Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose

Abstract

To investigate the effects of p53 upregulated modulator of apoptosis (PUMA) on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms. H9C2 cardiomyocytes were treated with 5.5mmol/L (control group) or 35 mmol/L glucose (HG group) for 6 h, 12 h, 24 h or 48 h respectively to induce apoptosis, each group sets 5 multiple wells. Apoptosis was tested by TUNEL assay. PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay. The mitochondrial membrane potential was detected by JC-1 method. The expressions of cleaved caspase-3 and cytochrome C (Cyt C) protein in mitochondria and cytoplasm were determined by Western blot assay. H9C2 cardiomyocytes were randomly divided into four groups, control group (5.5 mmol/L), HG (35 mmol/L) group, HG+si-scramble group(si-scramble treatment for 24 h, then 35 mmol/L high glucose treatment for 24 h) and HG-si-PUMA group (si-PUMA treatment for 24 h, then 35mmol/L high glucose treatment for 24 h). Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied. Compared with the control group, high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly (P＜0.05 or P＜0.01). Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group. The following experiment used high glucose 24 h as the stimulation time. The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3, increased mitochondria membrane potential and decreased Cyt C release (P＜0.05 or P＜0.01). There were no significant differences between the HG+si-scramble group and the high glucose group (P＞0.05). PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.