Abstract Physical exercise is a cornerstone for preventing diet-induced obesity, while it is unclear whether physical exercise could offset high-fat, high-calories diet (HFCD)-induced cardiac dysfunction. Here, mice were fed with HFCD and simultaneously subjected to physical exercise. As expected, physical exercise prevented HFCD-induced whole-body fat deposition. However, physical exercise exacerbated HFCD-induced cardiac damage. Further metabolomic analysis results showed that physical exercise induced circulating lipid redistribution, leading to excessive cardiac lipid uptake and lipotoxicity. Our study provides valuable insights into the cardiac effects of exercise in mice fed with HFCD, suggesting that counteracting the negative effect of HFCD by simultaneous physical exercise might be detrimental. Moreover, inappropriate physical exercise may damage certain organs even though it leads to weight loss and overall metabolic benefits. Of note, the current findings are based on animal experiments, the generalizability of these findings beyond this specific diet and mouse strain remains to be further explored. Subject terms: Weight management, Cardiovascular diseases __________________________________________________________________ While the metabolic benefits of physical exercise are well known, it is less clear whether exercise can prevent cardiac damage in diet-induced obesity. Here, the authors show that while exercise prevented weight gain in mice, it surprisingly worsened heart damage due to excessive lipid uptake and lipotoxicity. Introduction The prevalence of obesity has reached epidemic proportions worldwide during the last few decades. The epidemiological data show that more than 40% of adults in the USA were obese in 2018^[52]1. A high-calorie diet combined with a lack of physical exercise is the major cause of obesity, and leads to metabolic disorders, including hyperglycemia, hyperlipidemia, fatty liver diseases, hypertension, and cardiovascular diseases^[53]2,[54]3. Multiple studies have identified diet-induced obesity (DIO) as an independent risk factor for cardiomyopathy^[55]4,[56]5. Indeed, obesity imposes negative effect to the hearts through multiple mechanisms, including the alterations in cellular insulin sensitivity and metabolic reprogramming^[57]6–[58]8. Nutrition intervention and physical exercise are the two major approaches for the management of DIO^[59]9. Recent study showed that periods of fasting have a therapeutic effect on DIO and prevent cardiometabolic risk increasing due to high-fat diet^[60]10. Likewise, as another cornerstone for weight losing, physical exercise has also been recognized as an effective strategy for improving cardiovascular health. Clinical data showed exercise training was associated with a significantly lower risk of cardiovascular diseases, and the risk decreased by 30–40% in the most active individuals^[61]11,[62]12. Physical exercise also provided dramatic cardiac protection affection against myocardial ischemia and ischemia/reperfusion(I/R) injury in rodent models^[63]13,[64]14. It is worth mentioning that specific intensities of physical exercise to produce health benefits remains controversial. Flockhart and colleagues reported that mitochondrial function was impaired in the muscle homogenate from healthy volunteers who underwent excessive physical training, and this impairment coincided with disrupted glucose tolerance and insulin secretion^[65]15. In addition, vigorous intensity exercise was reported to be associated with an increased progression of coronary artery calcification and atherosclerotic plaques^[66]16. These evidences suggested that excessive or prolonged exercise may have deleterious effects on the cardiovascular system. Although physical exercise is now a popular non-pharmacologic approach to avoid DIO, it is still unclear whether it would also offer a cardiac protective effect while combating weight gain. In this work, we explore the whole-body and cardiac effects of physical exercise of different intensities in high-fat, high-calories diet (HFCD)-fed mice. As expected, three different intensities of physical exercise prevent HFCD-induced weight gain and fat deposition in the white adipose tissue (WAT) and the liver. However, a striking further reduction of cardiac function and abnormal cardiac lipid accumulation are observed in HFCD-fed mice subjected to moderate or higher intensity exercise. Further metabolic analysis results show that physical exercise induces circulating lipid redistribution from WAT and liver to the myocardium, leading to excessive cardiac lipid uptake and further cardiac lipotoxicity. Our results indicates that although physical exercise is able to prevent diet-induced weight gain, it might be not a feasible strategy to offset HFCD-induced cardiac damage. Results MIE prevented obesity and metabolic syndrome in HFCD-fed mice Wild-type C57BL/6J mice were fed ad libitum a chow diet (CD; 10% calories from fat, nutritional composition and ingredient list see Tables [67]S1 and [68]2) or an HFCD (60% calories from fat, nutritional composition and ingredient list see Tables [69]S1 and [70]2) for 8 weeks. CD- or HFCD-fed 8-weeks-old wild-type male C57BL/6J mice were randomly divided into sedentary (Sed) and moderate-intensity exercise (MIE) groups to test the effects of physical exercise on them (Fig. [71]1A). The MIE protocol was based on the previously reported protocols showing cardioprotective effect^[72]17. Serum corticosterone levels and lactate level were determined to reflect the stress state and fatigued state in mice of each group. As shown in the Fig. [73]S1A, B, MIE induced significant elevation of serum corticosterone levels and lactate level in both CD- and HFCD-fed mice, suggesting that MIE was able to induce indicating a stressful and fatigue condition. Serum lactate level of each mouse was listed in the Table [74]S9. Food intake and calories intake of mice was recorded and analyzed in each group. As shown in the Fig. [75]S3, there was no significant difference in the content of food intake in CD- and HFCD-fed sedentary mice, while HFCD-fed sedentary mice eat more calories than CD-fed sedentary mice. HFCD feeding induced a more significant time-dependent alteration in body weight compared with CD feeding, indicating the development of obesity in C57BL/6J mice (Fig. [76]1B, C and Table [77]S3). HFCD-fed mice also showed significantly higher concentrations of blood glucose, serum triglycerides (TAG), total cholesterol (Chol), HDL, and LDL and an increase in the weight of WAT compared with CD-fed mice (Fig. [78]1D–J and Table [79]S3). Although MIE elevated the content of food intake and calories intake of CD- and HFCD-fed mice, mice subjected to MIE showed significantly decreased body weight and blood glucose, TAG, and Chol concentrations (Fig. [80]1C–G and Table [81]S3). Therefore, MIE prevented the development of obesity, hyperglycemia, and hyperlipidemia in HFCD-fed mice .Moreover, MIE also diminished the weight of WAT in HFCD-fed mice (Fig. [82]1J). Overall, these findings suggested that MIE was sufficient to prevent DIO. Fig. 1. Moderate-intensity exercise (MIE) prevented obesity and metabolic syndrome in HFCD-fed mice. [83]Fig. 1 [84]Open in a new tab A Experimental design: C57BL/6J mice were fed ad libitum a normal chow diet (CD, 10% calories from fat) or an HFCD diet (60% calories from fat) for 8 weeks. CD and HFCD-fed mice were randomly divided into sedentary (Sed) and MIE groups. All elements of this image sourced by figdraw.com. B Body weight changes in CD- and HFCD-fed mice (n = 10 per group). C Final body weight of 16-week-old mice (n = 8 per group). D Blood glucose concentrations in the indicated groups of mice (n = 6 per group). E Final blood glucose concentrations in 16-weeks-old mice (n = 6 per group). F–I Serum triglycerides (TAG) (F), total cholesterol (Chol) (G), high-density lipoprotein (HDL) (H), and low-density lipoprotein (LDL) concentrations (I) in the indicated groups of mice (n = 6 per group). J Weight of white adipose tissue (WAT) (n = 6 per group). K, L Results of intraperitoneal glucose tolerance test (K), and glucose area under the curve (AUC) analysis during 120 min of follow-up (L) (n = 6 per group). M, N Results of intraperitoneal insulin tolerance test (M), and area under the curve (AUC) analysis during 120 min of follow-up (N) (n = 6 per group). O Serum insulin level in the indicated groups of mice (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file. DIO induces impaired insulin sensitivity in rodent models^[85]18. In this study, insulin sensitivity was determined using an intraperitoneal glucose tolerance test and insulin stimulation experiments. Eight weeks of HFCD feeding impaired insulin sensitivity, as indicated by increased area under the curve (AUC) in IPGTT and ITT test (Fig. [86]1K–N). However, the data showed that the AUC in IPGTT and ITT test showed no significant change in