Cardiac Shock Wave Therapy Attenuates H9c2 Myoblast Apoptosis by Activating the AKT Signal Pathway

Abstract

Background: Previous studies have demonstrated that Cardiac Shock Wave Therapy (CSWT) improves myocardial perfusion and cardiac function in a porcine model of chronic myocardial ischemia and also ameliorates myocardial ischemia in patients with severe coronary artery disease (CAD). Apoptosis plays a key role in ischemic myocardial pathogenesis. However, it remains unclear whether CSWT is beneficial for ischemia/hypoxia (I/H)-induced myocardial cell apoptosis and by which mechanism CSWT could improve heart function. We put forward the hypothesis that CSWT might protect heart function during ischemia/hypoxia by decreasing apoptosis. Methods: We generated ischemia/hypoxia (I/H)-induced apoptosis in the H9c2 myoblast cell line to examine the CSWT function and possible mechanisms. H9c2 cells were treated under hypoxic serum-starved conditions for 24 h and then treated with or without CSWT (500 shots, 0.06, 0.09, 0.12mJ/mm²). The apoptotic cell rate was determined by flow cytometry assay, cell viability was examined by the MTT assay, nuclear fragmentation was detected by Hoechst 33342 staining, and the mitochondrial-mediated intrinsic pathway of apoptosis was assessed by the expression of Bax and Bcl-2 protein and Caspase3 activation. Results: First, apoptosis could be induced by ischemia/hypoxia in H9c2 cells. Second, CSWT attenuates the cell death and decreases the H9c2 cell apoptosis rate induced by ischemia and hypoxia. Third, CSWT suppresses the expression of apoptosis molecules that regulate the intrinsic pathway of apoptosis in H9c2 cells. Fourth, CSWT increases the phosphorylation of AKT, which indicates the activation of the PI3K-AKT pathway. Conclusions: These results indicate that CSWT exerts a protective effect against I/H-induced cell death, potentially by preventing the activation of components of the mitochondrial-dependent intrinsic apoptotic pathway. We also demonstrate that the PI3K-Akt pathway may be involved in the CSWT effects on apoptosis.

Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia

“…Previous studies have used H9c2 cells instead of cardiomyocytes to investigate the occurrence of autophagy under hypoxia and have reached conclusions consistent with those of in vivo studies [26, 27]. Our in vitro study using the hypoxic model of H9c2 [11] demonstrated that CSWT could inhibit hypoxia-induced apoptosis, which was further verified in an animal study (unpublished). Thus, the model used in this study may be reliable, but further in vivo studies must be conducted in the future.…”

“…Compared with the Modulith SLC system with a complex water sac that was used in our previous study [11], the Duolith VET system used in the present study was designed with an independent probe, and the cells could be directly treated in the water bath. The attenuation of ultrasonic energy was markedly avoided, and relatively lower energies (0.02, 0.05, and 0.10 mJ/mm2) were chosen to evaluate the effect of SW treatment on hypoxic H9c2 cells.…”

“…In this manner, the optimal SW energy with the best therapeutic effect was chosen. After SW treatment, the cells were cultured in the incubator with 5% CO 2 and 95% air (v/v) for another 24 h before being harvested [11]. …”

“…Equivalent proteins (10-20 µg) were separated by SDS-polyacrylamide gels (12%) and electrically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) [11]. The membranes were probed with rabbit primary antibodies in a 1: 1000 dilution, including anti-LC3B (Sigma), anti-GAPDH (internal reference, CST), anti-Beclin1 (CST), anti-Phospho-AMPK (Thr172, CST), anti-AMPK (CST), anti-Phospho-mTOR (Ser2448, CST), anti-mTOR (CST), anti-Sirt1 (CST), and anti-HIF-1α (Abcam).…”

“…The mechanisms of heart protection after CSWT are still unclear but may include promotion of the recruitment of endothelial progenitor cells [9], stimulation of neovascularization [10], and reduction of oxidative stress, inflammation [5], and fibrosis [6]. Our recent in vitro [11] and in vivo studies (unpublished data) demonstrated that CSWT could inhibit apoptosis in ischemic/hypoxic myocardium and consequently enhance cell vitality and heart function. However, the mechanism of CSWT appears to be multifactorial and needs to be further elucidated.…”