Cardioprotection Attributed to Aerobic Exercise-Mediated Inhibition of ALCAT1 and Oxidative Stress-Induced Apoptosis in MI Rats

Figures

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  Figure 1. Schematic diagram of the animal experimental design and protocol. One week after the operation, male Sprague-Dawley rats in the MI + EX group and EX group were subjected to exercise on a motorized rodent treadmill for 4 weeks. All rats were euthanized for cardiac tissue and serum sample collection after a 4-week exercise protocol.

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  Figure 2. The expression of ALCAT1 mRNA and protein. (A) Quantitative reverse transcription polymerase chain reaction (RTq–PCR) analysis of ALCAT1 in the heart (n = 3). (B) Western blot images and densitometric quantitative analysis of ALCAT1 protein expression in the heart (n = 4). Values presented are the means ± SEM. CTL, normal control; sham; MI-1 h, 1 h after myocardial infarction; MI-6 h, 6 h after myocardial infarction; MI-12 h, 12 h after myocardial infarction; MI-24 h, 24 h after myocardial infarction; MI-5d, 5 days after myocardial infarction (** p < 0.01 vs. sham).

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  Figure 3. Effect of MI on myocardial injury. ELISA kit analysis of myocardial injury level, CK (A), CKMB (B), LDH and BNP (C,D) in rat serum (n = 6). Values presented are the means ± SEM. CTL, normal control; sham; MI-1 h, 1 h after myocardial infarction; MI-6 h, 6 h after myocardial infarction; MI-12 h, 12 h after myocardial infarction; MI-24 h, 24 h after myocardial infarction; MI-5d, 5 days after myocardial infarction (* p < 0.05, ** p < 0.01 vs. sham).

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  Figure 4. Tests of myocardial fibrosis after MI. Blue collagen, a fibrosis marker, red cardiac muscle fibers, and nuclei in dark brown emerged in the microscopic images. (A) Masson-negative staining portions (cardiac muscle fibers) and Masson-positive staining portions (blue collagen) were digitally measured by collagen volume of fraction (CVF %) (n = 3). (B) Values presented are the means ± SEM. CTL, normal control; sham; MI-1 h, 1 h after MI; MI-6 h, 6 h after MI; MI-12 h, 12 h after MI; MI-24 h, 24 h after MI; MI-5d, 5 days after MI (** p < 0.01 vs. sham).

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  Figure 5. Tests of myocardial function after MI. The cardiac hemodynamic parameters measured included systolic and diastolic function of the left ventricle measured with (A) LVSP, (B) LVEDP, (C) +dp/dt, maximum pressure increasing rate, and (D) −dp/dt, maximum pressure decreasing rate (n = 6.7). Values presented are the means ± SEM. CTL, normal control; sham; MI-1 h, 1 h after myocardial infarction; MI-6 h, 6 h after myocardial infarction; MI-12 h, 12 h after myocardial infarction; MI-24 h, 24 h after myocardial infarction; MI-5d, 5 days after myocardial infarction (* p < 0.05, ** p < 0.01 vs. sham).

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  Figure 6. Exercise training mitigates cardiac dysfunction after MI. Blue-stained collagen, a fibrosis marker, red-stained cardiac muscle fibers, and dark brown nuclei visualized by microscopy (scale bar = 0.61 µm). (A,B) The areas of no Masson’s trichrome staining (cardiac muscle fibers) and Masson’s trichrome-positive staining (blue-stained collagen) were digitally measured to determine the collagen volume fraction (CVF %) (n = 3). The systolic and diastolic function of the left ventricle was assessed by measuring cardiac hemodynamic parameters including (C) LVSP, (D) LVEDP, (E) −dp/dt, maximum pressure decreasing rate, and (F) +dt/dt, maximum pressure increasing rate (n = 6.7). The values are presented as the mean ± SEM. CTL, normal control; MI, myocardial infarct; EX, CTL + exercise; MI + EX, MI + exercise. * p < 0.05, ** p < 0.01.

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  Figure 7. mRNA and protein expression of ALCAT1. (A) Quantitative reverse transcription polymerase chain reaction (RTq-PCR) analysis of ALCAT1 expression in the heart (n = 6). (B,C) Western blot images of ALCAT1 protein in the serum and heart and densitometric analysis of the bands (n = 3). The values are presented as the mean ± SEM. CTL, normal control; MI, myocardial infarct; EX, CTL + exercise; MI + EX, MI + exercise. * p < 0.05, ** p < 0.01.

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  Figure 8. Effect of exercise and no exercise on myocardial oxidative stress following MI. ELISA of antioxidant levels. T-AOC (A,B), GSH-PX levels (C,D), CAT levels and SOD levels (E–H) in serum and tissue. (I) Representative blots of superoxide dismutase 1 and 2 (SOD1 and SOD2) and quantification of these data. (J,K) The level of MDA, an indicator of lipid peroxidation, was determined in rat serum and heart tissue samples with a TBARS kit. The values are presented as the mean ± SEM (n = 3, 6, 7). CTL, normal control; MI, myocardial infarct; EX, CTL + exercise; MI + EX, MI + exercise. * p < 0.05, ** p < 0.01.

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  Figure 9. Effect of exercise and no exercise on cardiomyocyte apoptosis following MI. (A) Representative blots of the apoptotic proteins, p53, Cyt C, Bcl-2, and Bax, in tissue (n = 3). (B) Bcl-2/BAX ratio. (C) The activity of caspase 3 in the myocardium was measured using a spectrophotometer (n = 6). The values are presented as the mean ± SEM. CTL, normal control; MI, myocardial infarct; EX, CTL + exercise; MI + EX, MI + exercise. * p < 0.05, ** p < 0.01.

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  Figure 10. Proposed mechanism by which exercise-induced cardioprotection protects against myocardial infarction (MI)-induced injury in the heart. Exercise training inhibits the production of ALCAT1 in circulation and cardiac tissues and exerts cardioprotective effects against MI-induced injury by suppressing myocardial apoptosis and enhancing antioxidant activity.