HFCD-fed mice subjected to MIE, indicating that MIE could not improve insulin sensitivity in HFCD-fed mice. Serum insulin level was also determined. As shown in Fig. [87]1O, when compared with that of CD-fed sedentary mice, serum insulin level of HFCD-fed sedentary mice significantly increased. MIE decreased the serum insulin level in the HFCD-fed mice. Taken together, these data suggested that MIE was able to prevent DIO and metabolic syndrome in HFCD-fed mice. MIE did not improve cardiac function in HFCD-fed mice and exacerbated HFCD-induced cardiac dysfunction Since our results revealed that MIE prevented the development of whole-body metabolic disorders, we further determined whether MIE could protect against HFCD-induced cardiac dysfunction. Cardiac function was detected using echocardiograph after 4 or 8 weeks of MIE. Figure [88]2A, B showed that sedentary mice who were fed HFCD for 4 weeks showed no change in cardiac function indicators compared with those who were fed CD, indicating optimal cardiac function. However, mice who were fed HFCD for 8 weeks showed impaired cardiac diastolic and systolic function (Fig. [89]2A, B and Table [90]S4), and MIE exacerbated HFCD-induced cardiac dysfunction. Compared with sedentary HFCD-fed mice, HFCD-fed mice who underwent 4 weeks of MIE showed a decreased E/A ratio, indicating impaired cardiac diastolic function (Fig. [91]2A, B). Moreover, the cardiac function impairment was aggravated in HFCD-fed mice who underwent 8 weeks of MIE. In contrast, MIE showed no significant effect on cardiac function in CD-fed mice. Further, a PV catheter was used to accurately detect the cardiac function, and the results were consistent with those obtained using echocardiography. Compared with CD-fed sedentary mice, the dp/dt[min] and ESPVR were significantly decreased and Tau and EDPVR were increased in HFCD-fed sedentary mice, suggesting impaired cardiac diastolic and systolic function (Fig. [92]2C–L and Table [93]S4). MIE further decreased dp/dt[min], dp/dt[max], and ESPVR, and increased Tau and EDPVR in these mice, suggesting an increased impairment of cardiac function (Fig. [94]2C–L and Table [95]S4). In contrast, cardiac function was unaffected in sedentary mice who underwent MIE (Fig. [96]2C–L and Table [97]S4). A greater exercise capacity relies on a better cardiac function. Hence, we performed additional experiments to detect exercise performance. As shown in the Fig. [98]S1C, D, the exercise performance in the HFCD-fed sedentary mice was significantly impaired, as indicated by decreased run distance and max run speed. In contrast, MIE showed a trend toward improvement in exercise performance CD-fed mice, while the difference was not statistically significant. However, MIE led to further impaired exercise performance in HFCD-fed mice. Taken together, these findings suggested that MIE could not prevent the development of cardiac dysfunction in HFCD-fed mice. Unlike the previously reported protective effect of MIE on ischemic heart^[99]19, our findings revealed that MIE further exacerbated cardiac systolic and diastolic function impairment in HFCD-fed mice. Fig. 2. MIE did not improve cardiac function in HFCD-fed mice, instead exacerbated HFCD-induced cardiac dysfunction. [100]Fig. 2 [101]Open in a new tab A Representative Doppler flow measurement of mitral inflow and quantitative analysis of E/A ratio (n = 8 per group). B Representative images and quantitative analysis (LVEF and LVFS) of M-mode echocardiography (n = 8 per group). C Representative LV pressure–volume loop traces (n = 6 per group). D–G Representative LV pressure–volume loops at vena cava during occlusion in mice as indicated (n = 6 per group). H Maximum descending rate of LV pressure (dp/dt[min]) (n = 6 per group). I End-diastolic pressure–volume relationship as a measure of LV diastolic stiffness (n = 6 per group). J Time constant of LV pressure decay (Tau) (n = 6 per group). K Maximum rising rate of LV pressure (dp/dt[max]) (n = 6 per group). L End-systolic pressure–volume relationship as a measure of LV systolic stiffness (n = 6 per group). M Ratio of heart weight to tibia length (n = 6 per group). N Representative wheat germ agglutinin (WGA) staining images, Scale bar, 50 μm, and quantitative analysis of cell area (n = 6 per group). O Quantitative analysis of the adult cardiomyocyte length and width (n = 6 per group). P Representative Masson staining images, Scale bar, 2 mm, and quantitative analysis of interstitial fibrosis (n = 6 per group). Q Serum BNP level (n = 6 per group). R Serum ANP level (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file. Consistent with the findings of a previous study^[102]5, HFCD feeding with or without MIE increased the heart weight to tibia length ratio, cross-sectional cardiomyocytes area (CSA), and cardiomyocytes length and width, indicating cardiac hypertrophy (Fig. [103]2M–O). However, MIE further deteriorated the cardiac hypertrophy in HFCD-fed mice (Fig. [104]2M–O). Cardiac fibrosis area and pathological hypertrophy biomarker were detected to examine pathological cardiac remodeling. As shown in the Fig. [105]2P–R, HFCD-feeding significantly increased cardiac fibrosis and pathological hypertrophy biomarker in sedentary mice. Moreover, MIE further elevated indices of cardiac fibrosis and pathological hypertrophy biomarker in HFCD-fed mice. Taken together, these data suggested that MIE exacerbates HFCD-induced cardiac pathologic changes and dysfunction. High- and moderate-intensity exercise exacerbated HFCD-induced cardiomyopathy, whereas low-intensity exercise improved cardiac health We determined whether the intensity of exercise influenced the cardiac effect of exercise on HFCD-fed mice. Therefore, different groups of HFCD-fed mice were subjected to LIE, MIE, or HIE for 8 weeks (Fig. [106]3A). As shown in the Fig. [107]S2, all the three intensities of exercise induced elevation of serum corticosterone and lactate level in HFCD-fed mice. Moreover, serum corticosterone and lactate level increased as the intensity of exercise elevated. As expected, mice from HIE group showed the highest level of serum corticosterone and lactate. Fig. 3. High- and moderate-intensity exercise exacerbated HFCD-induced cardiac dysfunction, whereas low-intensity exercise improved cardiac health. [108]Fig. 3 [109]Open in a new tab A Experimental design: C57BL/6J mice were fed ad libitum a normal chow diet (CD; 10% calories from fat) or an HFCD diet (60% calories from fat) for 8 weeks. CD and HFCD-fed mice were randomly divided into low-, moderate-, or high-intensity exercise (LIE, MIE, or HIE, respectively) groups. All elements of this image sourced by figdraw.com. B Body weight changes in HFCD-fed mice (n = 8 per group). C Final body weight of 16-weeks-old mice (n = 8 per group). D Blood glucose concentrations in the indicated groups of mice (n = 6 per group). E Final blood glucose concentrations in 16-weeks-old mice (n = 6 per group). F–I Concentrations of serum triglycerides (TAG) (F), total cholesterol (Chol) (G), high-density lipoprotein (HDL) (H), and low-density lipoprotein (LDL) (I) (n = 6 per group). J Weight of white adipose tissue (WAT) (n = 6 per group). K, L Intraperitoneal glucose tolerance was tested (K) and glucose area under the curve (AUC) was calculated during the 120 min follow-up (L) (n = 6 per group). M Intraperitoneal insulin tolerance was tested and area under the curve (AUC) was calculated during the 120 min follow-up. N Serum insulin level (n = 6 per group). O, P Representative Doppler flow measurement of mitral inflow and quantitative analysis of the E/A ratio (n = 8 per group). P–R Representative images and quantitative analysis (LVEF and LVFS) of M-mode echocardiography (n = 8 per group). S Ratio of heart weight to tibia length (n = 6 per group). T Representative wheat germ agglutinin (WGA) staining images, scale bar, 50 μm, and quantitative analysis of cell area (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by two-sided unpaired t-test without adjustment. Source data are provided as a Source Data file. In HFCD-fed mice, MIE and HIE, but not LIE, significantly increased the amount of food intake, as well as the calories intake (Fig. [110]S3C, D). All the three intensities of exercise ameliorated DIO (Fig. [111]3B, C and Table [112]S5). Compared with sedentary HFCD-fed mice, those who were subjected to different exercise regimens showed a relative gentle rising trends in body weight. MIE and HIE showed comparable weight loss effect, which were both better than that of LIE (Fig. [113]3B, C). Interestingly, LIE and MIE, but not HIE was able to decrease in blood glucose in HFCD-fed mice (Fig. [114]3D–E). The serum lipid level in the HFCD-fed mice subjected to three intensity exercise was comparable, which was all lower than sedentary HFCD-fed mice (Fig. [115]3F–I). In contrast, HIE showed the most pounced effect in reducing the weight of WAT (Fig. [116]3J). Insulin sensitivity