Cardiac shock wave therapy protects cardiomyocytes from hypoxia‑induced injury by modulating miR‑210

Abstract

Cardiac shock wave therapy (SWT) has been described as a novel therapeutic strategy that is able to alleviate myocardial ischemic injury. microRNA (miRNA/miR)‑210 plays a cytoprotective role in cardiomyocytes in response to hypoxia by regulating cell apoptosis. The aim of the present study was to investigate whether cardiac SWT could protect cardiomyocytes from hypoxia‑induced injury by regulating miR‑210 expression. The murine adult cardiomyocyte cell line HL‑1 was incubated for 5 h in hypoxic conditions, followed by reoxygenation for 12 h and treatment with SWT immediately following hypoxia in the present study. The cell viability was determined using an MTS assay. Western blot analyses were performed in order to detect cell signaling changes. Reactive oxygen species production was detected using dihydroethidium staining, and malondialdehyde levels were measured using the thiobarbituric acid method. miRNA and mRNA expression levels were confirmed via reverse transcription‑quantitative PCR. Apoptosis was evaluated by means of flow cytometry. HL‑1 cells were then transfected with miR‑210 mimics or inhibitors in order to alter miR‑210 expression levels, and the effects on HL‑1 cells were determined. Hypoxia led to elevated oxidative stress, enhanced cell apoptosis and upregulated miR‑210 expression levels in HL‑1 cells, while SWT could alleviate hypoxia‑induced cell injury and further promote miR‑210 expression. miR‑210 overexpression decreased apoptosis and oxidative stress during hypoxic stress in HL‑1 cells, whereas inhibition of miR‑210 increased cell apoptosis and promoted oxidative stress. Furthermore, miR‑210 inhibition could reverse the effects of SWT on HL‑1 cells. Finally, the mRNA analysis revealed that SWT significantly attenuated apoptosis‑inducing factor mitochondrion‑associated 3 and caspase 8 associated protein 2 mRNA expression levels in cardiomyocytes exposed to hypoxia, which were two targets of miR‑210. SWT could exert cardioprotective effects against hypoxia‑induced cardiac injury by modulating miR‑210.

Keywords: microrna-210; hypoxia; shock wave therapy; apoptosis; oxidative stress.

Figures

Figure 1.

Hypoxia induces cell injury in HL-1 cells, which can be reversed by SWT. (A) Cell viability was determined by MTS assay (n=4). (B) Annexin V/PI double staining was used to assess the apoptotic rates of HL-1 cells in the NC, NC + SWT, hypoxia and hypoxia + SWT groups (n=4). *P

Figure 2.

Western blotting and average data for Bcl-2, Bax, Akt, p-Akt, p38MAPK and p-p38MAPK levels in HL-1 cells exposed to hypoxia and SWT (n=4). *P

Figure 3.

SWT alleviates hypoxia-induced oxidative stress in HL-1 cells. (A) ROS production, as measured using dihydroethidium staining, in the NC, NC + SWT, hypoxia and hypoxia + SWT groups (n=3; scale bar, 100 µm). (B) MDA levels were assessed using the thiobarbituric acid method in HL-1 cells exposed to hypoxia and SWT (n=4). *P

Figure 4.

miR-210 protects cardiomyocytes against hypoxia and SWT enhances miR-210 expression in HL-1 cells exposed to hypoxia. (A) miR-210 expression in HL-1 cells exposed to hypoxia as demonstrated by RT-qPCR in the NC, NC + SWT, hypoxia, and hypoxia + SWT groups (n=4). (B) miR-210 expression in HL-1 cells exposed to normoxic and hypoxic conditions using RT-qPCR following the transfection of miR-210 mimics. Data are expressed as the relative fold change in comparison with the negative control (n=3). (C) miR-210 expression in HL-1 cells exposed to normoxic and hypoxic conditions as determined by RT-qPCR following the transfection of miR-210 inhibitors. Data are expressed as the relative fold change in comparison with the negative control (n=3). (D) Cell viability was assessed following the transfection of miR-210 mimics or miR-210 inhibitors into HL-1 cardiomyocytes compared with negative controls (n=4). (E) MDA levels were detected following the transfection of miR-210 mimics or miR-210 inhibitors into HL-1 cardiomyocytes (n=4). *P#P<0.05. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; SWT, shock wave therapy; ns, not significant; H, hypoxia; Inh, inhibitors; MDA, malondialdehyde.

Figure 5.

Western blotting of Bcl-2 and Bax. Levels of Bcl-2 and Bax in HL-1 cells exposed to hypoxia and transfected with miR-210 mimics or miR-210 inhibitors (n=3). #P<0.05. miR, microRNA; ns, not significant; NC, negative control; H, hypoxia; Inh, inhibitors.

Figure 6.

miR-210 inhibition blocks the protective effect of SWT. (A) Cell viability was determined using an MTS assay (n=4). (B) MDA levels were assessed using the thiobarbituric acid method (n=4). (C) Expression of apoptosis-associated proteins demonstrated by western blot analysis (n=3). *P#P<0.05. miR, microRNA; SWT, shock wave therapy; ns, not significant; NC, negative control; H, hypoxia; Inh, inhibitors; MDA, malondialdehyde.

Figure 7.

SWT led to a significant decrease in Casp8ap2 and AIFM3 mRNAs levels, as assessed by reverse transcription-quantitative PCR, in HL-1 cardiomyocytes exposed to hypoxia (n=3). Data were normalized to GAPDH and expressed relative to the normal control. *P