Moderate Exercise in Spontaneously Hypertensive Rats Is Unable to Activate the Expression of Genes Linked to Mitochondrial Dynamics and Biogenesis in Cardiomyocytes

Abstract

Hypertension (HTN) is a public health concern and a major preventable cause of cardiovascular disease (CVD). When uncontrolled, HTN may lead to adverse cardiac remodeling, left ventricular hypertrophy, and ultimately, heart failure. Regular aerobic exercise training exhibits blood pressure protective effects, improves myocardial function, and may reverse pathologic cardiac hypertrophy. These beneficial effects depend at least partially on improved mitochondrial function, decreased oxidative stress, endothelial dysfunction, and apoptotic cell death, which supports the general recommendation of moderate exercise in CVD patients. However, most of these mechanisms have been described on healthy individuals; the effect of moderate exercise on HTN subjects at a cellular level remain largely unknown. We hypothesized that hypertension in adult spontaneously hypertensive rats (SHRs) reduces the mitochondrial response to moderate exercise in the myocardium. Methods: Eight-month-old SHRs and their normotensive control-Wistar-Kyoto rats (WKYR)-were randomly assigned to moderate exercise on a treadmill five times per week with a running speed set at 10 m/min and 15° inclination. The duration of each session was 45 min with a relative intensity of 70-85% of the maximum O₂ consumption for a total of 8 weeks. A control group of untrained animals was maintained in their cages with short sessions of 10 min at 10 m/min two times per week to maintain them accustomed to the treadmill. After completing the exercise protocol, we assessed maximum exercise capacity and echocardiographic parameters. Animals were euthanized, and heart and muscle tissue were harvested for protein determinations and gene expression analysis. Measurements were compared using a nonparametric ANOVA (Kruskal-Wallis), with post-hoc Dunn's test. Results: At baseline, SHR presented myocardial remodeling evidenced by left ventricular hypertrophy (interventricular septum 2.08 ± 0.07 vs. 1.62 ± 0.08 mm, p < 0.001), enlarged left atria (0.62 ± 0.1 mm vs. 0.52 ± 0.1, p = 0.04), and impaired diastolic function (E/A ratio 2.43 ± 0.1 vs. 1.56 ± 0.2) when compared to WKYR. Moderate exercise did not induce changes in ventricular remodeling but improved diastolic filling pattern (E/A ratio 2.43 ± 0.1 in untrained SHR vs. 1.89 ± 0.16 trained SHR, p < 0.01). Histological analysis revealed increased myocyte transversal section area, increased Myh7 (myosin heavy chain 7) expression, and collagen fiber accumulation in SHR-control hearts. While the exercise protocol did not modify cardiac size, there was a significant reduction of cardiomyocyte size in the SHR-exercise group. Conversely, titin expression increased only WYK-exercise animals but remained unchanged in the SHR-exercise group. Mitochondrial response to exercise also diverged between SHR and WYKR: while moderate exercise showed an apparent increase in mRNA levels of Ppargc1α, Opa1, Mfn2, Mff, and Drp1 in WYKR, mitochondrial dynamics proteins remained unchanged in response to exercise in SHR. This finding was further confirmed by decreased levels of MFN2 and OPA1 in SHR at baseline and increased OPA1 processing in response to exercise in heart. In summary, aerobic exercise improves diastolic parameters in SHR but fails to activate the cardiomyocyte mitochondrial adaptive response observed in healthy individuals. This finding may explain the discrepancies on the effect of exercise in clinical settings and evidence of the need to further refine our understanding of the molecular response to physical activity in HTN subjects.

Keywords: cardiac remodeling; exercise; heart; hypertension; mitochondrial dynamics.

Figure 1

Hypertensive rat model validation. (A) Systolic and diastolic blood pressure (SBP and DBP) and (B) Heart rate values in WKYR and SHR 8 months old. (C) Heart weight/tibia length ratio as heart size parameter at protocol ending. (D) H&E staining, cardiomyocyte area and (E) myosin heavy chain 7 (Myh7) and (F) Titin (Ttn) expression as hypertrophy parameters in WKYR compared with SHR. (G) Masson's trichrome staining to determine interstitial fibrosis in SHR compared with WKYR after training. (H,I) Systolic and diastolic blood pressure at beginning (Baseline) and ending (Post-training) in WKYR and SHR. Groups: WYKR untrained (n = 8), WYKR trained (n = 8), SHR untrained (n = 10) and SHR trained (n = 10). All values are mean ± SD. Statistical significance was calculated using ANOVA, and group comparisons were performed using Tukey's test. *p < 0.05 untrained vs. trained group, **p < 0.01 untrained vs. trained group, ^#p < 0.05, ^###p < 0.001, and ^####p < 0.0001 WKYR vs. SHR. Circles and triangles correspond to outlier data in WKYR and SHR, respectively.

Figure 2

Ecochardiographic evaluation after completing the 8-week training period. LAD, Left atrial diameter; IV septum, Interventricular septum, LVEDd, Left ventricular end-diastolic diameter; LVESd, Left ventricular end systolic diameter; LVEF, Left ventricular ejection fraction; E/A ratio, Trans-mitral E wave/A wave ratio. Groups: WYKR untrained (n = 8), WYKR trained (n = 8), SHR untrained (n = 10) and SHR trained (n = 10). All values are mean ± SD. Statistical significance was calculated using ANOVA, and group comparisons were performed using Tukey's test. *p < 0.05 untrained vs. trained group, ^#p < 0.05 WKYR vs. SHR and ^##p < 0.01 WKYR vs. SHR.

Figure 3

Mitochondrial modulators expression in WKYR and SHR. (A) Ppargc1a, Opa1, Mfn2, Drp1, and Mff mRNA levels were determined by RT-qPCR in tibial and soleus muscles total RNA extract at ending of exercise protocol. Values were normalized to Pabpn1 mRNA expression. (B) Ppargc1a, Opa1, Mfn2, Drp1, and Mff mRNA levels were determined by RT-qPCR in heart total RNA extract at ending of exercise protocol. Values were normalized to Pabpn1 mRNA expression. (C) s-OPA1/l-OPA1 protein ratio was determined by Western blot in total protein extracts of WKYR and SHR hearts and normalized by GAPDH levels. Groups: WYKR untrained (n = 8), WYKR trained (n = 6), SHR untrained (n = 10), and SHR trained (n = 10). All values of each group are presented as mean ± SD. Statistical significance was calculated using ANOVA, and group comparisons were performed using Tukey's test, ^#p < 0.05 and ^##p < 0.01 WKYR vs. SHR. Circles and triangles correspond to outlier data in WKYR and SHR, respectively.

Figure 4

Metabolic and mitochondrial modulators activation in WKYR and SHR product of training program (A) phospho-AKT, (B) phospho-AMPK, and (C) phospho-mTOR was determined through Western blot in total protein extracts of WKYR and SHR hearts as an activation parameter and normalized by total AKT, AMPK, and mTOR, respectively. Additionally, in (C) we determined the total protein level of mTOR normalized by GAPDH levels. Images show two representative samples of each group and graphs include to the whole of samples for group. Groups: WYKR untrained (n = 8), WYKR trained (n = 8), SHR untrained (n = 10), and SHR trained (n = 10). All values are mean ± SD. Statistical significance was calculated using ANOVA, and group comparisons were performed using Tukey's test, *p < 0.05 untrained vs.f trained group, ^###p < 0.001 WKYR vs. SHR. Circles and triangles correspond to outlier data in WKYR and SHR, respectively.