was also determined. As shown in the Fig. [117]3K–N, all the three intensities of exercise failed in improving insulin sensitivity in the HFCD-fed mice. These data suggested that all the three intensities of exercise were able to prevent the development of obesity and metabolic syndrome in HFCD-fed mice, with HIE being the most effective. Echocardiograph was performed to further determine the effect of LIE, MIE, and HIE on the cardiac function in HFCD-fed mice (Fig. [118]3O–R). MIE and HIE significantly reduced the E/A ratio, LVEF, and LVFS in HFCD-fed mice after 4 weeks of exercise, whereas these parameters remained unchanged in HFCD-fed mice after 4 weeks of LIE (Fig. [119]3O–R and Table [120]S6). HFCD-fed mice who underwent LIE for 8 weeks showed improved cardiac function. LVEF, LVFS, and E/A ratio were increased in these mice compared with sedentary mice, suggesting that LIE protected against HFCD-induced impairment in cardiac function. However, 8 weeks of MIE or HIE induced severe cardiac dysfunction in HFCD-fed mice. Moreover, MIE and HIE led to a further increase in the heart weight to tibia length ratio, cardiomyocyte CSA and cardiac fibrosis, indicating that these exercise regimens aggravated HFCD-induced pathologic cardiac hypertrophy (Figs. [121]3S–T and [122]S4). Exercise performance results also indicated that LIE significantly increased exercise performance, while MIE and HIE led to impaired exercise performance in HFCD-fed mice (Fig. [123]S2C, D). Overall, our data suggested that the cardiac effect of exercise was significantly associated with its intensity in HFCD-fed mice. LIE showed cardioprotective effects, whereas MIE and HIE aggravated cardiac injury. HFCD-fed female mice underwent MIE showed delayed onset of cardiac injury To investigate whether there are gender differences in the effect of exercise. Eight-weeks old female mice were fed with CD or HFCD and subjected to Sed or MIE. As shown in the Fig. [124]S5, consistent with our observation in male mice, 8 weeks’ HFCD led to a significant elevation in body weight, blood glucose and serum lipid level (Fig. [125]S5A–H and Table [126]S7). Insulin sensitivity was also damaged by HFCD-feeding in the female mice, as indicated by impaired IPGTT and ITT performance (Fig. [127]S5I–L). MIE prevented HFCD-induced body weight elevation. Also, blood glucose and serum lipid level in HFCD-fed female mice were significantly decreased by MIE. However, MIE failed in improving insulin sensitivity of HFCD-fed female mice. Cardiac function and cardiac hypertrophy in the female mice were also determined. Four weeks’ HFCD-feeding showed no evident impact in the systolic and diastolic function, as indicated by comparable LVEF, LVFS, and E/A ratio (Fig. [128]S6A–E and Table [129]S8). In contrast, 8 weeks’ HFCD feeding impaired cardiac diastolic function, as evidenced by decreased E/A ratio (Fig. [130]S6A, B). These observations were consistent with our results in the male mice. Different from the systolic dysfunction observed in the 4 weeks’ MIE + HFCD group male mice, 4 weeks’ MIE showed no significant impact on the cardiac systolic function in the female mice (Fig. [131]S6C, D). Cardiac function of female mice in the HFCD + MIE group remains unchanged until 8 weeks (Fig. [132]S6A–E). The detrimental impact of HFCD combined with MIE on cardiac function was significantly delayed in the female mice than that of male mice. MIE aggravated HFCD-induced cardiac lipid accumulation To investigate the potential metabolic changes in the heart which was involved in MIE-induced cardiac injury in the heart of HFCD-fed mice, cardiac tissue of HFCD-fed sedentary mice and HFCD-fed MIE mice was harvested and subjected to metabolomic analysis. A variety of metabolic substrates was changed in the heart of HFCD-fed MIE mice when compared with sedentary HFCD-fed mice (Fig. [133]4A). Among all of these metabolic substrates, lipid and lipid-like molecular accounted for a highest ratio of 36.63% (Fig. [134]4B). GSEA enrichment plot also showed that metabolic substrates change was mainly enriched in glycerophospholipid metabolism and arachidonic acid metabolism (Fig. [135]4C). Heatmap regarding the changes of specific metabolites was shown in Fig. [136]S7 and Supplementary Data [137]1. Fig. 4. MIE aggravated HFCD-induced cardiac lipid accumulation. [138]Fig. 4 [139]Open in a new tab A Volcano plot shows the differential metabolites in the heart tissue samples between the HFCD + MIE and HFCD + Sed groups. Red circles represent upregulated proteins [P  <  0.05 and log2(fold change) > 1], and blue circles indicate downregulated proteins [P  <  0.05 and log2(fold change) < –1]. B Relative proportion of differential metabolites. C Gene Set Enrichment Analysis (GSEA) of the differential metabolites. D Representative images of Bodipy 493/503 staining showing the accumulation of neutral lipid droplets (vivid green dots) in the adult cardiomyocytes, Scale bar, 2 μm (n = 6 per group). Three times experiment was repeated with similar results. E The number of lipid droplets per cell (n = 6 per group). F Area of lipid droplets per cell (n = 6 per group). G, H Representative transmission electron microscopic images of the myocardium (G) and the number of lipid droplets per μm^2 (H) Scale bar, 2 μm (n = 6 per group). I Lipid composition in the heart of mice in the indicated groups. All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file. LDs are the main intracellular organelles for lipid storage and reflect the degree of lipid accumulation^[140]20. In this study, adult cardiomyocytes were isolated from mouse hearts and stained with Bodipy 493/503 to visualize LDs. HFCD feeding increased LD deposition in mouse hearts, and MIE further increased LD accumulation in the hearts of HFCD-fed mice (Fig. [141]4D–F). Moreover, the accumulation of LDs in the mouse heart was also detected using TEM (Fig. [142]4G–H). Consistent with the Bodipy staining images, TEM images also showed that MIE aggravated the intramyocardial LD deposition in HFCD-fed mice (Fig. [143]4G–H). We further analyzed the composition of lipid in the heart of mice in the four groups. As shown in the Fig. [144]4I, lipid in the CD-fed sedentary mice was mainly PC and PE, while the TAG only accounted for a small proportion. MIE further decreased the proportion of TAG in the CD-fed mice. However, in the heart of HFCD-fed sedentary mice, TAG became the first class and accounted for 15.59%, suggesting neutral lipids deposition. MIE further increased the proportion of TAG in the heart of HFCD-fed heart. Interestingly, PE became the first class in the heart of HFCD-fed MIE mice, which might be attributed to pathological hypertrophy. TAG is the predominant form of stored lipids, whereas diglycerides (DAG) and ceramides are the most common causes of lipotoxicity. Therefore, we quantified TAGs, DAGs, cholesterol, and ceramides using LC/MS-based lipidomic analysis (Fig. [145]S8). HFCD increased the concentration of TAGs and DAG content in mice heart, whereas cholesterol and ceramide content remained unchanged. However, 8 weeks of MIE further increased the concentration of TAG, DAG, ceramide, and cholesterol in the heart of HFCD-fed mice. HIE induced a more severe lipid accumulation in the heart of HFCD-fed mice compared with MIE (Fig. [146]S9). Taken together, MIE aggravated HFCD-induced intramyocardial lipid accumulation, which might be responsible for the impaired cardiac function. MIE led to lipid redistribution to the heart Having seen that MIE exacerbated HFCD-induced lipid deposition in the heart, we wondered if MIE changed the fate of circulating lipid in the HFCD-fed mice. Circulating lipid were traced with ^13C-labeled palmitate and bodipy 558/568 C[12] before exercise training. Thereafter, tissue sample of heart, liver, WAT and skeletal muscle were harvested (Fig. [147]5A). As shown in the Fig. [148]5B, C, in sedentary CD-fed mice, circulating lipid were primarily taken by the liver, with a less portion distributing in the heart and skeletal muscle. HFCD-feeding made the lipid accumulated in WAT besides the liver. In contrast, MIE led to an obvious reduction of both ^13C-labeled palmitate and fluorescence-labeled FAs in the liver tissue and WAT (Fig. [149]5B, C). Meanwhile, the content of ^13C-labeled palmitate as well as fluorescence-labeled FAs in the heart showed significant elevation, indicating FAs redistribution to the heart (Fig. [150]5B, C). Lipid deposition in the liver, WAT and skeletal muscle was also detected by oil-red O staining. As shown in Fig. [151]5D–I, HFCD-feeding mice exhibited significant lipid deposition in the liver, WAT and skeletal muscle. MIE ameliorated HFCD-induced fat deposition in the liver and WAT. However, physical exercise failed in decreasing oil-red O positive area in skeletal muscle. Taken together, these data suggested that MIE-induced lipid deposition in the heart of HFCD-fed mice was attributed to lipid redistribution