H9C2(2-1)

Effects of Rap, Baf, or Z-Asp on Apoptosis, Autophagy, and Cell Viability in DOX-Treated H9c2(2-1) Cells

Doxorubicin (DOX) is a potent chemotherapeutic agent; however, it causes severe heart injury via apoptosis induction in many patients. DOX-induced cardiotoxicity is attenuated by activated autophagy in the heart. Kanno et al. previously found that programmed cell death 1 (Pdcd1), an immune checkpoint receptor, inhibits DOX-induced cardiomyocyte apoptosis. In this study, they examined whether autophagy is involved in the anti-apoptotic effect of Pdcd1 against DOX.

DOX activates the executioner caspases-3/7, the key mediators of apoptosis. First, in a preliminary experiment, they determined the time-course of caspase-3/7 activation in H9c2(2-1) cells treated with 0.1-1 μM DOX (Fig. 1). These DOX concentrations were in the range of physiologically relevant cardiotoxic concentrations. At 4 h, significant caspase activation was detected with 1 μM, and at 8 h with ≥0.3 μM DOX. Therefore, they chose 1 μM DOX for further experiments to ensure early caspase activation. In Fig. 2, they showed the effects of Rap (autophagy inducer), Baf (autophagy inhibitor), and Z-Asp (pan-caspase inhibitor) on apoptosis, autophagy, and cell viability in DOX-treated H9c2(2-1) cells. Without any of these modulators, DOX (1 μM) robustly induced apoptosis after 8 h and decreased cell viability after 18 h. Interestingly, viability only decreased after the onset of apoptosis. The rate of apoptosis and cell viability in DOX-treated cells was 251.4% and 57.0% of that of untreated cells, respectively (Fig. 2a, c), suggesting that DOX initiates caspase-dependent apoptosis and subsequently induces cell death.

Overexpression Of Klotho Markedly Inhibits the H/R-Induced Apoptosis of H9C2(2-1) Cells

Early reperfusion is the most effective and important treatment for acute myocardial infarction. However, reperfusion therapy is often associated with a certain degree of myocardial damage. In the present study, Hu's team aims to identify the role of klotho, and the molecular mechanism underlying its effects, in myocardial damage in a model of myocardial hypoxia injury.

They first detected klotho protein levels by western blotting in H9c2(2-1) cells after H/R. As shown in Fig. 3A, H/R injury significantly reduced klotho expression. To further explore the role of klotho in apoptosis, they performed Hoechst 33258 nuclear staining. H/R-treated cells showed nuclear shrinkage, condensed chromatin with bright fluorescence, nuclear fragmentation, and apoptotic bodies. However, klotho-overexpressing cells had smooth, intact, and uniformly stained nuclei, which suggested that klotho overexpression alleviated H/R-induced apoptosis (Fig. 3B). To quantitatively detect the anti-apoptotic effect of klotho, H9c2(2-1) cells were infected with klotho lentivirus or control vector before H/R exposure, and apoptosis was measured by Annexin V-FITC/PI staining. As shown in Fig. 3C, H/R significantly increased apoptosis in control cells, but klotho overexpression markedly reduced the apoptotic rate. The results demonstrate that klotho inhibited H/R-induced apoptosis in H9c2(2-1) cells.