Exercise Training Reveals Inflexibility of the Diaphragm in an Animal Model of Patients With Obesity‐Driven Heart Failure With a Preserved Ejection Fraction

Figures

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  Figure 1. Schematic outline summarizing the present study. Aim 1 of the study evaluated at 20 weeks the effects of an obese cardiometabolic rat model of heart failure with a preserved ejection fraction (HFpEF) on the diaphragm compared with lean controls (Con), while aim 2 of the study evaluated at 28 weeks whether 8 weeks of exercise training (either high‐intensity interval training [HIIT] or moderate‐continuous training [MCT]) could reverse diaphragm alterations as a secondary prevention compared with sedentary (Sed) HFpEF rats. Dashed lines indicate the time point when animals were evaluated (20 or 28 weeks of age), which included assessment of exercise capacity, cardiac function (echocardiography/invasive hemodynamics), muscle function, and subsequent tissue analyses. The sample size (n) for each group are provided in parentheses.

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  Figure 2. Animal model characteristics at 20 weeks, which demonstrates that a typical heart failure with a preserved ejection fraction (HFpEF) phenotype was developed in obese rats compared with lean controls (Con). HFpEF was confirmed by measures of diastolic dysfunction (A and B) in the presence of normal systolic function (C) in parallel with a cardiac hypertrophy (D). In the absence of an increase in systolic blood pressure (SBP) (E), other comorbidities were developed in HFpEF rats including hyperglycemia (F) and obesity (G), as well as an impaired exercise capacity (H). Data are presented as mean±SEM, with n=8 for Con and n=12 for HFpEF, analyzed with unpaired t test. *P<0.05 vs Con. E/E′ indicates early mitral flow to tissue velocity; LVEF, left ventricular ejection fraction; LVEDP, left ventricular end‐diastolic pressure; LV/TL, left ventricle/tibial length; V˙O2peak, peak oxygen uptake.

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  Figure 3. Representative immunofluorescence sections of the diaphragm from control (Con; A) and heart failure with a preserved ejection fraction (HFpEF) (B) rats at 20 weeks, where sections were incubated with an antibody against myosin slow type I fibers to allow detection of type I (bright green) and type II (dark green) fiber proportion and size, with cell nuclei stained (blue) to establish fiber boundaries (scale bars represent 100 μm). The fiber cross‐sectional area (CSA) was increased in type I fibers in the diaphragm from HFpEF rats (C), with no effects observed for the type II isoform (D). Protein expression of MuRF1 (E) and MuRF2 (F) was also lower in HFpEF, with both proteins implicated in cooperating to regulate fiber size. The abbreviations C and H for Western blot lanes represent loaded samples from Con and HFpEF, respectively, with GAPDH used as a loading control. Data are presented as mean±SEM, analyzed with unpaired t test or Mann‐Whitney (type I CSA), with n=8 for Con and n=12 for HFpEF. *P<0.05 vs Con.

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  Figure 4. Heart failure with a preserved ejection fraction (HFpEF) induced a shift towards an oxidative phenotype in the diaphragm compared with controls (Con), with a greater proportion of type I (A) and lower proportion of type II fibers (B) at 20 weeks (see Figure 3A and 3B for representative diaphragm sections). This was associated with a greater peroxisome proliferator‐activated receptor gamma coactivator 1‐α (PGC1‐α) protein expression (C) as well as citrate synthase (D) and catalase enzyme activity (E). In contrast, in situ mitochondrial respiration revealed functional deficits in the diaphragm of HFpEF rats, with an increased leak state (LCI) (F) and impaired mitochondrial coupling efficiency (respiratory control ratio [RCR]) (G). Respiration states assessed, in turn, included: LCI, complex I leak respiration following addition of malate and glutamate; PCI, complex I phosphorylated state following addition of ADP; PCI + II, complex II phosphorylated state following addition of succinate. RCR (ie, PCI/LCI) and substrate control ratio (SCR; ie, PCI/PCI + II). The abbreviations C and H for Western blot lanes represent loaded samples from Con and HFpEF, respectively, with GAPDH used as…

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  Figure 5. Animal model characteristics at 28 weeks, which demonstrates the heart failure with a preserved ejection fraction (HFpEF) phenotype in sedentary (Sed) obese rats was generally not influenced by 8 weeks of either high‐intensity interval training (HIIT) or moderate‐continuous training (MCT). Compared with sedentary HFpEF rats, exercise training did not influence cardiac measures of diastolic (A and B) or systolic function (C), cardiac hypertrophy (D), or multiple comorbidities such as hypertension (E), hyperglycemia (F), and obesity (G). Importantly, however, both exercise training interventions of HIIT and MCT improved exercise capacity compared with the sedentary HFpEF condition (H). Data are presented as mean±SEM, with n=13 for Sed, n=11 for HIIT, and n=11 for MCT, analyzed with 1‐way ANOVA or Kruskal‐Wallis test with Dunn post hoc (glucose and V˙O2peak). *P<0.05 vs Sed, § P<0.05 vs HIIT. E/E′ indicates early mitral flow to tissue velocity; LVEF, left ventricular ejection fraction; LVEDP, left ventricular end‐diastolic pressure; LV/TL, left ventricle/tibial length; SPB, systolic blood pressure; V˙O2peak, peak oxygen uptake.