from the liver and WAT to the heart. Fig. 5. MIE led to lipid redistribution to the heart. [152]Fig. 5 [153]Open in a new tab A Schematic figure showing in-vivo lipid tracing procedure with ^13 C or fluorescence-labeled FAs. All elements of this image sourced by figdraw.com. B The organs were harvested and ex vivo imaged. Results are representative of three different experiments. C Relative ^13C-labeled lipid metabolites content in the heart, skeletal muscle, liver and blood (n = 4 per group). D Representative images of Oil red O staining of the liver tissue; Scale bar, 50 μm. E Representative images of Oil red O staining of the skeletal muscle tissue; Scale bar, 50 μm. F Representative images of Oil red O staining of the white adipose tissue; Scale bar, 50 μm. G–I Quantitative analysis of positive Oil red O area of liver, skeletal muscle and white adipose tissue (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file. MIE led to increased cardiac lipid uptake and decreased lipid oxidation in the hearts of HFCD-fed mice Multiple metabolic processes, including lipid uptake and oxidation, are involved in aberrant lipid deposition in the heart. Therefore, we combined transcriptomic and proteomic analysis to reveal the potential mechanisms by which MIE leads to cardiac lipid accumulation in HFCD-fed mice. Differentially expressed genes (DEGs) at transcript and protein levels induced by HFCD-feeding or MIE were determined. RNA-seq analysis of heart tissues obtained from CD + Sed and CD + MIE mice identified 365 downregulated and 23 upregulated DEGs [defined using log2(fold change) >1 and P value < 0.05] (Fig. [154]S10A). Comparative Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene set enrichment analysis (GSEA) revealed that these DEGs were significantly enriched for oxidative phosphorylation and mitochondrial respiratory pathways (Fig. [155]S10A–D). In particular, MIE increased the expression of genes involved in fatty acid (FA) uptake, FA degradation, and mitochondrial respiration in the hearts of CD-fed mice, indicating that MIE promoted FA metabolism in the mouse hearts. The RNA-seq analysis of heart tissues obtained from HFCD + Sed and CD + Sed mice identified multiple DEGs (Fig. [156]S11A) enriched in FA degradation and mitochondrial respiratory pathways (Fig. [157]S11B, C). KEGG analysis of DEGs revealed that the genes involved in cardiac lipid uptake (fat digestion and absorption) were significantly upregulated and those involved in fatty acid degradation pathways were downregulated in the hearts of MIE + HFCD-fed mice compared with Sed + HFCD-fed mice (Fig. [158]6A, B). Figure [159]6C, D shows a heatmap of these genes in the mouse heart, including CD36, Fabp1, Cpt1b, Cpt2, Hadha, and Hadhb. Next, we performed a proteomic analysis between the Sed + HFCD-fed and MIE + HFCD-fed groups (Fig. [160]6E, F). KEGG pathway analysis further validated the upregulation of proteins involved in fatty acid uptake (e.g., CD36 and Fabps) and downregulation of those involved in fatty acid degradation (e.g., Cpt1a, Cpt1b, and Hadh) (Fig. [161]6G, H). We then performed western blotting to detect the expression of major proteins associated with lipid metabolism and validate the proteomic data. The results showed that MIE further increased the levels of proteins involved in lipid uptake (CD36) in the heart of HFCD-fed mice, whereas the levels of proteins involved in lipolysis were decreased (e.g., CPT1a, CPT2, CPT1b, and HADHA) (Fig. [162]6I–M). Transcriptomic analysis of the GEO datasets revealed that genes associated with mitochondrial metabolism in the heart of HFCD-fed mice were maximally affected by MIE (Fig. [163]6N). Overall, these data suggested that the increased cardiac lipid uptake accompanied by decreased FA oxidation (FAO) was responsible for the aberrant lipid deposition in the hearts of HFCD-fed mice induced by MIE. Specifically, mitochondrial injury may be the major cause of decreased FAO. Fig. 6. MIE increased cardiac lipid uptake and decreased lipid oxidation in the hearts of HFCD-fed mice. [164]Fig. 6 [165]Open in a new tab A Volcano plot shows the differentially expressed genes in the heart tissue samples between the HFCD + MIE and HFCD + Sed groups. Red circles represent upregulated proteins [P  <  0.05 and log2(fold change) > 1], and blue circles indicate downregulated proteins [P  <  0.05 and log2(fold change) < –1]. B Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in the mouse hearts extracted from the HFCD + MIE and HFCD + Sed groups. C, D Heatmap of RNA-seq-based expression of genes involved in fat digestion, fat absorption, and fatty acid degradation pathways (n = 4 per group). E Differentially expressed proteins (n = 4 per group). F Volcano plot shows the difference in proteins of the heart tissues between the HFCD + MIE and HFCD + Sed groups. Red circles represent upregulated proteins, and green circles indicate downregulated proteins [P  <  0.05 and log2(fold change) < –1]. G KEGG pathway enrichment analysis of differentially expressed proteins in the mouse hearts extracted from the HFCD + MIE and HFCD + Sed groups. H Heatmap shows the difference in the protein abundance in the context of fat digestion, fat absorption, and fatty acid degradation pathways (n = 4 per group). I–M Representative western blots and quantitative analysis of CD36 (I), CPT1a (J), CPT2 (K), CPT1b (L), and HADHA (M) (n = 6 per group). Three times experiment was repeated with similar results. N Gene ontology (GO) pathway enrichment analysis of differentially expressed genes in the mouse hearts extracted from the HFCD + MIE and HFCD + Sed groups. All studies were carried out with male mice. Data presented as mean ± SEM in (I–L). P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file. MIE further impaired FAO and mitochondrial function in the hearts of HFCD-fed mice Mitochondria are the major site for lipid oxidation, and toxic lipids cause oxidative damage and mitochondrial respiratory defects^[166]21. High-resolution respirometry analysis was performed to determine the effect of exercise on mitochondrial metabolism. We observed a significant decrease in basal oxygen consumption rate in the hearts of HFCD-fed sedentary mice when compared with CD-fed sedentary mice, indicating impaired basal mitochondrial respiration in the hearts of HFCD-fed mice (Fig. [167]7A, B). Interestingly, respiration stimulation of the fatty acid oxidation (FAO) pathway in the presence of malate and octanoylcarnitine was significantly increased in the hearts of HFCD-fed sedentary mice, which might be attributed to a compensatory increase (Fig. [168]7A, C). MIE improved basal mitochondrial respiration and FAO in CD-fed mice (Fig. [169]7A, C). However, the mitochondrial respiratory function and FAO were further deteriorated by MIE and HIE in HFCD-fed mice (Figs. [170]7A, C and [171]S13). Additional ^13C-labled metabolic flux experiments to measure fatty acid oxidation in the hearts. As shown in the Fig. [172]S12, ^13C[16]-palmitate (M + 16) and ^13C[16]-palmitoylcarnitine (M + 16) accumulated in the heart of HFCD-fed sedentary mice. Meanwhile, ^13C[2]-acetyl-CoA (M + 2) was significantly increased in the heart of CD-fed MIE mice when compared with CD-fed sedentary mice (Fig. [173]S12A–E). However, in HFCD-fed mice, MIE induced a more evident deposition of ^13C[16]-palmitate and ^13C[16]-palmitoylcarnitine, while decreased the level of ^13C[2]-acetyl-CoA (Fig. [174]S12A–E). These data further confirmed that MIE impaired FAO capacity in the heart of HFCD-fed mice. Fig. 7. MIE further impaired fatty acid oxidation (FAO) and mitochondrial function in the hearts of HFCD-fed mice. [175]Fig. 7 [176]Open in a new tab A Representative experiment to detect fatty acid oxidation capacity using the O2K instrument (n = 6 per group). B Basic respiratory measured by oxygen consumption (n = 6 per group). C FAO measured by oxygen consumption (6 biological replicates for each group). D Representative experiment for determining mitochondrial oxidative phosphorylation (OXPHOS) capacity using the O2K instrument (n = 6 per group). E Mitochondrial OXPHOS capacity of complex I (6 biological replicates for each group). F Mitochondrial OXPHOS capacity of complex II ((n = 6 per group)). G Mitochondrial OXPHOS capacity of complex IV (n = 6 per group). H Heatmap shows the difference in the protein abundance in the electron transport chain (n = 4 per group). I Gene ontology (GO) analysis of differentially expressed proteins in the hearts of mice from the HFCD + MIE and HFCD + Sed groups. J Gene Set Enrichment Analysis (GSEA) of the gene set for the OXPHOS pathway. K Heatmap of RNA-seq analysis of gene expression involved in the OXPOS pathway (n = 4 per group). L Representative transmission electron microscopic images of the myocardium; Scale bar, 1 μm. Three times experiment was repeated with similar results. M Ratio of cristae area to mitochondrial area (n = 6 per group) N Relative Mitochondrial