbolic meaning of the analysis results is the same as in Fig. 1 . 3.5 Diverse Impacts of Varying BCAAs Concentrations on Myocardial Tissue in Mice All mice were sacrificed after 24 weeks of intervention and cardiac organs were collected. We performed a comprehensive evaluation of the macroscopic morphology and histological features of cardiac tissues from mice across different experimental groups. Hearts from mice fed the high-fat diets (HF, LB, HB group) were observed to be larger compared to those from the C group (Fig. 5 A). Conversely, the heart weight to body weight ratio was found to be increased in the LB group relative to the HF and HB groups, indicating that a decreased intake of BCAAs was associated with an increase in the heart weight (Fig. 5 B). Cardiac transmission electron microscopy (TEM) findings delineated the structural changes across the experimental groups. The control group (C) displayed orderly myofibrillar alignment and mitochondria with preserved architecture and intact cristae (Fig. 5 C). The HF group presented with mild cellular distention, yet without overt damage to either myofibrils or mitochondria. The LB group exhibited moderate cytosolic edema, myofibrillar disarray, and mitochondrial swelling accompanied by fragmented cristae. The HB group was characterized by sparse myofiber arrangement, indistinct intercalated discs, and the emergence of mega-mitochondria, along with lipid droplets interspersed among mitochondria, suggesting perturbations in the lipid metabolic processes within cardiomyocytes. 4 Discussion Some studies have shown that increased intake of BCAAs or defects in BCAAs’ metabolism is associated with the onset and exacerbation of cardiovascular diseases [ 3 ] . Nevertheless, is reduced intake of BCAAs associated with a protective effect against cardiovascular diseases? In this study, we demonstrated that both increased and decreased levels of BCAAs can cause damage to the heart. Low-density lipoprotein receptor (LDLR) knockout mice can develop hypercholesterolemia after being fed a high-fat diet, so they are often used as a model for cardiovascular disease research. We found that LDLR −/− mice on a diet enriched with BCAAs experienced weight gain. The link between BCAAs and obesity was also highlighted by a previous study [ 13 ] . The mechanism could be that BCAAs catabolism is associated with adipocyte differentiation and lipogenesis [ 14 , 15 ] . Conversely, a diminished intake of BCAAs did not culminate in weight reduction in our study. Increasing BCAAs intake has been identified as a precipitating factor for type 2 diabetes [ 13 , 16 ] . Despite this, reducing BCAAs intake did not lower blood glucose levels in our study. The analogous results in body weight and blood glucose suggested that reducing BCAA intake did not protect against obesity and type 2 diabetes. In this study, the concentration of BCAAs in serum was significantly higher in mice fed the high-fat diet than in those fed the standard diet, which may be caused by obesity. Several previous studies have shown that serum BCAAs concentrations are elevated in obese individuals and that dietary BCAAs intake is positively associated with obesity [ 17 – 21 ] . However, there was no significant difference in serum BCAAs levels among the three groups of obese mice, even though their diets contained different concentrations of BCAAs. This may be related to the fact that we starved the mice for 8 hours before sacrifice, which caused the serum BCAAs levels to return to baseline. In healthy adults, serum BCAA levels increased immediately after BCAA intake was raised, reached their peak at 30 minutes post-meal, and returned to baseline 3 hours after the meal [ 22 ] . In our study, both increased and decreased BCAA intake resulted in cardiac structural damage, and the symptoms appeared sooner in mice that consumed more BCAAs. With longer intervention, the effects of diminished BCAA intake were more significant. Interestingly, higher and lower BCAAs intake exerted different impacts on cardiac structure. Reduced BCAAs consumption was associated with the development of cardiac edema and a constriction of the internal diameter, while increased BCAAs consumption results in myocardial thinning and dilation of the internal diameter. Correspondingly, the results of tissue staining and electron microscopy also showed that the myocardial fibers were swollen or loose. This suggests that increasing and decreasing BCAAs may damage the heart through different mechanisms. Regarding cardiac function, only a 12-week increase in BCAAs intake led to changes in ejection fraction and fractional shortening. Post 24 weeks’ intervention, an amelioration in cardiac function was noted in mice with elevated BCAAs intake, indicative of compensatory improvements in cardiac performance. The enlargement of the ventricular chamber and the absence of a corresponding increase in cardiac output indicate a decrease in cardiac contractility. By observing the myocardial tissue, we found that the myocardial fibers in the HB group were loose, which explained the cause of the decreased myocardial contractility. Several studies have reported that BCAAs affect lipid metabolism. The metabolism of valine yields 3-hydroxyisobutyrate (3-HIB), which enhances endothelial fatty acid transport, facilitates muscle fatty acid uptake in vivo , and encourages lipid deposition in muscle tissues [ 23 ] . Leucine is known to upregulate the expression of genes involved in lipid synthesis and serum fatty acid levels, thereby impacting lipid metabolism [ 24 ] . Deficiency of BCAAs leads to a blocked pathway of fatty acids into mitochondria [ 25 ] . Our study corroborated these findings, with the observation of lipid droplets in the mitochondria of cardiac muscle cells under conditions of high BCAAs intake. Then we explored the association of heart failure with BCAAs and their metabolites using bioinformatics techniques based on the GENE EXPRESSION OMNIBUS (GEO) database. The results showed that several genes related to BCAAs metabolism were up-regulated in heart failure mice, and fatty acid metabolism was the most significantly affected pathway (Fig. S1 ). Extensive research has detailed the mechanisms by which elevated levels of BCAAs and their metabolites result in cardiac damage. Activation of the mammalian target of rapamycin (mTOR) signaling by BCAAs resulted in augmented protein translation [ 26 ] , mitochondrial dysfunction [ 27 ] , perturbations in redox balance [ 28 ] , and post-myocardial infarction cardiac dysfunction and remodeling [ 15 ] . Pharmacologic activation of BCAAs catabolism was protective in models of heart failure. However, reduced plasma and cardiac BCAA levels do not offer significant protection, suggesting that other mechanisms may be at play [ 29 ] . Nonetheless, the mechanism of cardioprotection has remained unclear. Our study revealed that diminished BCAAs intake predominantly leads to cardiac edema, aligning with observations of increased heart weight and ventricular wall thickening. We offered a histological interpretation of the mechanisms by which low BCAAs intake induced cardiac injury. Additional exploration is necessary for a comprehensive understanding. There are a few limitations of the present work. While our research encompassed a 24-week observational time frame and identified substantial changes in cardiac architecture, the study did not record overt heart failure, resulting in a lack of significant inter-group disparities in cardiac functionality. The use of a murine model in our investigation might have introduced interspecies discrepancies that could affect the study outcomes. Additionally, our research only offered an initial foray into the mechanisms responsible for the cardiac structural variations associated with different BCAAs dosages. A comprehensive elucidation of these mechanisms requires further in-depth investigation, especially the impact of reduced BCAAs consumption. In conclusion, the intake of BCAAs at both elevated and reduced levels induces detrimental effects on the cardiac health of heart failure mice. Reduced BCAAs consumption is associated with the development of cardiac edema and a constriction of the internal diameter, while increased BCAAs consumption results in myocardial thinning and dilation of the internal diameter. Therefore, individuals with cardiac conditions is cautioned against the indiscriminate reduction of BCAAs in their diet without a thorough evaluation of the potential hazards. Declarations Data availability: Data is provided within the manuscript or supplementary information files. The data links from the GEO database are as follows: GSE136308: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136308 GSE137442: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137442 Acknowledgments: We thank the associate editor and the