The Protective Role Hsp60 Under Heat Stress In Rat Myocardial Cells

With the global warming,the impact of heat stress gradually highlighted on China's farming.Chicken show an unusually high sudden death phenomenon in the early stage of heat stress,causing people to associate with the failure of vital organs.And many studies seem to also confirm the impact heat stress factors on the chicken heart fatal injury.Though there are differences in the stress and damage to the animals during the survival process,there is a set of highly conserved protective proteins-heat shock proteins from the prokaryotic to eukaryotic cells in order to repair the damage.The exact function of heat shock proteins(HSPs)is not yet clear,but these highly conserved proteins indicate that they have a pervasive and important physiological function.HSPs are expressed under normal physiological conditions and can protect themselves from various stressful damage caused by adverse stressor stimulation by increasing their synthesis when the cells are in a state of emergency.At present,HSPs have become a hotspot topic in the field of international basic biology,medicine and environmental hygiene.The research is very prominent on the relationship between HSPs expression and myocardial ischemia(myocardial infarction)and tumor development in human medicine.However,little is known about the relationship between HSPs and stress injury and stress-related diseases(pig stress syndrome,etc.)in the field of animal husbandry and veterinary diseases at home and abroad.The aim of this study is to understand the effect of changes of Hsp60 expression on cardiomyocytes in heat stress,and to provide a theoretical basis for further understanding the protective effect of Hsp60 in heat stress.Four pairs of siRNA sequences were designed according to the conserved region sequences of Hspdl gene of rat,at the same time a pair of siRNA sequence with no homologous mRNA expression was designed as negative control,Lenti-siRNA-GFP recombinant lentivirus was successfully constructed by digestion and sequencing.The recombinant lentiviral interference plasmids were co-transfected with 293T cells of the lentiviral packaging system for recombinant lentiviral pseudovirus packaging.The concentrated recombinant lentivirus was obtained by infecting H9C2 cells and adding 1 ?g/mL of puromycin resistance to obtain resistant cells.The cells were cloned and identified by fluorescence quantitative PCR and Western Blotting.The expression of Hsp60 protein and its gene was detected by fluorescence quantitative PCR and Western Blotting.The results showed that the expression of Hsp60 protein and its gene were detected by fluorescence quantitative PCR and Western Blotting.The expression of Hsp60 in each genome showed low expression,which proved that the cell line with stable low expression of Hsp60 was successfully established.When exposed to heat stress,more apoptosis was observed with increasing exposure to heat stress,while necrosis was not affected.Furthermore,heat stress induced the loss of mitochondrial membrane potential(??m)and a significant increase of reactive oxygen species(ROS)produced in mitochondria as measured by TMRE and MitoSOXTM red.The loss of DJm indicated a change in inner mitochondrial function.Real-time Quantitative PCR was used to investigate the mechanism by detecting mRNA expression profile of the inner mitochondrial membrane,including CypD,ANT,and PIC.Results showed no differences between lenti-siRNA Hsp60 and control.However,bax in the cytoplasm translocated to mitochondrial during heat stress and regulated the permeability of outer mitochondrial membrane.We hypothesize that Hsp60 can protect cardiac myocytes against apoptosis involving in outer mitochondrial membrane not the inner mitochondrial membrane under heat stress.