size (n = 4 per group). O Relative mitochondrial diameter (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file. The activity of mitochondrial complexes I, II, and IV was also determined in the four groups. HFCD feeding increased the activity of mitochondrial complexes I in sedentary mice (Fig. [177]7D–G). MIE improved the activity of mitochondrial complexes I and IV in CD-fed mice (Fig. [178]7D–G). Consistent with the changes in FAO capacity, the activity of the mitochondrial complex was also pronouncedly inhibited in the hearts of MIE + HFCD-fed mice (Fig. [179]7D–G). Proteomic analysis revealed that the expression of 888 proteins was differentially affected by MIE in the HFCD-fed mice. Figure [180]7H shows the heatmap of 24 major proteins involved in mitochondrial respiration. The pathway enrichment analysis revealed that pathways in the mitochondrial electronic transport chain and ATP synthesis were significantly influenced by MIE in MIE + HFCD-fed mice compared with Sed + HFCD-fed mice (Fig. [181]7I). GSEA of the transcriptomes also indicated that the mitochondrial oxidative phosphorylation pathway was inhibited in MIE + HFCD-fed mice (Fig. [182]7J–K). We performed TEM analysis to observe the mitochondrial ultrastructure. The TEM images indicated altered mitochondrial morphology and abnormal cristae structure in HFCD-fed mice. We found that HFCD-feeding induced decreased mitochondrial cristae density. When compared with that of HFCD-fed sedentary mice, hearts of HFCD-fed MIE mice showed decreased mitochondrial size (Fig. [183]7L–O). In addition, proteins related to mitochondrial unfolded protein response (UPRmt) including ATF4, ATF5, and CHOP was also determined. As shown in Fig. [184]S14A, the expression of ATF5 was significantly increased in the heart of HFCD-fed mice, indicating mitochondrial unfolded protein response (UPRmt) and mitochondrial dysfunction. Interestingly, we found that MIE decreased the protein level of ATF4 in the heart of both CD- and HFCD-fed mice, suggesting that MIE was able to suppress UPRmt. Moreover, PGC1α, an indicator of mitochondrial biogenesis, was significantly increased by HFCD-feeding. LIE induced an elevation of PGC1α expression in the heart of HFCD-fed mice, while HIE and MIE decreased the expression of PGC1α in the heart of HFCD-fed mice (Fig. [185]S14B). These data suggested that HFCD-feeding was able to induce mitochondrial unfolded protein response in the mice heart. Both MIE and HFCD-feeding alone was able to promote mitochondrial biogenesis. However, a combination of HFCD-feeding and MIE or HIE would induce mitochondrial biogenesis suppression. Collectively, MIE aggravated the impairment of mitochondrial respiration and FAO in the heart of HFCD-fed mice, leading to lipid deposition and functional impairment in the hearts of MIE + HFCD-fed mice (Fig. [186]8). Fig. 8. A pictorial summary of exercise-induced metabolic change in whole-body and the heart of CD- and HFCD-fed mice. [187]Fig. 8 [188]Open in a new tab A, B In sedentary mice, HFCD-feeding leads to increased body weight, accompanied by aberrant lipid accumulation in liver and WAT. MIE prevented HFCD-induced weight gain, while exacerbated cardiac functional impairments by inducing lipid redistribution from liver and WAT to the heart. All elements of this image sourced by figdraw.com. Discussion In this study, we focused on the cardiac effect of the exercises of different intensities in an HFCD-fed mouse model. Regular moderate- or high-intensity interval exercise, previously reported to exert cardioprotective effects, exacerbated cardiac injury in HFCD-fed mice, whereas low-intensity exercise protected against HFCD-induced cardiac dysfunction. The negative cardiac effect of exercise was associated with aggravated intramyocardial lipid accumulation and impaired mitochondrial respiration. Specifically, circulating lipids redistributed from the liver and adipose tissue to the myocardium in HFCD-fed mice subjected to MIE. The abnormally increased cardiac lipid uptake exceeded the mitochondrial FAO capacity, leading to toxic lipid deposition and further cardiac damage in HFCD-fed mice. Although moderate- or high-intensity physical exercise showed reliable weight management effects in mice fed with HFCD, it impaired cardiac lipid metabolism and function. Therefore, for mice fed with HFCD, counteracting the negative effect of HFCD by simultaneous physical exercise might cause unexpected damage to the heart. Exercise training was recommended as a cornerstone in the management of DIO, while the intensity and mode of exercise seems to play a decisive role. However, the selection of exercise mode and intensity remains unclear and controversial. We assumed that exercise may be effective in preventing the development of cardiac injury in mice with HFCD. Therefore, the MIE protocol in this study was established according to the protocol previously reported beneficial to mice with metabolic syndrome or cardiac injury^[189]17,[190]22. Considering MIE as a midpoint, mice were subjected to lower or higher intensity of exercise defined as LIE and HIE, respectively. Although studies directly comparing the effects of different exercise intensities in treating metabolic disorders are limited, high-intensity training is considered to have a superior effect^[191]23,[192]24. For instance, HIE was reported to be more effective than MIE in improving aerobic capacity in non-obese subjects^[193]25–[194]27. In this study, we found that MIE and HIE showed a comparable effect, superior to LIE, in preventing HFCD-induced weight gain. In addition, HIE and MIE were better at ameliorating lipid deposition in the WAT and liver compared with LIE. Interestingly, we found that both MIE and HIE elevated the food intake and calories intake in mice, which could be attributed to the larger muscle energy consumption. Since obesity is the consequence of larger energy intake than energy expenditure, it can be considered that HIE and MIE trained mice did not exhibit whole-body overnutrition even feed with HFCD. Previous studies reported that HIE was better at improving glycemic control in patients with metabolic syndrome compared with MIE, which was attributed to the exercise-induced adaptation in the skeletal muscles^[195]22. However, our study suggested that exercises of all intensities could not improve insulin sensitivity in HFCD-fed mice. In this study, we introduced HFCD as a confounding factor. HFCD can directly induce insulin resistance in muscle tissues; therefore, it cannot be excluded that diet have limited the potential beneficial adaptations in the skeletal muscle due to MIE or HIE. Hafstad et al. reported that HIE, but not MIE, was able to induce metabolic and energetic adaptions in hearts, whereas MIE and HIE were equally effective in ameliorating LV diastolic and systolic function and improving LV mechanical efficiency in mice with DIO^[196]22. Moreover, Lew et al also reported that MIE protected cardiac function in diabetic mice^[197]17. In contrast, we found that both MIE and HIE were detrimental to the cardiac function in HFCD-fed mice. This discrepancy may occur due to the differences in experimental protocol and feeding strategies. In the previous conducted by Hafstad et al., mice were fed with HFCD to induce obesity and OIC in advance, and thereafter subjected to physical exercise regimens to investigate the potential rescue effect of exercise on OIC. In contrast, we explored whether physical exercise could offset HFCD-induced cardiac metabolic and functional disorders. Therefore, physical exercise and HFCD feeding was simultaneously performed. Although MIE and HIE showed reliable effect in preventing HFCD-induced metabolic syndromes and fat deposition in WAT and liver, a striking further disorder of cardiac lipid metabolism and abnormal cardiac lipid accumulation were observed in the mice heart and skeletal muscle. Similar to our results, recent study also showed that exhaustive exercise increased damage to the muscles, including skeletal muscle and myocardium^[198]15. Notably, although we found that MIE or HIE would exacerbate cardiac damage in HFCD-fed mice, LIE conferred protective effect and ameliorated cardiac dysfunction in HFCD-fed mice. These findings in LIE-trained mice provided experimental evidence for the recommendations from professional organizations^[199]28,[200]29, which suggested minimum moderate exercise intensity to be beneficial to health improvements. The present study recommended that the effect of physical exercise should be meticulously assessed in particular populations. Our data indicate that the cardiac effect should be considered during the development of exercise strategies to avoid potential exercise-induced cardiac injury. More than 70% of cardiac ATP generation relies on lipid oxidation. However, excessive lipid storage in the heart causes lipotoxicity^[201]30. Intramyocardial lipid accumulation (detected using