Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained beta-adrenergic hyperactivity

Abstract

Myocardial hypertrophy and dysfunction occur in response to excessive catecholaminergic drive. Adverse cardiac remodelling is associated with activation of proinflammatory cytokines in the myocardium. To test the hypothesis that exercise training can prevent myocardial dysfunction and production of proinflammatory cytokines induced by beta-adrenergic hyperactivity, male Wistar rats were assigned to one of the following four groups: sedentary non-treated (Con); sedentary isoprenaline treated (Iso); exercised non-treated (Ex); and exercised plus isoprenaline (Iso+Ex). Echocardiography, haemodynamic measurements and isolated papillary muscle were used for functional evaluations. Real-time RT-PCR and Western blot were used to quantify tumour necrosis factor alpha, interleukin-6, interleukin-10 and transforming growth factor beta(1) (TGF-beta(1)) in the tissue. NF-B expression in the nucleus was evaluated by immunohistochemical staining. The Iso rats showed a concentric hypertrophy of the left ventricle (LV). These animals exhibited marked increases in LV end-diastolic pressure and impaired myocardial performance in vitro, with a reduction in the developed tension and maximal rate of tension increase and decrease, as well as worsened recruitment of the Frank-Starling mechanism. Both gene and protein levels of tumour necrosis factor alpha and interleukin-6, as well as TGF-beta(1) mRNA, were increased. In addition, the NF-B expression in the Iso group was significantly raised. In the Iso+Ex group, the exercise training had the following effects: (1) it prevented LV hypertrophy; (ii) it improved myocardial contractility; (3) it avoided the increase of proinflammatory cytokines and improved interleukin-10 levels; and (4) it attenuated the increase of TGF-beta(1) mRNA. Thus, exercise training in a model of beta-adrenergic hyperactivity can avoid the adverse remodelling of the LV and inhibit inflammatory cytokines. Moreover, the cardioprotection is related to beneficial effects on myocardial performance.

Figure 1. Effects of exercise training on the myocardial hypertrophy induced by isoprenaline

A, heart weight/body weight (HW/BW) ratio for non-trained (Con, n= 12; Iso, n= 13) and exercise-trained rats (Ex, n= 8; Iso+Ex, n= 8) determined after 13 weeks of follow-up. B, left ventricular weight/body weight (LVW/BW) ratio for all groups. C, right ventricular weight/body weight (RVW/BW) ratio. Significant differences in ANOVA, †P < 0.001 vs. other groups.

Figure 2. Exercise training increases the myocardial performance, even after β-adrenergic hyperactivity

Data were obtained at muscle lengths corresponding to 100% of L_max from non-trained (Con, n= 8; Iso, n= 13) and exercise-trained rats (Ex, n= 8; Iso+Ex, n= 6), as described in the Methods. A, peak developed tension (DT). B, maximal positive time derivative of developed tension (+dT/dt). C, maximal negative time derivative of developed tension (−dT/dt). D, resting tension (RT). Significant differences in ANOVA, *P < 0.05 vs. Con group, †P < 0.05 vs. other groups.

Figure 3. Left ventricular papillary muscle developed and resting length–tension curves obtained from Con (○), Iso (▴), Ex (•) and Iso+Ex groups (▵) as described in the Methods

A, straight lines were fitted to the developed length–tension relationships using linear regression analysis. The resulting mean slopes, corresponding to developed tension, were compared between groups. ANOVA and post hoc Newman–Keuls test were used for multiple comparisons. *P < 0.01 when the developed tension was compared with Con and Iso groups for each stretching, †P < 0.001 when slope was compared with other groups. B, the resting length–tension curves for the four groups were fitted to monoexponential non-linear relationships. The resulting means β₁ corresponded to resting tension were compared between groups.

Figure 4. Gene expression by real-time RT-PCR in myocardium as described in the Methods

A, gene expression of proinflammatory (TNF-α and IL-6) and anti-inflammatory cytokines (IL-10). B, ratio of TNF-α/IL-10 and IL-6/IL-10. C, gene expression of TGF-β₁. All values were normalized for levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are from five rats in each group. ANOVA and post hoc Newman–Keuls test were used for multiple comparisons. *P < 0.05 vs. Con and Ex groups. †P < 0.05 vs. other groups.

Figure 5. The protein expression by Western blot in myocardium as described in the Methods

A, protein expression of TNF-α, IL-6 and IL-10. The upper panel is a representative Western blot. B, ratio of TNF-α/IL-10 and IL-6/IL-10. All values were normalized for levels of GAPDH. Specifics blots of the GAPDH for each cytokine protein are shown in A. Data are from five rats in each group. ANOVA and post hoc Newman–Keuls test were used for multiple comparisons. †P < 0.05 vs. other groups.

Figure 6. Representative photomicrographs showing localization of TNF-α and NF-κB in myocardium

Brown stain indicates positive immunoreactivity. Intense immunoreactivity was detected in sedentary isoprenaline-treated animals (B). It is noteworthy that TNF-α was readily positive and localized to areas of inflammatory cell infiltration and uninjured myocardial tissue (open arrows). However, in exercise-trained animals there was inhibition of the intense proinflammatory staining after isoprenaline overload. Positive immunoreactivity to TNF-α was rarely detected in myocardium of Con (A), Ex (C) and Iso+Ex groups (D). In the right-hand panels, the filled arrows indicate positive nuclear immunoreactivity for NF-κB in Con (F), Iso (G), Ex (H) and Iso+Ex groups (I). E and J, statistical analysis for TNF-α and NF-κB, respectively. Numbers of rats used in each group and fold-expression relative to the Con group are shown. *P < 0.05 in ANOVA. Magnification ×40 (Scale bar, 250 μm).