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  Figure 6. Representative immunofluorescence sections of the diaphragm at 28 weeks from heart failure with a preserved ejection fraction (HFpEF) rats that remained sedentary (Sed; A) or performed 8 weeks of high‐intensity interval training (HIIT; B) or moderate‐continuous training (MCT; C), where sections were incubated with an antibody against myosin slow type I fibers to allow detection of type I (bright green) and type II (dark green) fiber proportion and size, with cell nuclei stained (blue) to establish fiber boundaries (scale bar represents 100 μm). Exercise training in HFpEF rats that performed HIIT or MCT had no effect on fiber cross‐sectional area (CSA) in both type I (D) and II (E) isoforms compared with their sedentary counterparts. Similarly, exercise training had no effect on the protein expression of either MuRF1 (F) or MuRF2 (G). The abbreviations S, H, and M for Western blot lanes represent loaded samples from HFpEF in the sedentary (S), HIIT (H), or MCT (M) groups, respectively, with GAPDH used as a loading control. Data are presented as mean±SEM, analyzed with 1‐way ANOVA, with n=13 for Sed, n=11 for HIIT, and n=11 for MCT.

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  Figure 7. In heart failure with a preserved ejection fraction (HFpEF) rats at 28 weeks, fiber type proportion in the diaphragm for type I (A) and II (B) isoforms was not influenced following either high‐intensity interval training (HIIT) or moderate‐continuous training (MCT) when compared with sedentary (Sed) controls (see Figure 6A through 6C for representative diaphragm sections). Similarly, markers of mitochondrial content remained unchanged, including the protein expression of peroxisome proliferator‐activated receptor gamma coactivator 1‐α (PGC1‐α) (C) and enzyme activities of both citrate synthase (D) and catalase (E), as were functional measures of mitochondrial respiration (F) and the respective control ratios (G). Respiration states assessed, in turn, included: LCI, complex I leak respiration following addition of malate and glutamate; PCI, complex I phosphorylated state following addition of ADP; PCI + II, complex II phosphorylated state following addition of succinate. The abbreviations S, H, and M for Western blot lanes represent loaded samples from HFpEF rats in the sedentary (S), HIIT (H), or MCT (M) groups, respectively, with GAPDH used as a loading control. Data are…

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  Figure 8. In vitro diaphragm function at 28 weeks in heart failure with a preserved ejection fraction (HFpEF) rats assessed in isolated fiber bundles revealed that compared with sedentary (Sed) rats, the interventions of high‐intensity interval training (HIIT) or moderate‐continuous training (MCT) had no benefits on contractile function (A) or fatigue resistance (B). Data are presented as mean±SEM, analyzed with 2‐way repeated measures ANOVA, with n=13 for Sed, n=11 for HIIT, and n=11 for MCT.

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  Figure 9. Alterations induced by heart failure with a preserved ejection fraction (HFpEF) in the soleus limb muscle at 20 weeks (control [Con] vs HFpEF) and also following 8 weeks of exercise training at 28 weeks (HFpEF rats that were sedentary [Sed] or performed high‐intensity interval training [HIIT] or moderate‐continuous training [MCT]), where in vitro maximal force generation (A) and fatigability (B) were assessed, followed by tissue analyses including muscle wet‐weight normalized to tibia length (TL) (C), citrate synthase activity (D), and peroxisome proliferator‐activated receptor gamma coactivator 1‐α (PGC1‐α) protein expression (E) as indices of mitochondrial density, and the expression of proteins involved in the muscle remodeling process MuRF1 (F) and MuRF2 (G). Proteins of interest were normalized to loading control GAPDH, with representative Western blot data shown in (H). At 20 weeks, HFpEF induced no functional alterations (A and B) but did reduce muscle mass (C), which was associated with a higher citrate synthase activity (D) and a trend for atrogins MuRF1 and MuRF2 to be increased (F and G). At 28 weeks, exercise training in HFpEF tended to increase muscle mass (C) and…