cardiac imaging) was associated with impaired left ventricular (LV) diastolic function and increased LV mass^[202]31,[203]32. In this study, 60 kcal% fat diet was used to induce obesity and mimic obesity-induced metabolic syndrome and cardiac dysfunction in rodent model. Results from echocardiography and PV catheter measurements also showed that prolonged HFCD-feeding induced a significantly impaired cardiac function accompanied by elevated lipid level in both serum and myocardium. However, Tadinada et al. reported that no impairment in cardiac function even after 36 weeks of HFCD feeding^[204]33. The basis for these disparities is currently not understood. Additional mechanisms such as increased oxidative stress, apoptosis, fibrosis, and impaired Ca^2+ handling may contribute to cardiac remodeling and functional defects in these models. A healthy heart would make precise regulation to keep a dynamic balance between lipid uptake and oxidation, thereby avoiding toxic lipid deposition^[205]34–[206]36. We found that HFCD-induced an increase in lipid uptake in the hearts, leading to impaired diastolic and systolic function. In contrast, physical exercise also resulted in increased cardiac lipid uptake but led to nearly absent cardiac LDs deposition. This may be attributed to significantly elevated FAO capacity in the hearts of mice subjected to exercise. In consist, it was reported that physical exercise improved protein level in both free fatty acid uptake and oxidation, thereby improving lipid metabolic flexibility^[207]37,[208]38. However, in HFCD-fed mice, we found MIE induced a further lipid uptake accompanied by FAO capacity impairment, which could be attributed to the abnormal deposition of toxic lipid. More specifically, in the premise of circulating lipid overload, MIE led to the lipid redistribution from lipid storage organs (adipose tissue and liver) to lipid utilization organs (cardiac and skeletal muscles). Further increased cardiac lipid uptake triggered the transition to decompensated lipid deposition, which in turn exacerbated mitochondrial impairment and accelerated the occurrence of cardiac lipotoxicity. Mitochondria are the main site for lipolysis^[209]39. Due to the high metabolic demand and mitochondrial content in the myocardium, mitochondrial dysfunction was significantly correlated with myocardial contraction. The impact of physical exercise on mitochondrial metabolism has been well described in skeletal muscles^[210]40, while the effect of exercise on cardiac muscle remains less clear. In skeletal muscle, exercise induced an abrupt increased mitochondrial capacity of oxidation in both glucose and lipid^[211]41, which provided a potential explanation for exercise-induced cardio-protection in acute cardiac injury. Here, in this study, we found that physical exercise induced an obvious elevation in mitochondrial FAO capacity as well as mitochondrial respiratory chain complex activity in CD-fed mice, suggesting an improvement of cardiac energetic metabolism. Consistent with our results, exercise was previously reported to enhance the expression of electron transport chain components in mitochondria and further super-complexes assembly in human skeletal muscle^[212]42. Researchers also found that exercise training increased mitochondrial biogenesis in the healthy heart^[213]43, which could also contribute to the improved mitochondrial metabolism. However, under condition of HFCD feeding, the improved mitochondrial metabolism in the heart is no longer observed, with physical exercise leading to a significant impairment in FAO capacity. The decreased mitochondrial FAO was confirmed in multiple ways including results from respirometric, transcriptomic, lipidomic and proteomic analysis, which make the evidences more convincible. Further data showed that the mitochondrial respiratory chain complex activity was also decreased. Notably, short-term HFCD feeding increased cardiac mitochondrial biogenesis and mitophagy, which promoted the turnover of mitochondria and serve as an adaptive change to maintain mitochondrial quality in response to lipid overload^[214]44,[215]45. It is interesting to see that either exercise or short-term HFCD feeding was associated with adaptive changes in mitochondrial function respectively, while a combination of which causes severe mitochondrial damage. One possibility is that, under the condition of constant lipid overload, HFCD-induced lipid uptake led to a near saturation of the mitochondrial FAO capacity. The mixture of physical exercise worsens the delicate balance between FAO and lipid uptake, which triggers toxic lipid deposition and mitochondrial dysfunction as a “domino effect”. These also explains why HFCD-feeding mice underwent LIE showed preserved cardiac function. The potential reason might be that LIE-induced metabolic changes did not reach the threshold of triggering the adverse response. Our study still has some limitation. The findings of this study were based on experimental results of mice feeding with specific diet. The generalizability of the findings beyond this specific diet and mouse strain is still unknown. Future studies are needed to further investigate this subject and further refine the findings of this study. Moreover, data from proper populations will help guide exercise protocols for the clinical management of DIO in humans. In summary, our study revealed that moderate- or high-intensity exercise may cause severe decompensatory cardiac injury in HFCD-fed mice. The mechanism involves exercise-induced maladaptive changes and a vicious cycle of cardiac lipid metabolism. These findings emphasize that the effects of any exercise regimen should be considered separately on different organs based on their distinct role in energy metabolism, in addition to considering the whole-body effects. Inappropriate physical exercise modes might cause unexpected detrimental damage in certain organs even though their robust effect in weight losing and overall metabolic benefit. Methods Animals Eight-week-old male and female wile-type C57BL/6J mice were procured from the Shanghai Biomodel Organism Science & Technology Development Lab. Mice were housed in a pathogen-free room with a 12-h light/12-h dark cycle, a temperature of 24 °C ± 2 °C, and a relative humidity of 40–70% and allowed free access to water and food. Mice were fed ad libitum a chow diet (Research diets, D12450B) or an HFCD (Research diets, D12492). Mice were housed at 3–5 animals cage racks with individual ventilation, negative pressure, and air filtration (Allentown, Inc., Allentown, NJ). Male and female mice were randomly assigned into sedentary or exercise groups at 8 weeks of age. All animal experimental procedures were approved by the Fourth Military Medical University Animal Use and Care Committee. Exercise protocol Mice were subjected to involuntary treadmill running 5 days/week for 8 weeks. A 10-min warm-up before each training session was included in the regimen to ensure compliance with exercise protocol and minimized the risk of injury. A shock grid setting of 25 V, 0.34 mA, and 2 Hz was used to guarantee the treadmill running exercise according to the defined protocol. The classification of exercise intensity as low, moderate, and high was modified from the protocol described by Lew et al.^[216]17. Physical exercise was started 7 a.m. and 9 a.m. in the morning. All the three exercise regimens started with 10-min running for warm-up. Low-intensity exercise (LIE) involved gradually raising the running pace from 0 to 6 m/min followed by continuous running for 60 min (5° incline) at 6 m/min. Moderate-intensity exercise (MIE) involved gradually raising the running pace from 0 to 12 m/min followed by continuous running for 60 min (5° incline) at 12 m/min, which was equivalent to 65% to 70% maximum oxygen consumption^[217]17. High-intensity exercise (HIE) involved gradually raising the running pace from 0 to 18 m/min followed by continuous running for 60 min (5° incline) at 18 m/min, which was equivalent to 85% to 90% maximum oxygen consumption^[218]17. The mice subjected to different exercise regimens were separately housed for 30 min. All mice were housed in cages (50 cm × 50 cm × 20 cm), which enable the mice move freely in cages during the experiments. To avoid the potential interference caused by voluntary exercise, mice was not able to access to wheels and other equipment. Before tissue harvesting, mice were fasted overnight and euthanized 24 h after the last exercise. Food consumption and caloric intake measures Food intake was measured by manually weighing the food remaining every 14 days. For calculation of the caloric uptake, the consumed food in gram was multiplied with the calories per gram of the respective type of food. The specific feed ingredients and calories density of chow diet and HFCD are shown in the Tables [219]S1 and [220]2. Biochemical and anthropometric analysis in mice Mouse body and heart