reviewers for their useful feedback that improved this paper. Funding: This research was funded by Basic and Translational Research on Nutritional Diet and Atherosclerosis Based on Cohort Study, grant number ZF2023019. The APC was funded by the Finance Department of Hebei Province. Author Contributions : Conceptualization, Wenhua Ling and Yandong Deng; Methodology and writing, Qingxia Li; Visualization, Fen Chen; Software, Xian Gao, Huaxing Zhang and Ziyi Liu; Supervision, Xian Gao; Validation, Tianyi Zhao, Shiyao Liu, Ziyue Tang, Yuanyuan Liu, Tianao Ling, Yang Liu, Chunfei Dang, Yili Xu; Funding acquisition, Yandong Deng and Yuxia Ma; All authors have read and agreed to the published version of the manuscript. Corresponding authors ： Yandong Deng and Wenhua Ling. Competing Interests: The authors declare no conflicts of interest. Ethical statement: The animal study protocol was approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University ( Approval No. IACUC-Hebmu-2024045). Consent to Publish declaration: Not applicable. References Le Couteur DG, Solon-Biet SM, Cogger VC, Ribeiro R, de Cabo R, Raubenheimer D, Cooney GJ, Simpson SJ. Branched chain amino acids, aging and age-related health. Ageing Res Rev 2020 , 64: 101198. Neinast M, Murashige D, Arany Z. Branched chain amino acids. Annu Rev Physiol 2019 , 81(1): 139-164. Mcgarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease. Nat Rev Cardiol 2023 , 20(2): 77-89. Sun H, Olson KC, Gao C, Prosdocimo DA, Zhou M, Wang Z, Jeyaraj D, Youn J, Ren S, Liu Y, Rau CD, Shah S, Ilkayeva O, Gui W, William NS, Wynn RM, Newgard CB, Cai H, Xiao X, Chuang DT, Schulze PC, Lynch C, Jain MK, Wang Y. Catabolic defect of branched-chain amino acids promotes heart failure. Circulation (New York, N.Y.) 2016 , 133(21): 2038-2049. Murashige D, Jung JW, Neinast MD, Levin MG, Chu Q, Lambert JP, Garbincius JF, Kim B, Hoshino A, Marti-Pamies I, Mcdaid KS, Shewale SV, Flam E, Yang S, Roberts E, Li L, Morley MP, Bedi KC, Hyman MC, Frankel DS, Margulies KB, Assoian RK, Elrod JW, Jang C, Rabinowitz JD, Arany Z. Extra-cardiac bcaa catabolism lowers blood pressure and protects from heart failure. Cell Metab 2022 , 34(11): 1749-1764. Li T, Zhang Z, Kolwicz SC, Abell L, Roe ND, Kim M, Zhou B, Cao Y, Ritterhoff J, Gu H, Raftery D, Sun H, Tian R. Defective branched-chain amino acid catabolism disrupts glucose metabolism and sensitizes the heart to ischemia-reperfusion injury. Cell Metab 2017 , 25(2): 374-385. Li R, He H, Fang S, Hua Y, Yang X, Yuan Y, Liang S, Liu P, Tian Y, Xu F, Zhang Z, Huang Y. Time series characteristics of serum branched-chain amino acids for early diagnosis of chronic heart failure. J Proteome Res 2019 , 18(5): 2121-2128. Wang W, Zhang F, Xia Y, Zhao S, Yan W, Wang H, Lee Y, Li C, Zhang L, Lian K, Gao E, Cheng H, Tao L. Defective branched chain amino acid catabolism contributes to cardiac dysfunction and remodeling following myocardial infarction. Am J Physiol-Heart C 2016 , 311(5): H1160-H1169. Uddin GM, Zhang L, Shah S, Fukushima A, Wagg CS, Gopal K, Al BR, Pherwani S, Ho KL, Boisvenue J, Karwi QG, Altamimi T, Wishart DS, Dyck J, Ussher JR, Oudit GY, Lopaschuk GD. Impaired branched chain amino acid oxidation contributes to cardiac insulin resistance in heart failure. Cardiovasc Diabetol 2019 , 18(1): 86. Satomi S, Morio A, Miyoshi H, Nakamura R, Tsutsumi R, Sakaue H, Yasuda T, Saeki N, Tsutsumi YM. Branched-chain amino acids-induced cardiac protection against ischemia/reperfusion injury. Life Sci 2020 , 245: 117368. Björkegren JLM, Lusis AJ. Atherosclerosis: recent developments. Cell 2022 , 185(10): 1630-1645. Tzoulaki I, Castagne R, Boulange CL, Karaman I, Chekmeneva E, Evangelou E, Ebbels T, Kaluarachchi MR, Chadeau-Hyam M, Mosen D, Dehghan A, Moayyeri A, Ferreira D, Guo X, Rotter JI, Taylor KD, Kavousi M, de Vries PS, Lehne B, Loh M, Hofman A, Nicholson JK, Chambers J, Gieger C, Holmes E, Tracy R, Kooner J, Greenland P, Franco OH, Herrington D, Lindon JC, Elliott P. Serum metabolic signatures of coronary and carotid atherosclerosis and subsequent cardiovascular disease. Eur Heart J 2019 , 40(34): 2883-2896. Vanweert F, Schrauwen P, Phielix E. Role of branched-chain amino acid metabolism in the pathogenesis of obesity and type 2 diabetes-related metabolic disturbances bcaa metabolism in type 2 diabetes. Nutr Diabetes 2022 , 12(1): 35. Jersin RA, Sri PTD, Skartveit L, Bjune MS, Muniandy M, Lee-Odegard S, Heinonen S, Alvarez M, Birkeland KI, Andre DC, Pajukanta P, Mccann A, Pietilainen KH, Claussnitzer M, Mellgren G, Dankel SN. Impaired adipocyte slc7a10 promotes lipid storage in association with insulin resistance and altered bcaa metabolism. J Clin Endocrinol Metab 2023 , 108(9): 2217-2229. Green CR, Wallace M, Divakaruni AS, Phillips SA, Murphy AN, Ciaraldi TP, Metallo CM. Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis. Nat Chem Biol 2016 , 12(1): 15-21. White PJ, Mcgarrah RW, Herman MA, Bain JR, Shah SH, Newgard CB. Insulin action, type 2 diabetes, and branched-chain amino acids: a two-way street. Mol Metab 2021 , 52: 101261. Felig P, Marliss E, Cahill GJ. Plasma amino acid levels and insulin secretion in obesity. N Engl J Med 1969 , 281(15): 811-816. Zhang Y, Rao S, Zhang X, Peng Z, Song W, Xie S, Cao H, Zhang Z, Yang W. Dietary and circulating branched chain amino acids are unfavorably associated with body fat measures among chinese adults. Nutr Res 2024 , 128: 94-104. Asoudeh F, Salari-Moghaddam A, Keshteli AH, Esmaillzadeh A, Adibi P. Dietary intake of branched-chain amino acids in relation to general and abdominal obesity. Eat Weight Disord 2022 , 27(4): 1303-1311. Lu J, Gu Y, Liu H, Wang L, Li W, Li W, Leng J, Zhang S, Qi L, Yang X, Hu G. Daily branched-chain amino acid intake and risks of obesity and insulin resistance in children: a cross-sectional study. Obesity (Silver Spring) 2020 , 28(7): 1310-1316. Zhao S, Zhou L, Wang Q, Cao JH, Chen Y, Wang W, Zhu BD, Wei ZH, Li R, Li CY, Zhou GY, Tan ZJ, Zhou HP, Li CX, Gao HK, Qin XJ, Lian K. Elevated branched-chain amino acid promotes atherosclerosis progression by enhancing mitochondrial-to-nuclear h(2)o(2)-disulfide hmgb1 in macrophages. Redox Biol 2023 , 62: 102696. Newton-Tanzer E, Can SN, Demmelmair H, Horak J, Holdt L, Koletzko B, Grote V. Apparent saturation of branched-chain amino acid catabolism after high dietary milk protein intake in healthy adults. J Clin Endocrinol Metab 2024 . Jang C, Oh SF, Wada S, Rowe GC, Liu L, Chan MC, Rhee J, Hoshino A, Kim B, Ibrahim A, Baca LG, Kim E, Ghosh CC, Parikh SM, Jiang A, Chu Q, Forman DE, Lecker SH, Krishnaiah S, Rabinowitz JD, Weljie AM, Baur JA, Kasper DL, Arany Z. A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance. Nat Med 2016 , 22(4): 421-426. Zhou X, Chen J, Sun B, Wang Z, Zhu J, Yue Z, Zhang Y, Shan A, Ma Q, Wang J. Leucine, but not isoleucine or valine, affects serum lipid profiles and browning of wat in mice. Food Funct 2021 , 12(15): 6712-6724. Gotvaldova K, Spackova J, Novotny J, Baslarova K, Jezek P, Rossmeislova L, Gojda J, Smolkova K. Bcaa metabolism in pancreatic cancer affects lipid balance by regulating fatty acid import into mitochondria. Cancer Metab 2024 , 12(1): 10. Latimer MN, Sonkar R, Mia S, Frayne IR, Carter KJ, Johnson CA, Rana S, Xie M, Rowe GC, Wende AR, Prabhu SD, Frank SJ, Rosiers CD, Chatham JC, Young ME. Branched chain amino acids selectively promote cardiac growth at the end of the awake period. J Mol Cell Cardiol 2021 , 157: 31-44. Vanderstad LR, Wyatt EC, Vaughan RA. Excess branched-chain amino acids suppress mitochondrial function and biogenic signaling but not mitochondrial dynamics in a myotube model of skeletal muscle insulin resistance. Metabolites 2024 , 14(7). Sun H, Olson KC, Gao C, Prosdocimo DA, Zhou M, Wang Z, Jeyaraj D, Youn JY, Ren S, Liu Y, Rau CD, Shah S, Ilkayeva O, Gui WJ, William NS, Wynn RM, Newgard CB, Cai H, Xiao X, Chuang DT, Schulze PC, Lynch C, Jain MK, Wang Y. Catabolic defect of branched-chain amino acids promotes heart failure. Circulation 2016 , 133(21): 2038-2049. Murashige D, Jung JW, Neinast MD, Levin MG, Chu Q, Lambert JP, Garbincius JF, Kim B, Hoshino A, Marti-Pamies I, Mcdaid KS, Shewale SV, Flam E, Yang S, Roberts E, Li L, Morley MP, Bedi KJ, Hyman MC, Frankel DS, Margulies KB, Assoian RK, Elrod JW, Jang C, Rabinowitz JD, Arany Z. Extra-cardiac bcaa catabolism lowers blood pressure and protects from heart failure. Cell Metab 2022 , 34(11): 1749-1764. