Myosin 1b Participated in the Modulation of Hypoxia/Reoxygenation-Caused H9c2 Cell Apoptosis and Autophagy

Abstract

Myocardial ischemia/reperfusion (I/R) injury seriously threats the health and life of patients with ischemia heart disease. Herein, we probed the potential influence of myosin 1b (myo1b) on hypoxia/reoxygenation- (H/R-) stimulated cardiomyocyte H9c2 cell apoptosis and autophagy. After H/R stimulation, the myo1b mRNA level in H9c2 cells was tested via qRT-PCR. Myo1b overexpression plasmid (OE-myo1b) and small interfering RNA (siRNA) targeting myo1b (si-myo1b) were transfected into H9c2 cells to alter myo1b expression in H9c2 cells. Following H/R stimulation and/or OE-myo1b (or si-myo1b) transfection, H9c2 cell apoptosis, proliferation, and autophagy were detected, respectively. We found that H/R stimulation reduced the mRNA level of myo1b in H9c2 cells and resulted in H9c2 cell apoptosis, proliferation inhibition, and autophagy. Overexpression of myo1b reversed the H/R-resulted H9c2 cell apoptosis, proliferation inhibition, and autophagy. Silence of myo1b had opposite effects, which promoted H9c2 cell apoptosis, reduced cell proliferation, and accelerated cell autophagy. Taken together, Myo1b took part in the modulation of H/R-stimulated cardiomyocyte apoptosis and autophagy, which might be serve as a potential endogenous target for prevention and therapy of I/R injury.

Figure 1

Overexpression of myo1b reversed the H/R-resulted H9c2 cell apoptosis. (a) Following H/R stimulation, the myo1b mRNA level in H9c2 cells was tested via qRT-PCR. (b) Following vector or OE-myo1b transfection, the myo1b mRNA level in H9c2 cells was tested via qRT-PCR. After H/R stimulation and OE-myo1b transfection, H9c2 cell apoptosis was measured via TUNEL assay (c) and Annexin V-FITC/PI staining (d); the Bcl-2, Bax, and cleaved-caspase 3 protein levels in H9c2 cells were evaluated through western blotting (e). ^∗∗P < 0.01 vs. control group; ^##P < 0.01 vs. vector group; P < 0.01 vs. H/R + vector group.

Figure 2

Overexpression of myo1b weakened the H/R-resulted H9c2 cell proliferation inhibition and autophagy. After H/R stimulation and OE-myo1b transfection, H9c2 cell proliferation was detected via EdU staining (a). H9c2 cell autophagy was measured through LC3B and p62 protein levels via western blotting (b) and autophagy double-labeled adenovirus assay (c). ^∗∗P < 0.01 vs. control group; P < 0.01 vs. H/R + vector group.

Figure 3

Myo1b expression was silenced in H9c2 cells. Following si-NC or si-myo1b transfection, the mRNA (a) and protein (b) expression levels of myo1b in H9c2 cells were tested via qRT-PCR and western blotting, respectively. ^∗∗P < 0.01 vs. si-NC group.

Figure 4

Silence of myo1b promoted H9c2 cell apoptosis. After si-NC or si-myo1b transfection, H9c2 cell apoptosis was measured via TUNEL assay (a) and Annexin V-FITC/PI staining (b); the Bcl-2, Bax, and cleaved-caspase 3 protein levels in H9c2 cells were evaluated through western blotting (c). ^∗∗P < 0.01 vs. si-NC group.

Figure 5

Silence of myo1b reduced H9c2 cell proliferation and accelerated cell autophagy. After si-NC or si-myo1b transfection, H9c2 cell proliferation was detected via EdU staining (a); H9c2 cell autophagy was measured through LC3B and p62 protein levels via western blotting (b) and autophagy double-labeled adenovirus assay (c). ^∗∗P < 0.01 vs. si-NC group.