weights were measured in all groups. Low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglycerides were quantified using standard protocols^[221]46. Blood glucose concentrations were measured using a sip-in sampling glucose meter (Glucometer 580, Yuwell, China). Echocardiography Cardiac echocardiography was performed using a VEVO 3100 echocardiography system (Visual Sonics Inc., Toronto, Canada) as previously described^[222]46. For echocardiography, mice were fasted overnight and anesthetized 24 h after the last exercise. Body temperature was maintained (36.5–37.5 °C) using a rectal thermometer interfaced with a servo-controlled heat lamp. Mice were anesthetized with 2% isoflurane, maintained under anesthesia with 1.5% isoflurane. Ejection fraction (EF%) was calculated as stroke volume (SV)/diastolic volume × 100%. Left ventricular diameters during diastole (LVID, d), left ventricular diameters during systole (LVID, s). Heart rates were determined from long-axis M-modes. Relative wall thickness was calculated as (diastolic posterior wall thickness + diastolic anterior wall thickness)/LVID, d. Doppler echocardiography was obtained to determine diastolic trans-mitral blood flow velocities for peak early (E) and late (A) fillings. Transmission electron microscopy (TEM) Left ventricular tissue was dissected into 3-mm^3 pieces and fixed overnight in 2.5% glutaraldehyde. Images were obtained using a TEM (JEM-1230, JEOL Ltd., Japan) at 300 kV and analyzed using ImageJ software (version 2.0.0)^[223]46. Lipidomic analysis To capture the metabolic state of the adapted heart as accurately as possible, the hearts were freeze-clamped in situ 24 h after the final exercise bout. Before tissue harvesting, mice were fasted for 6 h, heart sample were collected and conserved in liquid nitrogen until lipid extraction. Samples were thawed on ice, and metabolites were extracted from 20 µL of each sample using 120 µL of precooled 50% methanol buffer. Then the mixture of metabolites was vortexed for 1 min and incubated for 10 min at room temperature, and stored at −20 °C overnight. The mixture was centrifugated at 4000 × g for 20 min, subsequently the supernatant was transferred to 96‐well plates. The samples were stored at −80 °C prior to the LC‐MS analysis using a TripleTOF 5600 Plus high-resolution tandem mass spectrometer (SCIEX, Warrington, UK) with both positive and negative ion modes. LipidSearch software (Thermofisher) was used to quantitative analyze the fatty acid profile of cardiac muscle. LC/MS and LC/MS/MS data was used to match the LipidSearch software database to make the results more accuracy. Transcriptomic analysis Mouse hearts were subjected to the transcriptomic analysis using the GeneChip Mouse Genome 430 2.0 Array (Affymetrix)^[224]47. Array data were analyzed using the affy and limma packages of R for Robust Multi-array Average for normalization and for moderate t-statistics to derive p-values, respectively. The p-values were adjusted using the Benjamini and Hochberg method for false discovery rate control. For the genes with multiple probes set IDs, the probes set with the highest standard deviation among all samples was kept for further analysis. Gene Ontology analysis was performed using [225]DAVID. Isolation of adult cardiomyocytes Adult mouse cardiomyocytes were isolated using the protocol described previously^[226]48. For isolation of adult cardiomyocytes, mice were anesthetized and heparinized, and hearts were removed. Mice hearts were then submerged in oxygenated perfusion buffer, cannulated via the aorta under a microscope, and connected to a standard Langendorff retrograde perfusion system adapted to mice. Left ventricular and cardiomyocytes were isolated by using a modified protocol. Briefly, the heart was perfused at 2.2 ml/min for 10 min with a modified Joklik’s minimum essential medium (Life Technologies) mixed in 2000 ml deionized water containing (in mM) 1.2 MgSO[4], 1.0 dl-carnitine (Sigma Chemical), and 23.8 NaHCO[3]. The buffer was equilibrated with 5% CO[2]–95% O[2] for 30 min (37 °C, pH 7.2). Then, 150 μ/ml collagenase type II (Worthington) and 0.1% (wt/vol) bovine serum albumin (Sigma Chemical) were added, and the heart was perfused until it became palpably flaccid. The heart was then cut down into the Joklik’s medium containing 0.8% (wt/vol) bovine serum albumin (Sigma Chemical) and 0.75 mM CaCl[2]. The left ventricle was separated and minced and was shaken for 15–20 min in the collagenase type II and bovine serum albumin buffer (37 °C, 5% CO[2]–95% O[2], 100 rpm). The solution with the myocytes and unsolved tissue was then gently filtered through a nylon mesh (250 μm); added to HEPES buffer containing (in mM) 135 NaCl, 5 KCl, 1 MgCl[2] · 6H[2]O, 1.2 CaCl[2] · 2H[2]O, 10 HEPES, 8 C[6]H[12] · H[2]O (37 °C, pH 7.2, 5% CO[2]–95% O[2]); and centrifuged (600 rpm, 45 s, 21 °C). The supernatant was then removed, and HEPES buffer was added. Fluorescent imaging of lipid droplets (LDs) Bodipy 493/503 (200 ng/ml; Life Technology) was added to cardiomyocytes, and fluorescent images were immediately acquired using a Nikon A1 plus confocal laser-scanning microscope (Nikon, Japan) to visualize lipid droplets. Measurement of mitochondrial function Mitochondrial function was estimated as oxygen consumption using high-resolution respirometry (Oxygraph-2k, Oroboros Instruments)^[227]49. The Oroboros was calibrated with respiration medium MiR05 (500 µM EGTA, 3 mM MgCl[2].6H[2]O, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH[2]PO[4], 20 mM HEPES, 110 mM sucrose, 1 g/L fatty acid free BSA, adjusted to pH 7.1 with KOH) at 37 °C for approximately 1 h. The medium was stirred at 540 rpm until calibration at air saturation was attained, as evidenced by a stable oxygen flux. Approximately 5 mg myocardial tissue were obtained and mechanically homogenized in cold respiration media. After homogenation, the tissue homogenate was added to a 2 ml chamber to assess oxygen flux. To examine the activity of respiratory chain complexes, Glutamate (10 mM) and malate (2 mM) were introduced to the chamber to provide NADH for complex I (state 2 respiration), followed by saturating concentrations of ADP (2 mM) (state 3 respiration). Complex I was inhibited with 500 nM rotenone before succinate (10 mM) was added to support complex II respiration (state 3). Complex II was inhibited with malonate (10 mM) and glycerol-3-phosphate added (10 mM) to support complex III respiration. Inhibition of complex III with antimycin A (5 µM) was followed by stimulation of complex IV with 100 µM TMPD and 400 µM ascorbate. For measurement of fatty acid oxidation, ADP (5 mM), malate (2 mM) and octanoylcarnitine (Oct) (0.5 mM) were added. PV catheter Cardiac function was assessed using a pressure–volume (PV) catheter (Millar Instruments Inc.). A PV catheter was inserted just inside the ventricular wall, and a PV loop was recorded at the baseline. The catheter was calibrated, and the indices of systolic and diastolic function were determined. These indices were ejection fraction (EF), peak rate of pressure rise-end diastolic volume relation (dP/dt[max]–EDV), end-systolic pressure–volume relation (ESPVR), stroke work-end diastolic volume relation (preload recruited stroke work), peak rate pressure decline (−dP/dt[min]), relaxation time constant (Tau), and end diastolic pressure–volume relation (EDPVR). Western-blot analysis Mice heart and liver tissues were analyzed by western-blotting as previously described. Primary antibodies against the following proteins were used: CD36 (Abcam, #ab252923 dilution: 1:10,000), CPT1α (Proteintech, #15184-1-AP, dilution: 1:5000), GAPDH (Proteintech, #10494-1-AP, dilution: 1:5000), HADHA (Proteintech, #10758-1-AP, dilution: 1:2000), CPT1b (Proteintech, #22170-1-AP, dilution: 1:5000), CPT2 (Proteintech, #26555-1-AP, dilution: 1:5000), ATF4 (Proteintech, 60035-1-Ig, dilution: 1:4000), ATF5 (Proteintech, 67066-1-Ig, dilution: 1:5000), CHOP (Proteintech, 66741-1-Ig, dilution: 1:5000) and PGC1α (Proteintech, 66369-1-Ig, dilution: 1:5000). Glucose tolerance and insulin tolerance test An intraperitoneal glucose tolerance test (IPGTT) and test insulin tolerance (ITT) was used to test the insulin sensitivity of mice. ITT and IPGTT was performed 24 h after the final exercise bout. For IPGTT, after fasting for 12 h, mice were injected intraperitoneally with glucose(2 g/kg). Blood glucose was measured at 0, 15, 30, 60, 90, and 120 min using tail clippings. Insulin (0.5 U/Kg) was injected hypodermically after fasting for 6 h to test insulin tolerance (ITT). Then, the blood glucose was measured at 0, 15, 30, 60, 90, and 120 min. Immunohistochemical (IHC) staining Mice were anesthetized with 5% isoflurane, hearts were removed, fixed with 4% formaldehyde, embedded in paraffin and sectioned 7 μm thickness for IHC analysis. After paraffin sections dewaxing to water, the sections were placed in PBS (PH7.4) and washed by shaking on the decolorizing