Influence of diet-induced obesity and voluntary exercise training on cardiac lipids and mitochondrial function in mice

Figures

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  Fig. 1. VET attenuates the HFD feeding-induced obesity phenotype in mice. (A) Cumulative body weight measured weekly and (B–C) fat and lean mass measured monthly. (D) Absolute heart weight at study endpoint. (E) Running wheel rotations and (F) total distance run over a 24-h cycle averaged over 3 weeks in male (voluntary exercise training) VET mice fed a high fat or chow diet. Data taken at 17–19 weeks of age. (G) Fasted blood glucose levels at study endpoint. Non-fasted plasma profiles of (H) non-esterified fatty acids, (I) triglycerides and (J) cholesterol at study endpoint. Analysis was performed using (A–C) repeated measures two-way analyses of variance and (D and J) two-way analyses of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. (A–G) n: 11–12 per group; (H–J) n: 7–9 samples per group. (A and B) * p < 0.05, *** p < 0.001 for HFD sedentary mice and chow sedentary mice; ^ p < 0.05, ^^ p < 0.01 for HFD VET and HFD sedentary mice. (C–J) * p < 0.05, ** p < 0.01, **** p < 0.0001. HFD = high fat diet; NEFA = non-esterified fatty acid; VET = voluntary exercise training.

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  Fig. 2. High fat feeding induces diastolic dysfunction, and voluntary exercise promotes a hypertrophy phenotype. Diastolic function readouts of ventricular filling (A) peak E, (B) peak A, (C) E/A ratio, (D) deceleration time. (E) isovolumic contraction time, and (F) isovolumic relaxation time. (G–H) Pre and post exercise measurements of area diastole and systole. (I–J) Pre and post exercise measurements of volume diastole and systole. (A–F) Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. n: 9–11 per group. (B and D) * p < 0.05, ** p < 0.01, *** p < 0.001. (G–J) Analysis was performed using three-way analysis of variance with Tukey’s post hoc test for multiple comparisons. ** p < 0.01, *** p < 0.001, **** p < 0.0001 for pre and post VET in chow VET group; ^ p < 0.05, ^^^ p < 0.001 for pre and post VET in HFD VET group; #p < 0.05 for chow VET vs. chow sedentary at post VET timepoint. A = A wave; E = E wave; HFD = high fat diet; VET = voluntary exercise training.

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  Fig. 3. Impact of high fat feeding and voluntary exercise on left ventricle morphology in male mice. (A) Representative images of Picrosirius red staining for measurement of interstitial and perivascular fibrosis with 20× magnification. (B) Representative images for Hematoxylin and Eosin for measurement of cardiomyocyte width and area with 40× magnification. (C) Representative images of Oil red O staining for measurement of interstitial lipid deposition with 40× magnification. Quantitative analysis of percent area of (D) interstitial and (E) perivascular fibrosis expressed as fold change from sedentary chow group. Quantitative analysis of (F) cardiomyocyte width and (G) cardiomyocyte area. (H) Quantitative analysis of percent area of lipid deposition expressed as fold change from sedentary chow group left ventricle mRNA expression of fatty acid transporters (I) FABP3, (J) CD36, and hypertrophy markers (K) β-MHC and (L) ANP in male VET or sedentary mice fed an HFD or chow diet. Values were calculated relative to 18S housekeeper. Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the…

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  Fig. 4. High fat feeding reduces left ventricle cardiolipin lipid expression levels and is attenuated with voluntary exercise. (A) Principal component analysis of left ventricle lipidome in male VET or sedentary mice fed an HFD or chow diet. Volcano plots of log2 fold change vs. –log10p values depicting significant changes in cardiac lipids of (B) HFD sedentary vs. chow sedentary mice and (C) HFD exercise vs. HFD sedentary mice. (D) Lipidomic quantitation of total cardiolipin content in left ventricle samples. (E) The most abundant cardiolipin species within mitochondria, cardiolipin CL(18:2)4 tetra-linoleic form, in left ventricle samples. (F) Intermediary cardiolipin lipid class, phosphatidylglycerol, measured in left ventricle samples. Hierarchical clustering analysis (heat map) of individual cardiolipin lipid analytes in left ventricles (G) of HFD sedentary vs. chow sedentary mice and (H) HFD exercise vs. HFD sedentary mice. All lipid species were normalized to phosphatidylcholine for each sample. Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. n = 10 per group. **…

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  Fig. 5. Left ventricle mitochondria complex activity is reduced in male mice fed a high fat diet. (A–C) Seahorse XF96 analysis profile to assess left ventricle mitochondrial complex activity in male VET or sedentary mice fed an HFD or chow diet. Seahorse XF96 analysis profile quantitation of left ventricle OCR for (D) complex I, (E) complex II, and (F) complex IV at study endpoint. Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. n = 7 per group. * p < 0.05, *** p < 0.001, **** p < 0.0001. HFD = high fat diet; OCR = oxygen consumption rate; VET = voluntary exercise training.

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  Fig. 6. Voluntary exercise alters left ventricle gene and protein expression related to mitochondrial biogenesis and dynamics in high fat- and chow-fed mice. (A) Western blot analysis to assess purity of crude mitochondria isolation where left ventricle mitochondrial pellet expresses mitochondrial marker TOM70 and COXIV and supernatant expresses calnexin. Protein expression of (B) LCLAT1 and (C) MFN2 in left ventricle-isolated crude mitochondria and (D) LCLAT1, (E) PGC-1α, and (F) MFN2 in left ventricle tissue from male VET or sedentary mice fed an HFD or chow diet. Left ventricle mRNA expression of (G) OPA1 and (H) DRP1 in male VET or sedentary mice fed an HFD or chow diet. Values were calculated relative to 18S housekeeper. Calnexin and β-actin were used as internal controls of protein loading in left ventricle samples and COXIV as internal control of protein loading in crude mitochondria samples. Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. (B–C) n: 5–6 per group. (D–F) n = 7 per group. (G and H) n = 9 per group. CE = chow exercise; CS = chow sedentary; COXIV =…