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted 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-6995118","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516532255,"identity":"e0aa0982-537a-436b-9b61-15d4d4ef717e","order_by":0,"name":"Qingxia Li","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingxia","middleName":"","lastName":"Li","suffix":""},{"id":516532256,"identity":"cf810954-5efb-4dd7-9e67-0f23498886aa","order_by":1,"name":"Xian Gao","email":"","orcid":"","institution":"the Second Affiliated Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Gao","suffix":""},{"id":516532257,"identity":"7ca89a6a-f30c-4b50-afef-3858eec111ba","order_by":2,"name":"Fen Chen","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Chen","suffix":""},{"id":516532258,"identity":"312f334b-d87d-475f-8f80-1248afe5574f","order_by":3,"name":"Huaxing Zhang","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huaxing","middleName":"","lastName":"Zhang","suffix":""},{"id":516532259,"identity":"574768c5-1940-458d-b0d9-00526c5f1ef1","order_by":4,"name":"Tianyi Zhao","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianyi","middleName":"","lastName":"Zhao","suffix":""},{"id":516532260,"identity":"e0ca91b1-2e4f-4669-8baf-f8830e7ed572","order_by":5,"name":"Shiyao Liu","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shiyao","middleName":"","lastName":"Liu","suffix":""},{"id":516532261,"identity":"0b6562f5-0a4c-45fe-af8d-5abeb0a2f598","order_by":6,"name":"Ziyi Liu","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziyi","middleName":"","lastName":"Liu","suffix":""},{"id":516532262,"identity":"87db6bf8-a8f3-468f-8551-e996f6259993","order_by":7,"name":"Ziyue Tang","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziyue","middleName":"","lastName":"Tang","suffix":""},{"id":516532263,"identity":"c0cdbbf3-a8dd-45e7-9d6a-235ebd3eda94","order_by":8,"name":"Yuanyuan Liu","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Liu","suffix":""},{"id":516532264,"identity":"d173935d-d857-4c53-8823-25d7b71553be","order_by":9,"name":"Tianao Ling","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianao","middleName":"","lastName":"Ling","suffix":""},{"id":516532265,"identity":"41df516a-a7bf-409e-906c-4fe2f96cd0c4","order_by":10,"name":"Yang Liu","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""},{"id":516532266,"identity":"7ecc733d-d6c6-45cf-ad78-53fd5c565c2e","order_by":11,"name":"Chunfei Dang","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunfei","middleName":"","lastName":"Dang","suffix":""},{"id":516532267,"identity":"b08febbf-dd4b-42f3-9f2b-eb299fb07b34","order_by":12,"name":"Yili Xu","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yili","middleName":"","lastName":"Xu","suffix":""},{"id":516532268,"identity":"857700b7-071b-47b6-9d52-13773bc409f3","order_by":13,"name":"Yuxia Ma","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxia","middleName":"","lastName":"Ma","suffix":""},{"id":516532269,"identity":"9c795dab-0742-46cc-9ed4-887d922f3f8a","order_by":14,"name":"Yandong Deng","email":"","orcid":"","institution":"the First Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yandong","middleName":"","lastName":"Deng","suffix":""},{"id":516532270,"identity":"ac4dfe69-2fea-4545-b855-056628e2664b","order_by":15,"name":"Wenhua Ling","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBACPmYgwdhgw8DAzNwAEzTAq4UNoiUNqIWRWC0MYC2HwSSRWth5D7/m3XE+mr+dsYHxZ1tdYgN78zYJhpo7eBzGl2bNe+Z27ozDjA3MvG2HExt4jpVJMBx7hkcLj5lxbtvt3AaQFsa2A4kNEjlmEhCn4tVyLnf+YZjD5N8Q1GL8OLftQO4GoBYG3jZmoC08hG1h/tuWnLsRqOUwz7nDxm08acUWCcdwa+HnP2P8cWabXe6884cPPvxRVifbz354440PNbi1gCySgLEOMLJBYyoBnwZgtH9AsP/gVzoKRsEoGAUjEwAAJg1RZFdrUkoAAAAASUVORK5CYII=","orcid":"","institution":"Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Wenhua","middleName":"","lastName":"Ling","suffix":""}],"badges":[],"createdAt":"2025-06-28 03:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6995118/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6995118/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91624421,"identity":"c2d2ee11-161a-4f5d-8402-e7bdcb1edc17","added_by":"auto","created_at":"2025-09-18 12:01:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178023,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes in body weight and serum BCAAs in mice fed different diets.\u003c/p\u003e\n\u003cp\u003eA: Schematic design of the animal experiment: C57BL/6J LDLR\u003csup\u003e-/-\u003c/sup\u003e mice were randomly divided into four groups after a one-week acclimation period. ND: normal diet; HFD: high-fat diet; C: control, normal diet; HF: high-fat diet with normal BCAAs; LB: high-fat diet with low (1/2) BCAAs; HB: high-fat diet with high (twice as much) BCAAs. B: The line chart results showed the change in the average daily food intake of mice in each group with week. Two mice were housed in each cage. The weight of food consumed per cage was calculated once a week. C: Body weight of mice in each group after 24 weeks of intervention (n=6). D: Fasting blood glucose for each mouse after 24 weeks of intervention (n=6). E: Concentrations of total branched-chain amino acids in the serum of mice in each group (n=6). F: Concentrations of valine, leucine and isoleucine in each group (n=6). A black dot represents a mouse. Error bars represent standard deviation. # represents compared to the control group. #: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; ##: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ###: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. * represents the comparison between the two groups as marked by the broken line below it. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/e20e5b9b27513f8ca53382b1.png"},{"id":91625495,"identity":"fcda9b04-90e3-455b-bd92-4c164ad43f1f","added_by":"auto","created_at":"2025-09-18 12:09:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332095,"visible":true,"origin":"","legend":"\u003cp\u003eEchocardiographic results of heart mass and ventricular thickness in mice treated with different concentrations of BCAAs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/522bfa2277b2a323101a5d05.png"},{"id":91624425,"identity":"40d56f71-12a1-4250-8699-e09aec691888","added_by":"auto","created_at":"2025-09-18 12:01:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":270580,"visible":true,"origin":"","legend":"\u003cp\u003eEchocardiographic results of cardiac internal diameters and volume in mice treated with different concentrations of BCAAs.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/91936692e5b68586af1ed5ef.png"},{"id":91625496,"identity":"b5332788-755a-4f28-90b5-ddb203079b58","added_by":"auto","created_at":"2025-09-18 12:09:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":340359,"visible":true,"origin":"","legend":"\u003cp\u003eEchocardiographic results of cardiac function in mice treated with different concentrations of BCAAs.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/5d547d4b2cdcfdb99d8f729c.png"},{"id":91626712,"identity":"f97c33dd-7d03-4eac-bca4-8fcceaf85dc6","added_by":"auto","created_at":"2025-09-18 12:17:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":655384,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the effects of different concentrations of BCAAs on mouse heart tissue.\u003c/p\u003e\n\u003cp\u003eA: Pictures of mouse hearts. Mice were sacrificed and hearts were harvested after 24 weeks of intervention. Each scale represents 1 mm. B: Heart Index = heart weight/body weight*100%. C: HE staining and TEM view of cardiac tissues. The ruler is in the lower right corner of each image. Damaged mitochondria are indicated by red arrows, giant mitochondria by blue arrows, and lipid droplets by orange arrows.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/651a1ec8f5c23b6185a473f5.png"},{"id":94489857,"identity":"f156c1c0-e5fe-4301-a8a9-510badb282a6","added_by":"auto","created_at":"2025-10-27 17:06:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2840879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/7962593b-3978-4d8b-ab55-da8ac29da891.pdf"},{"id":91624426,"identity":"d21be6e2-7ef2-4b93-ba04-da0be0128627","added_by":"auto","created_at":"2025-09-18 12:01:54","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":177466,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6995118/v1/9bb50c7d14d222dbc463283f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLower and higher dietary intake of branched-chain amino acids impact cardiac structure and functions in high-fat fed LDLR\u003csup\u003e -/-\u003c/sup\u003e mice\u003c/p\u003e","fulltext":[{"header":"Key summary points","content":"\u003cp\u003e1.Elevation of dietary branched-chain amino acids (BCAAs) is regarded as a risk factor for heart failure. However, exposure to lower or higher levels of BCAAs on cardiac structure and function remained unclear.\u003c/p\u003e\n\u003cp\u003e2.In the present study, we explored the influence of dietary BCAAs supplementation at low or high doses for different durations on cardiac structure and function in mice.\u003c/p\u003e\n\u003cp\u003e3.Either decreased or increased dietary BCAAs intake induced negative effects on heart structure and function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.Patients with cardiovascular disease should not blindly reduce dietary BCAAs intake.