shaker for 3 times, 5 min each time. The sections were placed in 3% hydrogen peroxide solution, incubated at room temperature away from light for 25 min, and the slides were placed in PBS (PH7.4) and washed three times on a decolorizing shaking table for 5 min each time. The sections were uniformly covered with 3%BSA in the tissue chemical circle and closed at room temperature for 30 min. The sections were incubated with primary antibodies at 4 °C for overnight. The slices were placed in PBS (PH7.4) and washed by shaking on the decolorizing shaker for 3 times, 5 min each time. After the slices were slightly dried, the tissue was covered with the secondary antibody of the corresponding species of the primary antibody, and incubated at room temperature for 50 min. the freshly prepared DAB color developing solution was added into the circle. The color developing time was controlled under the microscope. The positive color was brown and yellow, and the sections were rinsed with tap water to terminate the color development. Enzyme-linked immunosorbent assay (ELISA) Mice were anesthetized with 5% isoflurane, and blood was collected from the carotid artery. After centrifugation at 3000 rpm for 10 min at 25 °C, the supernatant was collected as serum. BNP, ANP, insulin, lactate, and corticosterone level were measured using the commercial ELISA Kit (Elabscience Biotechnology, China) according to the manufacturer’s instructions. Real-time RT-PCR Total RNA was extracted using the trizol RNA extraction reagent. First strand cDNA was transcribed from total RNA using a first strand synthesis kit (Takara, Japan). Real-time RT-PCR was performed using SYBR green reagent and specific primers (Table [228]S10). Oil red staining According to manufacturer’s instructions, oil red staining was performed using the Lipid (Oil Red O) staining kit from Biovision. Dihydroethidium (DHE) staining Intracellular superoxide anion (O2•−) levels in the mice hearts tissue were detected by DHE staining. Images were obtained with a confocal laser-scanning microscope (Nikon A1R MP+ Confocal Microscope, Nikon,Japan). The images were analyzed with ImageJ image analysis software. Measurement of MDA level in heart tissue MDA level of mice heart tissue was detect using lipid peroxidation MDA assay kit (S0131, Beyotime Biotechnology, Jiangsu, China) according to manufacturer’s protocols. Run to exhaustion treadmill testing The exhaustion test was conducted as previously described. Mice started the run at 10 m/min for 10 min, and the speed was increased in 2-m/min increments every 10 min until exhaustion. Mice were motivated to run using bristled brushes at the back of the treadmill and were considered exhausted when they could no longer run in response to this stimulus. The experimenter was blinded to the genotype of the mice during the run tests. In vivo ^13C-labeled FAs tracing For in vivo 13C-labeled FAs tracing. Mice was injected intraperitoneally with the ^13C-labeled palmitate (100 mg/Kg), After injection for 4 h, animals were sacrificed and extracted blood, heart, liver, skeletal muscle to analyze. Fatty acid uptake in vivo To measure fatty acid uptake in the different organs using fluorescent dyes, each mouse was injected appropriate doses (0.5 mg per kg body weight) of BODIPY fluorescent-conjugated fatty acids (558/568 C12, Invitrogen, D3835) through the tail vein. After injection for 1 h, heart, skeletal muscle, liver and WAT were collected to observe distributions of fatty acid by live tissue imaging. Metabolomic profiling and analyses Mice hearts were subjected to metabolomic analysis. non-targeted metabolomic analysis was performed by Biotree on a 1290 Infinity series UHPLC System (Agilent Technologies) coupled to a Triple time-of-flight (TOF) 6600 mass spectrometer (AB Sciex) equipped with an electrospray ionization source. Briefly, chromatographic separation was achieved using a Waters BEH Amide column (2.1 × 100 mm, 1.7 µm) at 25 °C. The mobile phase consisted of water with 25 mM ammonium acetate and 25 mM ammonium hydroxide (A) and acetonitrile (B). The flow rate was 0.5 ml min^−1. Mass spectrometry analysis was operated in information-dependent basis mode under positive and negative modes. The 12 most abundant ions were selected, fragmented and sent to a TOF mass analyser to acquire MS2 information. Data pretreatment including peak deconvolution, alignment and integration were achieved by XCMS (v.3.2) implemented in R language. Metabolite identification was based on an in-house MS2 database. MetaboAnalyst was employed for pathway analysis. Heatmaps were generated by Complex Heatmap. Proteomics Mice heart proteins were extracted using RIPA lysis buffer at 4 °C and quantified using the BCA assay (Biyutian, China). The samples were then centrifuged at 13,000 rpm at 4 °C for 10 min and the supernatant was collected. The proteins were redissolved, and DTT and indole-3-acetic acid were added, followed by protein digestion using trypsin. Equal amounts of proteins were labeled with Tandem Mass Tags (TMT) according to the protocol of the TMT label kit (Thermo Fisher Scientific). For nano–LC-MS/MS analysis, total peptides were separated and analyzed with a nano-UPLC (EASY-nLC1200) coupled to a Q Exactive HFX Orbitrap instrument (Thermo Fisher Scientific) with a nano–electrospray ion source. Separation was performed using a reversed-phase column (100 μm inside diameter ×15 cm, Reprosil-Pur 120 C18-AQ, 1.9 μm; Dr. Maisch). The mobile phases were H2O with 0.1% formic acid (FA), 2% ACN (phase A) and 80% ACN, 0.1% FA (phase B). Sample separation was executed with a 90-min gradient at a 300 nl/min flow rate. Gradient B was 2 to 5% for 2 min, 5 to 22% for 68 min, 22 to 45% for 16 min, 45 to 95% for 2 min, and 95% for 2 min. Data-dependent acquisition was performed in profile and positive mode with an Orbitrap analyzer at a resolution of 120,000 (at 200 m/z) and an m/z range of 350 to 1600 for MS1; for MS2, the resolution was set to 45k with a fixed first mass of 110 m/z. The automatic gain control target for MS1 was set to 3 × 106 with max injection time (IT) 30 ms, and 1 × 105 for MS2 with max IT 96 ms. The top 20 most intense ions were fragmented by HCD with an NCE of 32% and an isolation window of 0.7 m/z. The dynamic exclusion time window was 45 s; single-charged peaks and peaks with charge exceeding six were excluded from the data-dependent acquisition procedure. For the proteome discoverer database search, Vendor’s raw MS files were processed using Proteome Discoverer software (version 2.4.0.305) and the built-in Sequest HT search engine. MS spectra lists were searched against their species-level UniProt FASTA databases, with carbamidomethyl (C), TMT 6 plex (K), and TMT 6 plex (N-term) as fixed modifications and oxidation (M) and acetyl (protein N-term) as variable modifications. Trypsin was used as the protease. A maximum of two missed cleavages was allowed. The FDR was set to 0.01 for both peptide-spectrum match and peptide levels. Peptide identification was performed with an initial precursor mass deviation of up to 10 ppm and a fragment mass deviation of 0.02 Da. Unique peptides and razor peptides were used for protein quantification and total peptide amount for normalization. All other parameters were reserved as defaults. Histology Following euthanasia, the heart tissue was excised, flushed with PBS solution, and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 µm. Heart cross-sections were stained with 4'6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA) and wheat germ agglutinin (WGA; ThermoFisher, Waltham, MA) for quantification of cardiomyocyte cross-sectional area. Quantitative measurements were determined using Nikon Elements software, with no less than 100 myocytes with centrally located nuclei assessed for area measurements. To prevent bias, the individual analyzing all histology was unaware of the group assignments. A subset of slides from each group was used based on power calculations to detect a 20% difference in cross-sectional area: myocyte ratio at α < 0.05. Statistical analysis Differences between any two groups were examined using the Student’s t-test. Differences between groups were determined by two-way ANOVA followed by Tukey’s post hoc test or Bonferroni post hoc test, and by one-way ANOVA followed by Tukey’s post hoc test or Bonferroni post hoc test as appropriate. All data are expressed as mean ± SEM. P < 0.05 was considered statistically significant. Reporting summary Further information on research design is available in the [229]Nature Portfolio Reporting Summary linked to this article. Supplementary information [230]supplementary information^ (3.5MB, pdf) [231]41467_2025_55917_MOESM2_ESM.docx^ (19KB, docx) Description of Additional Supplementary Files [232]Supplementary Data 1^ (341.4KB, xlsx) [233]Reporting Summary^ (106.3KB, pdf) [234]Peer Review file^ (3.8MB, pdf) Source data [235]Source Data^ (82.2KB, xlsx) Acknowledgements