| BackgroundsExperimental studies in the field of myocardial ischemia-reperfusion injury(IRI)have used in vivo,ex vivo,and in vitro models.It is generally believed that in vivo and ex vivo models of myocardial IRI have a proven success rate by occluding and releasing vessels in specific animals.However,there have been diverse methods for generating in vitro models when it comes to the types of cells used,the stimulation environment and the stimulation time.The experiment was designed to assess the feasibility of 12 in vitro cardiomyocyte models simulating IRI commonly used in previous literatures.Our main purpose was to determine whether these models could minic some of pathophysiological features of in vivo myocardial IRI and therefore could be used for researchs related to myocardial IRI.This study included three parts:Part One:Assessing the survival of cardiomyocytes by alone simulated ischemia and simulated ischemia-reperfusion treatments with 12 vitro cardiomyocyte modelsIn this part experiment,the simulated ischemia(SI)treatments and simulated ischemia-reperfusion(SIR)treatments were performed on the neonatal rat cardiomyocytes and H9c2 cardiomyocytes,respectively.The morphological changes,lactate dehydrogenase(LDH)release rate and adenosine triphosphate(ATP)levels were measured to determine whether the simulated reperfysion(SR)treatment could inflict further injury to cardiomyocytes that had undergone SI treatment.According to the purpose of the expeiment,for each studied model,both neonatal rat cardiomyocytes and H9c2 cardiomyocytes were randomly divided into:(1)control(C)group:cardiomyocytes were normally cultured;(2)SI group:cardiomyocytes were dealt with SI treatment;(3)SIR group:cardiomyocytes were dealt with SI treatment and then dealt with SR treatment.For the models 1-10,5 different durations of SI treatment,including 3 h,6 h,9h,12h and 15 h,were exerted.For the models 11 and 12,4 different durations of SI treatment,including 15 min,30 min,45 min and 60 min,were used.After each group received the assigned intervention,the morphological changes,LDH release rate,and ATP levels of cardiomyocytes were measured.The results showed that,firstly,SI treatment on neonatal rat cardiomyocytes and H9c2 cardiomyocytes lead to morphological damage of cardiomyocytes,and the morphological damage of cardiomyocytes was aggravated with the prolongation of SI treatment.Subsequent SR treatment did not cause further morphological damage of cardiomyocytes.Secondly,in the models 1,2,3,4,5,7,8,9 and 10,SI treatment resulted in a significantly increased LDH release rate of cardiomyocytes(compared with the C group,all P values<0.05),and the LDH release rate increased with the prolongation of SI treatments.Subsequent SR treatment did not further increase the LDH release rate,but significantly decreased the LDH release rate(compared with the corresponding SI group,all P values<0.05).In the model 6,SI treatment produced an increase of LDH release rate(compared with the C group,P<0.05),with statistic difference but relatively smaller change than other models,and the LDH release rate did not increase with the prolongation of SI treatment Subsequent SR treatment did not further increase the LDH release rate.In the models 11 and 12,SI treatment did not lead to an increased LDH release rate,even decreased the LDH release rate,and subsequent SR treatment resulted in a significantly increased LDH release rate(compared with the corresponding SI group,all P values<0.05).Finally,SI treatment resulted in a significantly decreased ATP level of cardiomyocytes(compared with the C group,all P valures<0.05),and the decreased extent of ATP was aggravated with the prolongation of SI treatment.Subsequent SR treatments significantly restored ATP level of cardiomyocytes(compared with the corresponding SI group,all P valures<0.05).Part two:Assessing the pathophysiological characteristics of myocardial ischemia-reperfusion injury by alone simulated ischemia and simulated ischemia-reperfusion treatments with 12 vitro cardiomyocyte modelsThe purpose of this part experiment was to observe the injury characteristics of neonatal rat cardiomyocytes and H9c2 cardiomyocytes treated with SIR,and to determine whether the injury of cardiomyocytes caused by SIR treatment had some pathophysiological characteristics of myocardial IRI in vivo.According to the purpose of experiment,for each model,both neonatal rat cardiomyocytes and H9c2 cardiomyocytewere randomly divided into:(1)C group:cardiomyocytes were normally cultured;(2)SI group:cardiomyocytes were dealt with SI treatment;(3)SIR-0.5h group:cardiomyocytes were dealt with SI treatment and then dealt with SR treatment for 0.5 h.For the models 1-10,the duration of SI treatment was 6 h;for the models 11 and 12,the duration of SI treatment was 30 min.After each group received the assigned intervention,the reactive oxygen species(ROS),intracellular Ca2+and mitochondrial membrane potential(MMP)of cardiomyocytes and inflammatory cytokine levels of culture supernatant were measured.The results showed that,firstly,for the neonatal rat cardiomyocytes,in the models 1-12,the ROS level was significantly increased in the SI groups compared with the C group(all P values<0.05).In the models 1,2,3,4,5,7,8,9,and 10,the ROS level was significantly lower in the SIR-0.5h groups than in the corresponding SI groups(all P values<0.05);in the models 6 and 11,there was no significant difference in ROS level between the SIR-0.5h and SI groups(all P values>0.05).For h9c2 cardiomyocytes,in the models 1,2,3,8,9,10,11 and 12,the ROS level was significantly higher in the SI groups than in the C group(all P values<0.05),while in the models 4,5,6 and 7,the ROS level was significantly lower in the SI groups than in the C group(all P values<0.05).In the models 3,8,9,10,11 and 12,the ROS level was significantly lower in the SIR-0.5h groups than in the corresponding SI groups(all P values<0.05);in the models 4,5 and 6,the ROS levels