\u003c/p\u003e\n"},{"header":"1. Introduction","content":"\u003cp\u003eBranched-chain amino acids (BCAAs), which encompass Leucine (Leu), Isoleucine (Ile), and Valine (Val), are essential amino acids\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. BCAAs constitute about 35% of the free essential amino acids in human plasma\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and play a crucial role in maintaining intestinal health, modulating immune function, providing energy, promoting nutrient metabolism, and facilitating protein synthesis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe heart is one of the major organs of BCAAs metabolism. The correlation between BCAAs (including their metabolites) and cardiac diseases has attracted considerable research attention. A substantial body of evidence points towards a potential detrimental effect of increased BCAAs levels on cardiac function. In a previous study, the expression of BCAAs catabolic genes is notably diminished in both human and murine-failing hearts, indicating a significant downregulation of this metabolic pathway\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Reduced BCAAs levels in extracardiac organs have also been linked to lowered blood pressure and a reduced risk of heart failure\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In a knockout mouse model lacking the 2C-type serine-threonine protein phosphatase (PP2Cm), defective BCAAs catabolism is associated with glucose metabolic disruptions and an enhanced vulnerability of the heart to ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Also, correlations between serum BCAAs levels and the severity of cardiac dysfunction have been identified in rats with chronic heart failure\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. An increase in cardiac BCAAs concentrations has also been reported in rats with heart failure post-myocardial infarction\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Similarly, elevated levels of BCAAs in the heart have been detected in patients with dilated cardiomyopathy\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In myocardial infarction model mice, dietary BCAAs supplementation has been implicated in exacerbating post-infarction contractile dysfunction and enlarging infarct size\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Contrarily, other research suggests a beneficial role for BCAAs in the heart, with short-term (8 weeks) administration demonstrating a reduction in infarct size and a cardioprotective effect in ischemia-reperfusion models \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough the prevailing research indicates that elevated intake of BCAAs could adversely affect the heart, the cardiac implications of reduced BCAAs levels remain equivocal. As essential amino acids, the impact of short-term fluctuations in BCAAs intake on the heart may differ from long-term effects. Current research in this area is sparse, highlighting the need to study the effects of increasing or decreasing BCAAs on cardiac structure and function and to explore whether the duration of intervention influences outcomes.\u003c/p\u003e\u003cp\u003eAtherosclerosis, a cardinal pathogenic factor in cardiovascular diseases (CVDs), exerts a profound impact on global health, necessitating vigilant study. The etiology of atherosclerosis encompasses inflammatory processes and lipid metabolism anomalies\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Initial insights have been gleaned into the nexus between BCAAs and oxidative stress, inflammatory pathways, coronary artery calcification, and carotid intima-media thickness\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, a comprehensive understanding of their role in atherosclerotic cardiovascular pathology, especially concerning the heart's structure and function, is lacking. Consequently, the present investigation seeks to delineate the effects of BCAAs dietary modulation on cardiac structure and function in atherosclerotic murine models induced by a high-fat diet over both short-term (12 weeks) and long-term (24 weeks). Furthermore, this study initially explored the investigation of the underlying mechanisms of BCAAs\u0026rsquo; action in the atherosclerotic heart.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal care and diets.\u003c/h2\u003e\u003cp\u003eThe animals used in this study were 24 male C57BL/6J LDLR\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice at 8 weeks of age (Changzhou Cavens Laboratory Animal Co., Ltd.). After a one-week acclimation period, they were randomly divided into four groups using the random number method: control group (C) with a standard diet, model group with high-fat diet (HF), low BCAAs group (LB) with high-fat and half dose of BCAAs diet, high BCAAs group (HB) with high-fat and 2 fold of BCAAs diet. The standard diet contained 0.87g leucine, 0.48g isoleucine, and 0.57g valine per 100g. Two mice were housed per cage. The mice were allowed to eat ad libitum and food intake was recorded twice a week. All mice had free access to water. The environmental conditions were maintained at a temperature of (20\u0026thinsp;\u0026plusmn;\u0026thinsp;5)\u0026deg;C and a relative humidity of (50\u0026thinsp;\u0026plusmn;\u0026thinsp;10)%, with a 12-hour light-dark cycle. The feed was purchased from Beijing Keao Xieli Feed Co., Ltd., and the feed formulation is detailed in Supplementary Material Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The animal study protocol was approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University ( Approval No. IACUC-Hebmu-2024045). We confirm that the study is reported by ARRIVE guidelines. Clinical trial number: not applicable.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Small Animal Ultrasound Imaging\u003c/h2\u003e\u003cp\u003eEchocardiography was performed on the hearts of the mice at 12 and 24 weeks of intervention. After a 4-hour fasting period, the ventral thoracic region of the mice was depilated using a hair removal cream. Subsequently, inhalation anesthesia was administered using an EZ-SA800 Single Animal System from E-Z Systems Inc., America, with isoflurane (Ruiwo De) maintained at a gas pressure of 0.1 Mpa. Once fully anesthetized, the mice were prepared with conductive gel for ultrasonographic imaging. They were immobilized on a heating platform ventral side up to maintain the body temperature at 37\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. The Vevo @2100 Imaging System by FUJIFILM VisualSonics Inc., Canada, was utilized, selecting a short-axis view at the level of the papillary muscle with an 11.4 MHz high-frequency cardiac ultrasound transducer. For each animal, three cardiac cycles were measured, and the average values were recorded. The measured parameters included the left ventricular (LV) anterior wall end-diastolic thickness (LVAWD), LV anterior wall end-systolic thickness (LVAWS), the LV end-diastolic dimension (LVEDD), the LV end-systolic dimension (LVESD), Other parameters such as the LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), ejection fraction (EF), fractional shortening (FS), stroke volume (SV), cardiac output (CO) and LV Mass were derived automatically by Vevo 2100 software (Version-3.2.0) from the following formulas:\u003c/p\u003e\u003cp\u003e(1) LVEDV = [7.0 / (2.4\u0026thinsp;+\u0026thinsp;LVEDD)] \u0026times; LVEDD\u003csup\u003e3\u003c/sup\u003e;\u003c/p\u003e\u003cp\u003e(2) LVESV = [7.0 / (2.4\u0026thinsp;+\u0026thinsp;LVESD)] \u0026times; LVEDD\u003csup\u003e3\u003c/sup\u003e;\u003c/p\u003e\u003cp\u003e(3) EF (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; [(LVEDV \u0026ndash; LVESV)/LVEDV];\u003c/p\u003e\u003cp\u003e(4) FS (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; [(LVEDD \u0026ndash; LVESD) / LVEDD];\u003c/p\u003e\u003cp\u003e(5) SV\u0026thinsp;=\u0026thinsp;LVEDV \u0026ndash; LVESV;\u003c/p\u003e\u003cp\u003e(6) CO\u0026thinsp;=\u0026thinsp;SV \u0026times; HR;\u003c/p\u003e\u003cp\u003e(7) LV Mass\u0026thinsp;=\u0026thinsp;1.053 \u0026times; ((LVIDD\u0026thinsp;+\u0026thinsp;LVPWD\u0026thinsp;+\u0026thinsp;LVAWD)\u003csup\u003e3\u003c/sup\u003e\u0026ndash;LVIDD\u003csup\u003e3\u003c/sup\u003e);\u003c/p\u003e\u003cp\u003e(8) Corrected LV Mass\u0026thinsp;=\u0026thinsp;LV Mass \u0026times; 0.8.