Physiological ischemic training improves cardiac function through the attenuation of cardiomyocyte apoptosis and the activation of the vagus nerve in chronic heart failure

Figures

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  Figure 1. Experimental flow chart.

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  Figure 2. Functional outcomes of the control group and experimental group. (A) Cardiac function (LVEF, LVEDD and LVESD). (B) Plasma BNP level in peripheral blood. (C) Cardiopulmonary fitness (VO2max and AT). (D) Exercise capacity (distance under the 6MWT). (E) Quality of life (MLHFQ scores, lower score indicating higher quality of life). ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001. CON, control group (regular treatments, including pharmaceutical treatment and health education); EXP, experimental group (additional PIT intervention after regular treatments).

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  Figure 3. Effects on LVEF and cardiac biomarkers after various PIT protocols in CHF rats. (A) Left ventricular ejection fraction (LVEF). (B) Plasma NT-proBNP level. (C) Plasma tumor necrosis factor-α (TNF-α) level. (D) Plasma interleukin 6 (IL-6) level. *p < 0.05, **p < 0.01, and ***p < 0.001. SO, sham operation; CHF, chronic heart failure; PIT, physiological ischemic training.

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  Figure 4. Effects of Ang II and PIT on programmed cell death of H9c2 cells and cardiomyocytes of CHF rats, respectively. (A) Apoptosis detected by flow cytometry in control and Ang II treated H9c2 cells. (B) Protein expression of Bcl-2, Bax, and Cleaved-caspase3 in control and Ang II treated H9c2 cells. (C) Protein expression of Bcl-2, Bax, and Cleaved-caspase3 in cardiomyocytes of SO, CHF and PIT rats. (D) Necrosis detected by YO-PRO-1/PI staining in control and Ang II treated H9c2 cells. (E) Protein expression of RIPK1 and MLKL in control and Ang II treated H9c2 cells. (F) Protein expression of RIPK1 and MLKL in cardiomyocytes of SO, CHF and PIT rats. (G) Ferroptosis detected by ROS assay in control and Ang II treated H9c2 cells. (H) Protein expression of SLC7A11 and GPX4 in control and Ang II treated H9c2 cells. (I) Protein expression of SLC7A11 and GPX4 in cardiomyocytes of SO, CHF and PIT rats. (J) Autophagy detected by MDC assay in control and Ang II treated H9c2 cells. (K) Protein expression of p62 and LC3B in control and Ang II treated H9c2 cells. (L) Protein expression of p62 and LC3B in cardiomyocytes of SO, CHF and PIT rats.

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  Figure 5. Effects of PIT on autonomic nervous activity in CHF rats. (A) Representative echocardiography images and quantitative results of LVEF. (B) Concentration of NE in circulation. (C) Activity of AChE in cardiac tissues. (D) Correlation between LVEF and NE concentration as wells AChE activity. Correlation coefficients and p values were obtained from Spearman’s correlation analysis. ns, not significant; **p < 0.01 and ***p < 0.001. SO, sham operation; CHF, chronic heart failure; PIT, physiological ischemic training.

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  Figure 6. Cardioprotective effect of PIT under vagus nerve stimulation and cut. (A) Representative echocardiography images and quantitative results of LVEF. (B) Distribution of cholinergic neurons in cardiac tissues by Karnovsky–Roots staining, and density of cholinergic neurons. (C) AChE activity in cardiac tissues. (D) Bcl-2, Bax, and Cleaved-caspase3 expression by western blot. *p < 0.05, **p < 0.01, and ***p < 0.001. SO, sham operation; CHF, chronic heart failure; PIT, physiological ischemic training; VNS, vagus nerve stimulation; VNC, vagus nerve cut.

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  Figure 7. Cardioprotective effect of PIT under muscarinic M2 receptor activation and inhibition. (A) Representative echocardiography images and quantitative results of LVEF. (B) Distribution of cholinergic neurons in cardiac tissues by Karnovsky and Roots staining, and density of cholinergic neurons. (C) AChE activity in cardiac tissues. (D) Proteins related to apoptosis by western blot. *p < 0.05, **p < 0.01, and ***p < 0.001. SO, sham operation; CHF, chronic heart failure; PIT, physiological ischemic training; M2R+, M2 receptor activation; M2R−, M2 receptor inhibition.