was significantly higher in the SIR-0.5h groups compared to the corresponding SI groups(all P values<0.05);in the models 1,2 and 7,there was no significant difference in the ROS levels between the SIR-0.5h and SI groups(all P values>0.05).Secondly,for the neonatal rat cardiomyocytes,in the models 1,2,3,4,5,7,8,9,10,11 and 12,the intracellular Ca2+ level was sigificantly higher in the SI groups than in the C group(all P values<0.05);in the model 6,there was no significant difference in the intracellular Ca2+ level between the SI and C groups(P value>0.05).In the models 11 and 12,the intracellular Ca2+ level was significantly lower in the SIR-0.5h groups compared to the corresponding SI grups(all P values<0.05);in the models 1,2,3,4,5,6,7,8,9 and 10,there was no significant difference in the intracellular Ca2+ levels between the SIR-0.5h and corresponding SI groups(all P values>0.05).For h9c2 cardiomyocytes,in the models 1,2,3,7,8,9,10,11 and 12,the intracellular Ca2+ level was significantly increased in the SI groups compared to the C group(all P values<0.05);in the models 4,5 and 6,there was no significant difference in the intracellular Ca2+level between the SI and C groups(all P values>0.05).In the model 11,the intracellular Ca2+level was significantly desreased in the SIR-0.5h group compared to the corresponding SI group(P value<0.05);in the models 1,2,3,4,5,6,7,8,9,10 and 12,there was no significant difference in the intracellular Ca2+ level between the SIR-0.5h and corresponding SI groups(all P values>0.05).Thirdly,for neonatal rat cardiomyocytes and h9c2 cardiomyocytes,there was almost no inflammatory cytokines in the culture suprnatant.After cardiomyocytes received the SI and SIR treatments,the inflammatory cytokine levels in the culture supernatan were not increased.The forth,for the neonatal rat cardiomyocytes,in the models 1-12,the MMP level was significantly lower in the SI grous than in the C group(all P values<0.05).In the models 6 and 9,the MMP level was significantly increased in the SIR-0.5h groups compared with the corresponding SI groups(all P values<0.05);in the models 1,2,3,4,5,7,8,10 and 11,there was no significant difference in the MMP levels between the SIR-0.5h and SI groups(all P values>0.05).For h9c2 cardiomyocytes,in the models 1,2,3,7,8,9,10,11 and 12,the MMP level was significantly lower in the SI groups than in the C group(all P values<0.05);in the models 4,5 and 6,the MMP level was significantly higher in the SI groups than in the C group(all P values<0.05).In the models 1,2,3,4,5,6 and 7,there was no significant difference in the MMP level between the SIR-0.5h and SI groups(all P values>0.05);in the models 8,9,10,11 and 12,the MMP levels were significantly increased in the SIR-0.5h groups compared to the corresponding SI groups(all P values<0.05).Part three:Assessing feasibility of constructing an in vitro cardiomyocyte models simulating ischemia-reperfusion injury by a cardiomyocyte-endothelial-macrophage co-culture systemIn this part experiment,a transwell chamber was used to construct a cardiomyocyte-endothelial-macrophage co-culture system.The SI and SIR treatments of 12 in vitro cardiomyocyte models were performed on this co-culture system.The proportion of viable cardiomyocytes was detected to determine whether the SIR treatment could cause reperfusion injury of cardiomyocytes.This part experiment aimed to determine whether the use of a cardiomyocyte-endothelial-macrophage co-culture system could successfully construct an in vitro model of myocardial reperfusion injury.According to the purpose of experiment,for each model,cardiomyocytes were randomly divided into:(1)C group:cells in the co-culture system were normally cultured;(2)SI group:cells in the co-culture system were dealt with SI treatment;(3)SIR-0.5h group:cells in the co-culture system were dealt with SI treatment and then dealt with SR treatment for 0.5 h.For the models 1-10.the duration of SI treatment was 6 h;for the models 11 and 12,the duration of SI treatment was 30 min.The results showed that the cardiomyocyte-endothelial-macrophage co-culture system could be successfully constructed using the transwell chamber,with cardiomyocytes in the lower chamber and endothelial cells and macrophages in the upper chamber.The proportion of viable cardiomyocytes was significantly reduced in the SI groups comared to the C group(all P values<0.05),and but were not decreased in the SIR-0.5h groups compared with the SI groups.Conclusions:By a comprehensive analysis of results of the above three-part experiments,we can draw the following conclusions.1.In the 12 models used in this study,SI treatment can cause morphological damage,a significant increase in LDH release rate and a significant decrease in ATP level to the neonatal rat cardiowocytes and H9c2 cardiomyocytes.However,subsequent SR treatment does not inflict further injury,demonstrated by no further morphological damage,no further increase in LDH release rate(even a significant decrease in LDH release rate),and a significant increase in ATP level.All the results indicate that the 12 commonly used in vitro models can not successfully simulate in vivo myocardial IRI.2.In the 12 models used in this study,SIR treatment on neonatal rat cardiomyocytes and H9c2 cardiomyocytes can not successfully simulate the typical pathophysiological characteristics of myocardial IRI,including large generation of ROS,Ca2+ overload,over inflammatory response and continuous opening of mitochondrial permeability transition pore.All the results indicate that the 12 commonly used in vitro models are not suitable means for studying the mechanisms of myocardial IRI and related interventions.3.Even the use of a cardiomyocyte-endothelial-macrophage co-culture system,the 12 in vitro models used in this study can only simulate in vivo myocardial ischemic injury,but can not simulate successfully in vivo myocardial reperfusion injury. |
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