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Animal Blood and Tissue Collection\u003c/h2\u003e\u003cp\u003eFollowing a 24-week BCAAs intervention and subsequent echocardiographic examination, animals were administered 2.5% sodium pentobarbital solution 50 mg/kg via intraperitoneal injection. Once deep anesthesia was achieved, the mice were euthanized by the cervical dislocation method and blood was obtained and centrifuged at 2500 g for 10 min at 4\u0026deg;C to obtain serum after coagulation. All the serum specimens were stored at -80℃. Then the heart was rapidly excised. Suitable tissue samples were then cut and either immersed in an electron microscopy preservative solution for subsequent scanning electron microscopy (SEM) analysis at Servicebio or fixed in 4% paraformaldehyde for hematoxylin and eosin (H\u0026amp;E) staining of the myocardial tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Transmission electron microscopy of cardiac tissue\u003c/h2\u003e\u003cp\u003eThe myocardial tissue was removed from the fixative, and then washed with 0.1 M PB (pH 7.4) 3 times, 15 min each. The next steps are briefly outlined as post-fix with 1% OsO4, dehydrate with a concentration gradient of ethanol, resin penetration, embedding with EMBed 812, and then polymerizing the resin blocks. The resin blocks were cut to 60-80nm thin on the ultramicrotome, and the tissues were fished out onto the 150-mesh cup rum grids with formvar film. After that, the tissue sections were stained with 2% uranium and 2.6% Lead citrate and observed under TEM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Branched-Chain Amino Acids Targeted Quantitative Analysis\u003c/h2\u003e\u003cp\u003eFrozen serum was thawed at 4\u0026deg;C, then internal standard was added and AccQ Tag amino acid derived reagent was used to prepare samples to be tested. Chromatographic separation was performed using Waters ACQUITY UPLC I-CLASS ultra-performance liquid chromatography. The column model was Waters UPLC HSS T3 (1.7 \u0026micro;m, 2.1 mm\u0026times;150 mm). The mobile phases were 0.1% formic acid (phase A) and acetonitrile (phase B), respectively. Mass spectrometry was then performed using a Waters XEVO TQ-S tandem quadrupole mass spectrometry system. The positive ion source voltage was 1.5kV and the cone voltage was 20V. The desolvation temperature was 600℃, and the desolvation gas flow rate was 1000 L/h. The gas flow rate of the cone hole was 10L/h. The peak area of the data was calculated by MassLynx quantitative software, the retention time allowed for an error of 15s, and the quantitative results were obtained by the standard curve method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using SPSS software version 27.0. Quantitative data conforming to a normal distribution were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;s). The comparison of means between the two groups was performed using the independent samples t-test. For quantitative data not conforming to a normal distribution, the comparison between the two groups was conducted using the Mann-Whitney U test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Increased Intake of BCAAs Leads to Increased Body Weight and Blood Glucose in LDLR\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Mice\u003c/h2\u003e\n \u003cp\u003eIn this study, dietary interventions were conducted on the mice as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. Food intake data of mice are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. In terms of body weight, those fed a high-fat diet (HF, LB, and HB group) were heavier than those on a standard diet (C group). Specifically, the mice of the HB group were heavier than those of the HF and LB groups (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). High-fat feeding induced an increase in blood glucose levels, and the HB group showed significantly higher fasting blood glucose levels compared to the other groups (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Content of Branched-Chain Amino Acids in Serum\u003c/h2\u003e\n \u003cp\u003eThe serum total BCAAs in HF, LB, and HB groups fed with a high-fat diet were about 40% higher than those in the control group fed with a standard diet (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). However, there were no statistical differences in leucine, isoleucine, and valine among HF, LB, and HB groups (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE, F).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Divergent Impacts of BCAAs Intake at Varying Concentrations on Cardiac Structure in Mice.\u003c/h2\u003e\n \u003cp\u003eFollowing a 12-week intervention with high-fat diets with different doses of BCAAs, the results showed a higher average corrected left ventricular mass (LV Mass) of high-fat fed mice (HF, LB, and HB group) relative to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Prolonging of the intervention for 24 weeks elevated the corrected LV Mass in LB group than that of the HF group. No significant differences in the corrected LV Mass were observed among mice fed with high-fat diets when comparing short-term to long-term BCAAs intervention periods (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The difference between the control group at 12 and 24 weeks, may be due to the physiological ventricular hypertrophy that occurred in the mice due to the longer rearing time.\u003c/p\u003e\n \u003cp\u003eWe examined ventricular wall thickness at the end of systole in the heart. The LB group was significantly higher than the HB group for the left ventricular anterior wall thickness at end-systole (LVAW-s) at 24 weeks (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). For the left ventricular posterior wall thickness at end-systole (LVPW-s) at 12 weeks (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), the LB group was higher than HB group, too. When the intervention time was prolonged from 12 weeks to 24 weeks, the ventricular wall thickness at the end of the systole of the LB group increased most significantly.\u003c/p\u003e\n \u003cp\u003eThe ventricular anterior and posterior wall thickness at end-diastole ((LVAW-d and LVPW-d ) was also examined. For 12 weeks intervention, the LVPW-d of the HB group was thinner than the HF group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE) and 15% lower than the LB group. While for 24 weeks intervention, both LVAW-d (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD) and LVPW-d (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE) the LB group were higher than the HB group, 14% and 13% respectively. At end-diastole, the ventricular wall thickness became thinner in the HB group and thicker in the LB group. However, no significant differences were observed between the groups under short-term and long-term intervention.\u003c/p\u003e\n \u003cp\u003eAfter 12 weeks and 24 weeks of BCAAs intervention, the small animal ultrasound was detected, and the results of the two stages were compared. A: Corrected LV Mass: corrected left ventricular mass. B: LVAW-s: left ventricular anterior wall thickness at end-systole. C: LVPW-s: left ventricular posterior wall thickness at end-systole. D:\u003c/p\u003e\n \u003cp\u003eLVAW-d: left ventricular anterior wall thickness at end-diastole. E: LVPW-d: left ventricular posterior wall thickness at end-diastole. The symbolic meaning of the analysis results is the same as in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe increased ventricular wall thickness observed in the LB group and the decreased thickness in the HB group indicate that intake of BCAAs at varying concentrations can induce structural remodeling of the murine heart.\u003c/p\u003e\n \u003cp\u003eThe internal diameter and volume of the left ventricle reflect the cardiac structure and are crucial for evaluating the heart\u0026apos;s pumping efficiency. After a 12-week intervention period, a comparative analysis between the HB group and the HF group demonstrated an enlargement in both the left ventricular systolic (LVSD) and diastolic (LVDD) internal diameters (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), 15% and 9% respectively. Correspondingly, there was a notable increase in the left ventricular end-systolic volume (LVESV) and end-diastolic volume (LVEDV) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). These observations implied that an elevated intake of BCAAs over the short term can result in the dilation of the ventricular chamber and induce structural modifications within the heart. For long-term intervention, aside from the HB group\u0026apos;s LVDD exceeding the LB group\u0026apos;s, no significant differences in dimensions or volume were noted. These results indicated that BCAAs surplus may augment cardiac volume for short-term intervention. Moreover, no statistical variances in left ventricular dimensions and volume were detected within groups between the 12-week and 24-week interventions. This suggests a potential stabilization or reversal of structural adaptations over time, as the heart may achieve a new equilibrium through intrinsic mechanisms.\u003c/p\u003e\n \u003cp\u003eIn summary, the influences of elevated BCAAs intake on cardiac architecture were markedly more evident in the short term, as indicated by decreased ventricular wall thickness and increased internal diameter and volume. The effects of reducing BCAAs intake are predominantly reflected in the thickening of the murine ventricular wall. The results of the intra-group comparison showed that the prolongation of the intervention period mainly increased the systolic ventricular wall thickness.