Inhibition Of Endoplasmic Reticulum Stress And Apoptosis In Cultured Hypoxic Cardiomyocytes By Sarcoplasmic Reticulum Calcium ATPase 2a Overexpression

Ischemic heart disease(IDH) is one of the leading contributors to the heart failure currently and the morbidity and mortality of IDH are increasing greatly. With the rapid development of in the field of cell biology and molecular biology, the mechanism of IDH was further elucidated and the effective prevention measures were discovered.As one of strategies for the treatment of IDH,Gen therapy is a new method.In recently years,the signaling and effect of endoplasmic reticulum stress(ERS) is extensively investigated.The apoptosis mediated by ERS play an important role in the progression of myocardial ischemia and heart failure.The heart's overloaded pressure by coarctation of aorta could induce ERS,and the more apoptosis of cardiac myocytes were discovered in myocardium as a consequence.The overexpression of molecular chaperones of endoplasmic reticulum such as glucose regulated protein 78(GRP78) and GRP94 could attenuate the level of ERS response,then more myocytes undergoing all kinds of stimuli survived.The homeostasis of calcium is one of very important inducers for ERS.In the regulatory proteins involving the cycling of calcium,the level and activity of sarcoplasmic reticulum calcium ATPase 2a(SERCA2a) plays a key role for the homeostasis of calcium.The reduced level and activity of SERCA2a is the to critical to the normal contraction and relaxation of heart.Previous studies showed that overexpression of SERCA2a by transgene technique improved the pumping function of heart and hemodynamics.Recent research identified that overexpression of SERCA2a delayed the occurrence of heart failure induced by cardio infarction,but the exact mechanism was not clarified until now.Here we supposed that overexpression of SERCA2a attenuate the effect of ERS induced by myocardial ischemia and protect the cardiac myocytes from stressors.We observed the process of ERS under the ischemic conditions and the influence of SERCA2a overexpression on ERS and the associated apoptosis in cardiomyocytes. Firstly,HEK293 cells were used to the amplification of recombinant adenovirus and the target gene was identified by polymerase chain reaction(PCR). The rAd-GFP vector and LDH was utilized to determine the appropriate multiplicity of infection.Secondly,cultured myocytes were treated under hypoxia.The protein immunoblotting was used to measure the change of expression of Glucose Regulated Protein 78 and CHOP and Caspase-12.The techniques of DNA ladder and flow cytometry were occupied to examine the apoptosis of cardiomyocytes. The results showed that the expression of GRP78 protein increased in 6 hours and approached the highest level in 24 hours in hypoxia.A decreased trend of GRP78 expression was observed after 24 hours.Those observations indicated that hypoxia could induce the time-limited expression of GRP78 in cardiomyocytes. With the prolonged hypoxia,the protein of CHOP and Caspase-12 expressed increasingly and the cell's apoptosis rate was higher and higher.We conclude that hypoxia could induce ERS in cardiomyocytes.The protective effect of ERS could not counteract the insulted effect with prolonged hypoxia.Finally,the apoptosis of cardiomyocytes was observed.Thirdly,although the level of GRP78 did not change significantly,CHOP and Caspase-3 decreased by 52.7%and 39.7%respectively in SERCA2a+hypoxia group compared with the hypoxia group.The apoptosis rate of cardiomyocytes decreased by 66%compared with the hypoxia group.Those results indicated that overexpression SERCA2a could attenuate the level of ERS induced by hypoxia. The role of overexpression of SERCA2a can protect the cardiomyocytes from stimulus such as hypoxia.Finally,the level of cellular free calcium was measured by Fluo-3 and confocal microscopy.The elevated calcium concentration was observed in 12 hours under hypoxia conditions.The overload of calcium in cardiac myocytes was decreased with overexpression of SERCA2a.Those results indicated that overexpression SERCA2a could augment the ability of endoplasmic reticulum to uptake calcium and the apoptosis was depressed.