H2 And H3 Relaxin Inhibit High Glucose-induced Apoptosis In Ventricular Myocytes

Objective: High concentrations of glucose induce apoptosis in cardiomyocytes,and contribute to diabetic cardiomyopathy.Relaxin-2 and relaxin-3 are two members of the relaxin peptide family that are cardioprotective.However,it remains unknown whether relaxin-2 or relaxin-3 regulates apoptosis in high glucose treated-neonatal rat ventricular myocytes(NRVMs),and the plasma level of relaxin-2 and relaxin-3 in patients with diabetes.Materials and methods:(1)We studied 33 newly diagnosed type 2 diabetes patients and 38 age-matched healthy subjects.The plasma levels of relaxin-3 and relaxin-2 were measured by Radioimmunoassay.(2)NRVMs were treated with 5.5mmol/l low glucose(NG)and 33mmol/l high glucose(HG)for 0?6?12?24?48 and 72 h,apoptotic rate were observed by flow cytometry and Hoechst staining,apoptosis related protein expression,endoplasmic reticulum stress markers and autophagy markers were determined by western blot,autophagosome formation was observed using electron microscopy.(3)NRVMs were treated with 5.5mmol/l low glucose(NG)and 33mmol/l high glucose(HG)for 0?6?12?24?48 and 72 h,relaxin-1?relaxin-3?RXFP1 and RXFP3 m RNA expression were determined by Real-time PCR.Then NRVMs were treated with HG,with or without 100ng/ml human recombinant relaxin-2(H2 relaxin)or 100ng/ml human recombinant relaxin-3(H3 relaxin)pretreatment,apoptotic rate were observed by flow cytometry and Hoechst staining,and apoptosis related protein expression,endoplasmic reticulum stress markers and autophagy markers were determined by western blot,autophagosome formation was observed using electron microscopy.Results:(1)Relaxin-2 levels were significantly lower in patients with diabetes than in controls(p?0.05),However,no differences in relaxin-3 levels were observed between the diabetes group and controls(p?0.05).There was no association between the plasma level of relaxin-2 and relaxin-3.(2)HG increased the apoptotic rate of NRVMs observed by flow cytometry and Hoechst staining in a time-dependent manner,and caspase-3 activation was observed;HG increased the protein expression of cleaved caspase-8 and-9,two initiators of the extrinsic and intrinsic pathways of apoptosis,which activated caspase-3;Furthermore,endoplasmic reticulum stress(ERS)markers CCAAT/enhancer-binding protein homologous protein(CHOP)and cleaved caspase-12 were markedly increased in HG-treated NRVMs,leading to apoptosis;Treatment of NRVMs with HG reduced the expression of autophagy marker(LC3-?)and the formation of autophagosome;(3)In cultured NRVMs,HG reduced relaxin-1 and RXFP1 m RNA in a time-dependent manner,did not affect the m RNA expression of relaxin-3 and RXFP3;HG-increased NRVMs apoptosis was attenuated by H2 relaxin or H3 relaxin;HG-increased the protein expressions of cleaved caspase-8 and-9 which were effectively attenuated by H2 relaxin or H3 relaxin;HG-induced ERS in NRVMs was inhibited by H2 relaxin or H3 relaxin,however,HG-reduced autophagy which cannot be adjusted by H2 relaxin or H3 relaxin.Conclusions: Relaxin-2 levels were significantly lower in patients with diabetes than in controls,However,no differences in relaxin-3 levels were observed between the diabetes group and controls.HG-induced apoptosis in NRVMs was mediated,in part,by the activation of the extrinsic and intrinsic pathways of apoptosis and ERS,which were inhibited by H2 relaxin or H3 relaxin.