\u003c/p\u003e\n \u003cp\u003eAfter 12 weeks and 24 weeks of BCAAs intervention, the small animal ultrasound was detected, and the results of the two stages were compared. A: LVSD: left ventricular systolic internal diameters. B: LVDD: left ventricular diastolic internal diameters. C: LVESV: left ventricular end-systolic volume. D: LVEDV: left ventricular end-diastolic volume. The symbolic meaning of the analysis results is the same as in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 The Impact of Varying BCAAs Intake on Murine Cardiac Function\u003c/h2\u003e\n \u003cp\u003eWe then detected the cardiac functions. For 12-week intervention, a tendency for decreased heart rate was noted across HF, LB, HB groups feeding with high-fat diets (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Interestingly, the trend reversed when the intervention was extended to 24 weeks (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The stroke volume of the mice exhibited no statistical differences in all groups under either short-term or long-term intervention (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Cardiac Output (CO), calculated as the product of heart rate and stroke volume, was consistent with the trend of heart rate (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eEjection Fraction (EF) and Fractional Shortening (FS) are pivotal indicators of cardiac systolic function. In our study, a 12-week short-term dietary intervention resulted in a decline in EF (15%) and FS (17%) within the HB group in contrast to the HF group. After a prolonged intervention, the trend was reversed, too. In contrast, a decreased intake of BCAAs does not appear to affect cardiac contractility, regardless of the intervention duration being short or long (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\n \u003cp\u003eCardiac function was measured by echocardiography at 12 and 24 weeks after BCAAs intervention, and the results of the two stages were compared. The detection results presented in figures A-E are shown in the figure notes. The calculation of each index is described in the Materials and Methods section. The symbolic meaning of the analysis results is the same as in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Diverse Impacts of Varying BCAAs Concentrations on Myocardial Tissue in Mice\u003c/h2\u003e\n \u003cp\u003eAll mice were sacrificed after 24 weeks of intervention and cardiac organs were collected. We performed a comprehensive evaluation of the macroscopic morphology and histological features of cardiac tissues from mice across different experimental groups. Hearts from mice fed the high-fat diets (HF, LB, HB group) were observed to be larger compared to those from the C group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Conversely, the heart weight to body weight ratio was found to be increased in the LB group relative to the HF and HB groups, indicating that a decreased intake of BCAAs was associated with an increase in the heart weight (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eCardiac transmission electron microscopy (TEM) findings delineated the structural changes across the experimental groups. The control group (C) displayed orderly myofibrillar alignment and mitochondria with preserved architecture and intact cristae (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). The HF group presented with mild cellular distention, yet without overt damage to either myofibrils or mitochondria. The LB group exhibited moderate cytosolic edema, myofibrillar disarray, and mitochondrial swelling accompanied by fragmented cristae. The HB group was characterized by sparse myofiber arrangement, indistinct intercalated discs, and the emergence of mega-mitochondria, along with lipid droplets interspersed among mitochondria, suggesting perturbations in the lipid metabolic processes within cardiomyocytes.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eSome studies have shown that increased intake of BCAAs or defects in BCAAs\u0026rsquo; metabolism is associated with the onset and exacerbation of cardiovascular diseases\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, is reduced intake of BCAAs associated with a protective effect against cardiovascular diseases? In this study, we demonstrated that both increased and decreased levels of BCAAs can cause damage to the heart.\u003c/p\u003e\u003cp\u003eLow-density lipoprotein receptor (LDLR) knockout mice can develop hypercholesterolemia after being fed a high-fat diet, so they are often used as a model for cardiovascular disease research. We found that LDLR\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on a diet enriched with BCAAs experienced weight gain. The link between BCAAs and obesity was also highlighted by a previous study\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The mechanism could be that BCAAs catabolism is associated with adipocyte differentiation and lipogenesis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Conversely, a diminished intake of BCAAs did not culminate in weight reduction in our study. Increasing BCAAs intake has been identified as a precipitating factor for type 2 diabetes\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Despite this, reducing BCAAs intake did not lower blood glucose levels in our study. The analogous results in body weight and blood glucose suggested that reducing BCAA intake did not protect against obesity and type 2 diabetes.\u003c/p\u003e\u003cp\u003eIn this study, the concentration of BCAAs in serum was significantly higher in mice fed the high-fat diet than in those fed the standard diet, which may be caused by obesity. Several previous studies have shown that serum BCAAs concentrations are elevated in obese individuals and that dietary BCAAs intake is positively associated with obesity\u003csup\u003e[\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. However, there was no significant difference in serum BCAAs levels among the three groups of obese mice, even though their diets contained different concentrations of BCAAs. This may be related to the fact that we starved the mice for 8 hours before sacrifice, which caused the serum BCAAs levels to return to baseline. In healthy adults, serum BCAA levels increased immediately after BCAA intake was raised, reached their peak at 30 minutes post-meal, and returned to baseline 3 hours after the meal\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our study, both increased and decreased BCAA intake resulted in cardiac structural damage, and the symptoms appeared sooner in mice that consumed more BCAAs. With longer intervention, the effects of diminished BCAA intake were more significant. Interestingly, higher and lower BCAAs intake exerted different impacts on cardiac structure. Reduced BCAAs consumption was associated with the development of cardiac edema and a constriction of the internal diameter, while increased BCAAs consumption results in myocardial thinning and dilation of the internal diameter. Correspondingly, the results of tissue staining and electron microscopy also showed that the myocardial fibers were swollen or loose. This suggests that increasing and decreasing BCAAs may damage the heart through different mechanisms.\u003c/p\u003e\u003cp\u003eRegarding cardiac function, only a 12-week increase in BCAAs intake led to changes in ejection fraction and fractional shortening. Post 24 weeks\u0026rsquo; intervention, an amelioration in cardiac function was noted in mice with elevated BCAAs intake, indicative of compensatory improvements in cardiac performance. The enlargement of the ventricular chamber and the absence of a corresponding increase in cardiac output indicate a decrease in cardiac contractility. By observing the myocardial tissue, we found that the myocardial fibers in the HB group were loose, which explained the cause of the decreased myocardial contractility.