Protection And Its Mechanisms Of Intermedin In Thapsigargin-induced Apoptosis Of Rat Cardiomyocyte

Intermedin（IMD）is a member of the calcitonin/calcitonin gene-related peptide family.Accumulating evidence has indicated that IMD inhibits cardiomyocyte injury induced by heart failure and myocardial infarction,the underlying protective mechanisms are still unclear.Thapsigargin（TG）is a specific and potent inhibitor for sarco/endoplasmic reticulum（ER）calcium ATPase（SERCA）.By inhibiting SERCAs,TG interferes with the ER lumen Ca²⁺flux that subsequently leads to endoplasmic reticulum stress（ER stress）.It can partly simulate the pathophysiological process of cardiovascular disease.The aim of this study was to investigate the effect of IMD on TG-induced cardiomyocyte apoptosis in vitro and in vivo and to explore its mechanism by detecting ER stress and sarcoplasmic reticulum calcium pump.It is divided into the following three parts.Part 1 TG induces cardiomyocyte apoptosis in a concentration-and time-dependent mannerObjective:The purpose of this study is to explore the effects of TG on cardiomyocytes in primary culture of neonatal rat cardiomyocytes.Methods:The cardiomyocytes were divided into 4 groups according to different concentrations of TG treatment:control group（control,TG 0μM）,TG 1.5μM group,TG3μM group,and TG 6μM group,and incubated for 24 h.The cardiomyocytes were further divided into 4 groups according to the different treatment time of TG:control group,TG 12 h group,TG 24 h group,and TG 48 h group.The inverted microscope was used to observe the morphology of cardiomyocytes.MTT method was used to detect the cardiomyocyte viability.The cell damage and cardiomyocyte apoptosis were detected by lactate dehydrogenase（LDH）release and flow cytometry and caspase-3 activity measurement,respectively.Results:1.Effects of different concentrations of TG on cardiomyocyte apoptosis（1）Compared with the control group,the morphological changes of cardiomyocytes were obvious after treatment with different concentrations of TG,especially in 6μM TG group.（2）The survival rate of cardiomyocytes treated with 1.5μM,3μM or 6μM TG were（90.02±5.93）%,（69.33±4.63）%and（44.52±6.88）%,respectively.There was significant difference compared with the control group（P<0.05 or P<0.01）;（3）The LDH content of cardiomyocytes in the control group was（488.3±12.86）U/L.After treatment with different concentrations of TG,the LDH content of cardiomyocytes were（622.7±53.11）U/L,（869.3±95）U/L and（1109±391）U/L respectively.There was significant difference compared with the control group（P<0.05 or P<0.01）;（4）The results of flow cytometry showed the apoptosis rate of cardiomyocytes treated with 1.5μM、3μM or 6μM TG were（9.81±7.55）%,（26.47±10.31）%and（50.87±13.51）%respectively,which was significantly higher than that of the control group（3.10±0.55）%（P<0.05 or P<0.01）.The caspase-3 activity increased significantly in TG-concentration dependent manner（P<0.05 or P<0.01）as compared with the control group.2.Effect of different time of TG treatment on cardiomyocyte（1）Cardiomyocytes were incubated with 3μM TG for 12h,24h or 48h.The morphological changes of cardiomyocytes were obvious as observed by inverted microscope,especially in TG 48 h group.（2）The survival rates of cardiomyocytes after 12h,24h or 48h TG treatment were（90.67±6.28）%,（64.43±7.68）%,（26.6±7.63）%.The survival rates in TG 24h group and48h group were significantly lower than those in the control group（P<0.01）.（3）The content of LDH in the control group was（475.30±33.25）U/L,and that in cardiomyocytes after 12h,24h or 48h TG treatment were（583±49.08）U/L,（923±86.66）U/L or（1040±227.20）U/L respectively.LDH in TG 24h and TG 48h groups were significantly higher than that in the control group（P<0.01）.The content of LDH in TG48h group was the highest.（4）The apoptosis rate of cardiomyocytes in the control group was（3.00±1.26）%,and the apoptosis rates of TG 12h group,24h group and 48h group were（8.77±3.25）%,（36.27±9.51）%,（68.87±12.43）%,respectively.The apoptosis rates of TG 24h group and TG 48h group were significantly higher than those in the control group（P<0.01）.The results of caspase-3 activity showed that compared with the control group,the caspase-3activity in TG group increased significantly,and the caspase-3 activity increased in a time-dependent manner（P<0.05 or P<0.01）.Conclusion:TG induced cardiomyocyte apoptosis in a concentration-dependent and time-dependent manner.In subsequent experiments,3μM TG was used to induce cardiomyocyte apoptosis.Part 2 Protection and its mechanisms of intermedin in thapsigargin-induced rat cardiomyocyte apoptosis in vitroSection I IMD inhibits TG-induced ER stress in a concentration-dependent mannerObjective To observe the effect of IMD on ER stress and ER stress-related apoptosis in primary cultured neonatal rat cardiomyocytes subjected to TG.Methods Cardiomyocytes were randomly divided into 6 groups:1.Control group,cardiomyocytes were incubated for 24h;2.IMD group,Cardiomyocytes were incubated with 100n M IMD for 24h;3.TG group,Cardiomyocytes were incubated with 3μM TG for 24 h;4.TG+IMD 1n M group,Cardiomyocytes were incubated with IMD 1 nm for 30 min,then incubated with 3μM TG for 24 h;5.TG+IMD 10n M group,Cardiomyocytes were incubated with IMD 10nm for 30min,then incubated with 3μM TG for 24 h;6.TG+IMD 100n M group,Cardiomyocytes were incubated with IMD 100 nm for 30min,then incubated with 3μM TG for 24 hRT-PCR and Westernblot assay were used to detect glucose regulatory protein 78（GRP78）,C/EBP homologous protein（CHOP）and caspase-12 m RNA and protein Expression.The flow cytometry and caspase-3 activity were used to detect cardiomyocyte apoptosis.Results1.Compared with the control group,the expressions of GRP78,CHOP,and caspase12 m RNA and protein in cardiomyocytes of the TG group were significantly increased（P<0.01）,and compared with the TG group,the expressions levels of GRP78,CHOP,and caspase12 m RNA and protein in TG+IMD 10n M group and 100n M group were significantly decreased.TG+IMD 100n M group had the most significant effect.2.The results of flow cytometry showed that:After 24 h incubation with 3μM TG,the apoptosis rate of cardiomyocytes was（34.73±6.83）%,which was significantly higher than that of the control group（4.07±2.48）%（P<0.01）;The apoptosis rates of cardiomyocytes in TG+IMD 10 n M group and TG+IMD 100 n M group were（16.13±2.54）%and（9.67±3.22）%respectively,which were significantly lower than those in TG group,especially in TG+IMD 100n M group（P<0.05 or P<0.01）.The results of caspase-3activity showed that the caspase-3 activity in TG group was（2.32±0.89）which was significantly higher than that in the control group（P<0.01）.There was no significant difference in caspase-3 activity between TG+IMD 1n M group（2.02±0.28）and TG group（P<0.05）.The caspase-3 activity in TG+IMD 10n M group（1.75±0.09）and TG+IMD100n M group（1.26±0.11）was significantly lower than that in TG group（P<0.05 or P<0.01）.TG+IMD 100 n M group had the most significant protective effect on cardiomyocyte apoptosis.Therefore,100 n M was selected in subsequent experiments.Conclusion IMD inhibits TG-induced ER stress in a concentration-dependent manner.Section II IMD attenuates SERCA suppression and[Ca²⁺]_i overload induced by TGObjective To observe the effect of IMD on the activity and expression of SERCA,and calcium overload in cardiomyocytes.Methods Cardiomyocytes were divided into 4 groups:1.Control group,cardiomyocytes were incubated for 24h;2.IMD group,Cardiomyocytes were incubated with 100n M IMD for 24h;3.TG group,Cardiomyocytes were incubated with 3μM TG for 24 h;4.TG+IMD 100n M group,Cardiomyocytes were incubated with IMD 100 n M for 30min,then incubated with 3μM TG for 24 h;SERCA activity,SERCA2 protein expression were measured.[Ca²⁺]_i was detected by immunofluorescence.Results 1.Compared with the control group（120.70±11.02）nmol Pi/mg prot^·min,the SERCA activity in the TG group was（46.00±13.01）nmol Pi/mg prot^·min（P<0.01）.Compared with the TG group,SERCA function is increased signigicantly in the TG+IMD group（94.00±13.45）nmol Pi/mg prot^·min（P<0.05）.2.After TG treatment,SERCA2a protein expression in cardiomyocytes was significantly lower than that in the control group（P<0.05）,and the expression level of SERCA2a protein in TG+IMD group was significantly higher than that in TG group（P<0.05）;3.Compared with the control group,the[Ca²⁺]_i of the TG group was significantly increased（P<0.01）.IMD decreased the cytoplasmic[Ca²⁺]_i significantly（P<0.01）.Conclusion The effect of IMD on ER stress may be related to the restoration of SERCA activity and the inhibition of[Ca²⁺]_ioverload.Section III IMD protected TG induced apoptosis through PKA/SERCA2a/ER stress pathwayObjective To investigate whether PKA pathway is involved in the protective effect of IMD.Methods Cardiomyocytes were devided into six group:1.Control group:cardiomyocytes were incubated for 24h;2.IMD group:cardiomyocytes were incubated with 100n M IMD for 24h;3.TG group:cardiomyocytes were