\u003c/p\u003e\u003cp\u003eSeveral studies have reported that BCAAs affect lipid metabolism. The metabolism of valine yields 3-hydroxyisobutyrate (3-HIB), which enhances endothelial fatty acid transport, facilitates muscle fatty acid uptake in \u003cem\u003evivo\u003c/em\u003e, and encourages lipid deposition in muscle tissues\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Leucine is known to upregulate the expression of genes involved in lipid synthesis and serum fatty acid levels, thereby impacting lipid metabolism\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Deficiency of BCAAs leads to a blocked pathway of fatty acids into mitochondria\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Our study corroborated these findings, with the observation of lipid droplets in the mitochondria of cardiac muscle cells under conditions of high BCAAs intake. Then we explored the association of heart failure with BCAAs and their metabolites using bioinformatics techniques based on the GENE EXPRESSION OMNIBUS (GEO) database. The results showed that several genes related to BCAAs metabolism were up-regulated in heart failure mice, and fatty acid metabolism was the most significantly affected pathway (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExtensive research has detailed the mechanisms by which elevated levels of BCAAs and their metabolites result in cardiac damage. Activation of the mammalian target of rapamycin (mTOR) signaling by BCAAs resulted in augmented protein translation\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, mitochondrial dysfunction\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, perturbations in redox balance\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and post-myocardial infarction cardiac dysfunction and remodeling\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Pharmacologic activation of BCAAs catabolism was protective in models of heart failure. However, reduced plasma and cardiac BCAA levels do not offer significant protection, suggesting that other mechanisms may be at play\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, the mechanism of cardioprotection has remained unclear. Our study revealed that diminished BCAAs intake predominantly leads to cardiac edema, aligning with observations of increased heart weight and ventricular wall thickening. We offered a histological interpretation of the mechanisms by which low BCAAs intake induced cardiac injury. Additional exploration is necessary for a comprehensive understanding.\u003c/p\u003e\u003cp\u003eThere are a few limitations of the present work. While our research encompassed a 24-week observational time frame and identified substantial changes in cardiac architecture, the study did not record overt heart failure, resulting in a lack of significant inter-group disparities in cardiac functionality. The use of a murine model in our investigation might have introduced interspecies discrepancies that could affect the study outcomes. Additionally, our research only offered an initial foray into the mechanisms responsible for the cardiac structural variations associated with different BCAAs dosages. A comprehensive elucidation of these mechanisms requires further in-depth investigation, especially the impact of reduced BCAAs consumption.\u003c/p\u003e\u003cp\u003eIn conclusion, the intake of BCAAs at both elevated and reduced levels induces detrimental effects on the cardiac health of heart failure mice. Reduced BCAAs consumption is associated with the development of cardiac edema and a constriction of the internal diameter, while increased BCAAs consumption results in myocardial thinning and dilation of the internal diameter. Therefore, individuals with cardiac conditions is cautioned against the indiscriminate reduction of BCAAs in their diet without a thorough evaluation of the potential hazards.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e Data is provided within the manuscript or supplementary information files. The data links from the GEO database are as follows:\u003c/p\u003e\n\u003cp\u003eGSE136308: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136308\u003c/p\u003e\n\u003cp\u003eGSE137442: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137442\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments: \u003c/strong\u003eWe thank the associate editor and the reviewers for their useful feedback that improved this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding: \u003c/strong\u003eThis research was funded by Basic and Translational Research on Nutritional Diet and Atherosclerosis Based on Cohort Study, grant number ZF2023019. The APC was funded by the Finance Department of Hebei Province. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eConceptualization, Wenhua Ling and Yandong Deng; Methodology and writing, Qingxia Li; Visualization, Fen Chen; Software, Xian Gao, Huaxing Zhang and Ziyi Liu; Supervision, Xian Gao; Validation, Tianyi Zhao, Shiyao Liu, Ziyue Tang, Yuanyuan Liu, Tianao Ling, Yang Liu, Chunfei Dang, Yili Xu; Funding acquisition, Yandong Deng and Yuxia Ma; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eYandong Deng and Wenhua Ling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests: \u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement: \u003c/strong\u003eThe animal study protocol was approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University ( Approval No. IACUC-Hebmu-2024045).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLe Couteur DG, Solon-Biet SM, Cogger VC, Ribeiro R, de Cabo R, Raubenheimer D, Cooney GJ, Simpson SJ. Branched chain amino acids, aging and age-related health. \u003cstrong\u003eAgeing Res Rev\u003c/strong\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 64: 101198.\u003c/li\u003e\n\u003cli\u003eNeinast M, Murashige D, Arany Z. Branched chain amino acids. \u003cstrong\u003eAnnu Rev Physiol\u003c/strong\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 81(1): 139-164.\u003c/li\u003e\n\u003cli\u003eMcgarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease. \u003cstrong\u003eNat Rev Cardiol\u003c/strong\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 20(2): 77-89.\u003c/li\u003e\n\u003cli\u003eSun H, Olson KC, Gao C, Prosdocimo DA, Zhou M, Wang Z, Jeyaraj D, Youn J, Ren S, Liu Y, Rau CD, Shah S, Ilkayeva O, Gui W, William NS, Wynn RM, Newgard CB, Cai H, Xiao X, Chuang DT, Schulze PC, Lynch C, Jain MK, Wang Y. Catabolic defect of branched-chain amino acids promotes heart failure. \u003cstrong\u003eCirculation (New York, N.Y.)\u003c/strong\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 133(21): 2038-2049.\u003c/li\u003e\n\u003cli\u003eMurashige D, Jung JW, Neinast MD, Levin MG, Chu Q, Lambert JP, Garbincius JF, Kim B, Hoshino A, Marti-Pamies I, Mcdaid KS, Shewale SV, Flam E, Yang S, Roberts E, Li L, Morley MP, Bedi KC, Hyman MC, Frankel DS, Margulies KB, Assoian RK, Elrod JW, Jang C, Rabinowitz JD, Arany Z. Extra-cardiac bcaa catabolism lowers blood pressure and protects from heart failure. \u003cstrong\u003eCell Metab\u003c/strong\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 34(11): 1749-1764.\u003c/li\u003e\n\u003cli\u003eLi T, Zhang Z, Kolwicz SC, Abell L, Roe ND, Kim M, Zhou B, Cao Y, Ritterhoff J, Gu H, Raftery D, Sun H, Tian R. Defective branched-chain amino acid catabolism disrupts glucose metabolism and sensitizes the heart to ischemia-reperfusion injury. \u003cstrong\u003eCell Metab\u003c/strong\u003e \u003cstrong\u003e2017\u003c/strong\u003e, 25(2): 374-385.\u003c/li\u003e\n\u003cli\u003eLi R, He H, Fang S, Hua Y, Yang X, Yuan Y, Liang S, Liu P, Tian Y, Xu F, Zhang Z, Huang Y. Time series characteristics of serum branched-chain amino acids for early diagnosis of chronic heart failure. \u003cstrong\u003eJ Proteome Res\u003c/strong\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 18(5): 2121-2128.\u003c/li\u003e\n\u003cli\u003eWang W, Zhang F, Xia Y, Zhao S, Yan W, Wang H, Lee Y, Li C, Zhang L, Lian K, Gao E, Cheng H, Tao L. Defective branched chain amino acid catabolism contributes to cardiac dysfunction and remodeling following myocardial infarction. \u003cstrong\u003eAm J Physiol-Heart C\u003c/strong\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 311(5): H1160-H1169.\u003c/li\u003e\n\u003cli\u003eUddin GM, Zhang L, Shah S, Fukushima A, Wagg CS, Gopal K, Al BR, Pherwani S, Ho KL, Boisvenue J, Karwi QG, Altamimi T, Wishart DS, Dyck J, Ussher JR, Oudit GY, Lopaschuk GD. Impaired branched chain amino acid oxidation contributes to cardiac insulin resistance in heart failure. \u003cstrong\u003eCardiovasc Diabetol\u003c/strong\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 18(1): 86.\u003c/li\u003e\n\u003cli\u003eSatomi S, Morio A, Miyoshi H, Nakamura R, Tsutsumi R, Sakaue H, Yasuda T, Saeki N, Tsutsumi YM. Branched-chain amino acids-induced cardiac protection against ischemia/reperfusion injury. \u003cstrong\u003eLife Sci\u003c/strong\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 245: 117368.\u003c/li\u003e\n\u003cli\u003eBj\u0026ouml;rkegren JLM, Lusis AJ. Atherosclerosis: recent developments. \u003cstrong\u003eCell\u003c/strong\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 185(10): 1630-1645.\u003c/li\u003e\n\u003cli\u003eTzoulaki I, Castagne R, Boulange CL, Karaman I, Chekmeneva E, Evangelou E, Ebbels T, Kaluarachchi MR, Chadeau-Hyam M, Mosen D, Dehghan A, Moayyeri A, Ferreira D, Guo X, Rotter JI, Taylor KD, Kavousi M, de Vries PS, Lehne B, Loh M, Hofman A, Nicholson JK, Chambers J, Gieger C, Holmes E, Tracy R, Kooner J, Greenland P, Franco OH, Herrington D, Lindon JC, Elliott P. Serum metabolic signatures of coronary and carotid atherosclerosis and subsequent cardiovascular disease. \u003cstrong\u003eEur Heart J\u003c/strong\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 40(34): 2883-2896.\u003c/li\u003e\n\u003cli\u003eVanweert F, Schrauwen P, Phielix E. Role of branched-chain amino acid metabolism in the pathogenesis of obesity and type 2 diabetes-related metabolic disturbances bcaa metabolism in type 2 diabetes. \u003cstrong\u003eNutr Diabetes\u003c/strong\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 12(1): 35.\u003c/li\u003e\n\u003cli\u003eJersin RA, Sri PTD, Skartveit L, Bjune MS, Muniandy M, Lee-Odegard S, Heinonen S, Alvarez M, Birkeland KI, Andre DC, Pajukanta P, Mccann A, Pietilainen KH, Claussnitzer M, Mellgren G, Dankel SN. Impaired adipocyte slc7a10 promotes lipid storage in association with insulin resistance and altered bcaa metabolism. \u003cstrong\u003eJ Clin Endocrinol Metab\u003c/strong\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 108(9): 2217-2229.\u003c/li\u003e\n\u003cli\u003eGreen CR, Wallace M, Divakaruni AS, Phillips SA, Murphy AN, Ciaraldi TP, Metallo CM. Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis. \u003cstrong\u003eNat Chem Biol\u003c/strong\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 12(1): 15-21.\u003c/li\u003e\n\u003cli\u003eWhite PJ, Mcgarrah RW, Herman MA, Bain JR, Shah SH, Newgard CB. Insulin action, type 2 diabetes, and branched-chain amino acids: a two-way street. \u003cstrong\u003eMol Metab\u003c/strong\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 52: 101261.\u003c/li\u003e\n\u003cli\u003eFelig P, Marliss E, Cahill GJ. Plasma amino acid levels and insulin secretion in obesity. \u003cstrong\u003eN Engl J Med\u003c/strong\u003e \u003cstrong\u003e1969\u003c/strong\u003e, 281(15): 811-816.\u003c/li\u003e\n\u003cli\u003eZhang Y, Rao S, Zhang X, Peng Z, Song W, Xie S, Cao H, Zhang Z, Yang W. Dietary and circulating branched chain amino acids are unfavorably associated with body fat measures among chinese adults. \u003cstrong\u003eNutr Res\u003c/strong\u003e \u003cstrong\u003e2024\u003c/strong\u003e, 128: 94-104.\u003c/li\u003e\n\u003cli\u003eAsoudeh F, Salari-Moghaddam A, Keshteli AH, Esmaillzadeh A, Adibi P. Dietary intake of branched-chain amino acids in relation to general and abdominal obesity. \u003cstrong\u003eEat Weight Disord\u003c/strong\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 27(4): 1303-1311.\u003c/li\u003e\n\u003cli\u003eLu J, Gu Y, Liu H, Wang L, Li W, Li W, Leng J, Zhang S, Qi L, Yang X, Hu G. Daily branched-chain amino acid intake and risks of obesity and insulin resistance in children: a cross-sectional study. \u003cstrong\u003eObesity (Silver Spring)\u003c/strong\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 28(7): 1310-1316.\u003c/li\u003e\n\u003cli\u003eZhao S, Zhou L, Wang Q, Cao JH, Chen Y, Wang W, Zhu BD, Wei ZH, Li R, Li CY, Zhou GY, Tan ZJ, Zhou HP, Li CX, Gao HK, Qin XJ, Lian K. Elevated branched-chain amino acid promotes atherosclerosis progression by enhancing mitochondrial-to-nuclear h(2)o(2)-disulfide hmgb1 in macrophages. \u003cstrong\u003eRedox Biol\u003c/strong\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 62: 102696.\u003c/li\u003e\n\u003cli\u003eNewton-Tanzer E, Can SN, Demmelmair H, Horak J, Holdt L, Koletzko B, Grote V. Apparent saturation of branched-chain amino acid catabolism after high dietary milk protein intake in healthy adults. \u003cstrong\u003eJ Clin Endocrinol Metab\u003c/strong\u003e \u003cstrong\u003e2024\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eJang C, Oh SF, Wada S, Rowe GC, Liu L, Chan MC, Rhee J, Hoshino A, Kim B, Ibrahim A, Baca LG, Kim E, Ghosh CC, Parikh SM, Jiang A, Chu Q, Forman DE, Lecker SH, Krishnaiah S, Rabinowitz JD, Weljie AM, Baur JA, Kasper DL, Arany Z. A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance. \u003cstrong\u003eNat Med\u003c/strong\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 22(4): 421-426.\u003c/li\u003e\n\u003cli\u003eZhou X, Chen J, Sun B, Wang Z, Zhu J, Yue Z, Zhang Y, Shan A, Ma Q, Wang J. Leucine, but not isoleucine or valine, affects serum lipid profiles and browning of wat in mice. \u003cstrong\u003eFood Funct\u003c/strong\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 12(15): 6712-6724.\u003c/li\u003e\n\u003cli\u003eGotvaldova K, Spackova J, Novotny J, Baslarova K, Jezek P, Rossmeislova L, Gojda J, Smolkova K. Bcaa metabolism in pancreatic cancer affects lipid balance by regulating fatty acid import into mitochondria. \u003cstrong\u003eCancer Metab\u003c/strong\u003e \u003cstrong\u003e2024\u003c/strong\u003e, 12(1): 10.\u003c/li\u003e\n\u003cli\u003eLatimer MN, Sonkar R, Mia S, Frayne IR, Carter KJ, Johnson CA, Rana S, Xie M, Rowe GC, Wende AR, Prabhu SD, Frank SJ, Rosiers CD, Chatham JC, Young ME. Branched chain amino acids selectively promote cardiac growth at the end of the awake period. \u003cstrong\u003eJ Mol Cell Cardiol\u003c/strong\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 157: 31-44.\u003c/li\u003e\n\u003cli\u003eVanderstad LR, Wyatt EC, Vaughan RA. Excess branched-chain amino acids suppress mitochondrial function and biogenic signaling but not mitochondrial dynamics in a myotube model of skeletal muscle insulin resistance. \u003cstrong\u003eMetabolites\u003c/strong\u003e \u003cstrong\u003e2024\u003c/strong\u003e, 14(7).\u003c/li\u003e\n\u003cli\u003eSun H, Olson KC, Gao C, Prosdocimo DA, Zhou M, Wang Z, Jeyaraj D, Youn JY, Ren S, Liu Y, Rau CD, Shah S, Ilkayeva O, Gui WJ, William NS, Wynn RM, Newgard CB, Cai H, Xiao X, Chuang DT, Schulze PC, Lynch C, Jain MK, Wang Y. Catabolic defect of branched-chain amino acids promotes heart failure. \u003cstrong\u003eCirculation\u003c/strong\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 133(21): 2038-2049.\u003c/li\u003e\n\u003cli\u003eMurashige D, Jung JW, Neinast MD, Levin MG, Chu Q, Lambert JP, Garbincius JF, Kim B, Hoshino A, Marti-Pamies I, Mcdaid KS, Shewale SV, Flam E, Yang S, Roberts E, Li L, Morley MP, Bedi KJ, Hyman MC, Frankel DS, Margulies KB, Assoian RK, Elrod JW, Jang C, Rabinowitz JD, Arany Z. Extra-cardiac bcaa catabolism lowers blood pressure and protects from heart failure. \u003cstrong\u003eCell Metab\u003c/strong\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 34(11): 1749-1764.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"branched-chain amino acids, cardiac structure, cardiac function, heart failure","lastPublishedDoi":"10.21203/rs.3.rs-6995118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6995118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e\u003cp\u003eElevation of dietary branched-chain amino acids (BCAAs) is regarded as a risk factor for heart failure. However, exposure to lower or higher levels of BCAAs on cardiac structure and function remained unclear.\u003c/p\u003e\u003ch2\u003eMthods:\u003c/h2\u003e\u003cp\u003eThe male C57BL/6J LDLR\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were divided into 4 groups (n\u0026thinsp;=\u0026thinsp;6) as feeding standard diet, high-fat diet, high-fat diet with low BCAAs, and high-fat diet with high BCAAs for 24 weeks. They underwent echocardiography after 12 and 24 weeks of dietary intervention. Then the mice were sacrificed and the heart organs were harvested for histological examination.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe results showed that low or high nutritional BCAAs intake possessed different phenotypes on cardiac structure and function. Reduced BCAAs intake caused edema and a smaller inner diameter of the hearts in mice, while high BCAAs intake caused thinning and a larger inner diameter of the hearts. Pathological results showed that reducing the intake of BCAAs caused swelling of cardiomyocytes, loose fibers, and fragmentation of myocardial mitochondria. In contrast, higher intake of BCAAs increased the formation of lipid droplets and giant mitochondria.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eEither decreased or increased dietary BCAAs intake induced negative effects on heart structure and function. The present research findings help the understanding of nutritional BCAAs intake at low or high levels related to pathological changes in the heart.\u003c/p\u003e","manuscriptTitle":"Lower and higher dietary intake of branched-chain amino acids impact cardiac structure and functions in high-fat fed LDLR -/- mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 12:01:49","doi":"10.21203/rs.3.rs-6995118/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6e647d0b-56ab-447f-aff2-2f584da52d58","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T15:06:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 12:01:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6995118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6995118","identity":"rs-6995118","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}