incubated with 3μM TG for 24 h;4.TG+IMD 100n M group:cardiomyocytes were incubated with IMD 100 n M for 30min,then incubated with 3μM TG for 24 h;5.TG+H-89 group:cardiomyocytes were incubated with H-89（10μM）for 30 min,then incubated with 3μM TG for 24 h;6.TG+H-89+IMD group:cardiomyocytes were incubated with IMD 100 n M and H-89（10μM）for 30 min,then incubated with 3μM TG for 24 h;Results 1.Compared with the control group,SERCA activity and SERCA2 protein expression in TG group were significantly decreased,which were restored by IMD（P<0.05 or P<0.01）,H-89 can block the protective effect of IMD（P<0.05）.2.Compared with the control group,p-PLB/PLB in TG group was decreased significantly.IMD improved PLB phosphorylation.H-89 inhibited the protective effect of IMD on PLB phosphorylation（P<0.05）,suggesting that IMD can improve SERCA activity by increasing PLB phosphorylation.3.Compared with the control group,the expression of GRP78,CHOP and caspase-12 protein and apoptotic rate in TG group were increased significantly,IMD significantly decreased the change（P<0.05 or P<0.01）,H-89 blocked the inhibitory effect of IMD on ER stress.4.The results of flow cytometry showed that the apoptosis rate of cardiomyocytes in the control group was（3.13±2.78）%,and that in the TG group was（30.37±5.71）%.Compared with the control group,the apoptosis rate of cardiomyocytes in the TG group was significantly increased（P<0.01）;Cardiomyocyte apoptosis in IMD group was（13.3±3.99）%,which was significantly decreased than that in TG group（P<0.01）;H-89blocked the protective effect of IMD on apoptosis（P<0.05）;The activity of Caspase-3 in TG group was significantly higher than that in control group（P<0.01）,and the activity of Caspase-3 in TG+IMD group was significantly lower than that in TG group（P<0.05）.H-89 blocked the protective effect of IMD on apoptosis（P<0.05）.Conclusion PKA/SERCA/ER stress signaling pathway is involved in the protective effect of IMD on TG induced apoptosis.Part 3 The Protective Effect and the Mechanisms of IMD on TG induced myocardial injury in VivoObjective To investigate the effect of IMD on apoptosis induced by TG in vivo.Methods SD rats were randomly divided into 5 groups:1.control group:saline（1ml/kg）intraperitoneal injection（ip）,2.TG group:TG（1mg/kg）ip3.TG+IMD group:IMD（1μg/kg）femoral vein injection（iv）+TG ip,4.TG+H-89 group:H-89（10μg/kg）iv+TG ip5.TG+H-89+IMD group:IMD iv+H-89 iv+TG ip.The cardiac function of rats was detected by the animal ultrasonic instrument.The heart was collected 48 hours later.Results:（1）Compared with the control group,the left ventricular end-diastolic diameter（LVEDD）was significantly increased（P<0.05）,fractional shortening（FS,%）and left ventricular ejectionn fraction,（LVEF,%）of TG were significantly decreased（P<0.05）,IMD improved the cardiac function as compared with the TG group（P<0.05）.In the control group,LVEDD was（4.70±0.73）mm,FS was（52.82±4.83）%,LVEF was（81.06±7.09）%.In the TG group,LVEDD was（7.19±0.70）mm,FS was（32.52±6.45）%,LVEF was（57.26±7.46）%,all of these parameters in TG group were significantly different from those in the control group（P<0.05）;In IMD group,LVEDD was（5.82±0.39）mm,FS was（47.96±5.34）%and LVEF was（4.76±5.75）%,all of which were increased than TG group（P<0.05）;H89 inhibited the protective effect of IMD（P<0.05）;（2）The SERCA activity and protein expression in TG group decreased significantly（P<0.05 or P<0.01）;Compared with the TG group,IMD improved the SERCA function significantly（P<0.05 or P<0.01）;H-89 blocked the protective effect of IMD on SERCA（P<0.05）.Compared with the control group,p-PLB/PLB in TG group decreased（P<0.05）,and IMD improved the phosphorylation of PLB（P<0.05）.The increase of PLB phosphorylation by IMD was inhibited by H-89（P<0.05）.It suggested that IMD could improve SERCA activity correlating with PLB phosphorylation.（3）Compared with the control group,the expression of GRP78,CHOP,caspase-12protein in TG group increased significantly（P<0.05 or P<0.01）;compared with TG group,IMD significantly reduced the upregulation（P<0.05 or P<0.01）,which was blocked by H-89.（4）The apoptic rate in control group is（2.86±1.69）%,and the apoptotic rate in TG group was（23.64±6.68）%.There was significant difference between two groups（P<0.01）;IMD significantly reduced myocardial apoptosis induced by TG（P<0.01）;the protection effect of IMD on apoptosis was blocked by H-89.Conclusion PKA/SERCA2a/ER stress pathway is involved in the protection of IMD on the myocardial cell injury induced by TG in vivo.