Effects Of Mitochondrial Dysfunction-mediated Endoplasmic Reticulum Stress On Cardiomyocytes Performance During Hypoxia/Reoxygenation Injury And Its Mechanism

Myocardial ischemia-reperfusion（I/R）represents a pathological condition that the structural damage and dysfunction of cells or tissues are progressively aggravated after the ischemic myocardium returns to normal perfusion.Current medical interventions to restore blood flow to the ischemic region have proven to be successful in reducing the progression of necrosis after ischemia.Paradoxically,however,the return of blood flow can result in additional cardiac damage and complications;this is referred to as reperfusion injury.Myocardial injuries induced by I/R are commonly occur in open heart surgery,coronary artery bypass grafting,coronary angioplasty and thrombolytic surgery.As I/R injury is a common clinical problem and is associated with serious complications,it is important to identify the mechanism and explore therapeutic approaches.There are numbers of studies focused on the I/R injury,however,the mechanisms are still unclear due to the difficulty in I/R model establishment and tens of pathways involved.A wide range of pathological processes such as reactive oxygen species production,calcium overload,inflammation contribute to ischemia and reperfusion injury.A lot of researches showed that endoplasmic reticulum（ER）stress and mitochondrial dysfunction played critical roles in I/R injury.However,most of them are predominantly focused on the apoptosis mediated by ER and mitochondrial dysfunction,few researchers are in-depth study on the association between ER,mitochondrial and cardiomyocyte function.In this study,we established a hypoxia/reoxygenation（H/R）cell model using adult rat cardiomyocytes which could highly simulate the real myocardial I/R injury in clinic.After that,we detected the changes of cardiomyocyte contraction/relaxation,calcium concentration and Ca²⁺sensitivity to better understand the important role of ER stress during the H/R injury.Herein,we explored the complex links among increased SDH activity,overproduced ROS,inflammation,mitochondrial dysfunction and ERS,along with an attempt to address the possible molecular mechanisms.What is more,we tested the effects of ROS scavenger on decreasing H/R injury.In this study,we tried to provide evidences for the prevention and treatment of clinical myocardial I/R injury in the future.Objectives1.To clarify the changes of cardiomyocyte relaxation-contraction function,Ca²⁺concentration and Ca²⁺sensitivity during H/R and explore the potential mechanisms.2.To clarify the changes of mitochondrial dynamics and function during H/R and explore effective protection schemes.3.To clarify the effects of mitochondrial dynamics and function on ERS activation and cardiomyocyte structure and function during H/R,and to explore possible mechanisms.Materials and methods1.Experimental study on the establishment of isolated cardiomyocytes H/R modelThe cardiomyocytes were isolated from SD rat using Langendorff system and randomly assigned to control（CTL）and H/R groups.Cells were incubated in M199 in a cell chamber and perfused with Tyrode solution for 30 min to allow stabilization before the experimental protocol was started.During this time the cardiomyocytes were electrically stimulated at a rate of 0.5 Hz using a pair of platinum electrodes controlled by a MyoPacer stimulator（IonOptix）.H/R group cardiomyocytes were initially exposed to normoxia（21%O₂）for 20 min followed by 30 min exposure to hypoxic solution（2.5%O₂）without glucose and finally 120 min exposure to reoxygenation（21%O₂）.For time-matched control,cardiomyocytes were maintained in normoxia at 37℃in the cell chamber for 150min.Trypan blue staining,Ion Optix system,inverted phase contrast microscopy,laser scanning confocal microscopy（LSCM）,immunofluorescence and oxygen concentration probe were used to study and verify the survival rate,morphology,purity and reliability of model construction of primary isolated adult rat cardiomyocytes.At the same time,relaxation-contraction function including sarcomere resting length（SRL）,sarcomere shortening length（SSL）,shortening and relaxing velocity,and the changes of intracellular[Ca²⁺]_cytyt concentration including basic/peak Ca²⁺concentration,and calcium sensitivity were being constantly monitored.The effects of H/R on the expression of cardiac troponin I（cTnI）and phosphorylated cTnI（p-cTnI）in rat cardiac myocytes were determined by immunofluorescence assay to explore the possible molecular biological mechanisms.2.Study on the effect of H/R injury on mitochondrial function and dynamicsThe cardiomyocytes were randomized to CTL group,H/Rgroup,Malonate group（30minutes incubation before hypoxia and reoxygenation with Malonate-an SDH inhibitor at5 mmol/L）and TEMPO group（30 min incubation with ROS scavenger TEMPO 1 mmol/L before hypoxia and reoxygenation）.Succinate abundance,SDH activity and ATP content were detected using commercial kits.ROS level and mitochondrial membrane potential were detected using confocal.Mitochondrial fission/fusion associated protein Drp1 and Mfn2 were detected by western blot.3.Study on the effects of mitochondirial protection on ER stress and cardiomyocytes functionThe experimental cells were randomly divided into CTL group,H/R group and TEMPO group.Western blot and hos-tag techniques were used to detect the three main pathways of ERS:inositol-requiring enzyme1（IRE1）-X-box binding protein 1（XBP1）pathway,protein kinase R-like endoplasmic reticulum kinase（PERK）-eukaryotic initiation factor 2（eIF2）pathway and activating transcription factor 6（ATF6）pathway.At the same time,the changes of relaxation-constraction,[Ca²⁺]_cytyt concentration,calcium sensitivity in rat cardiomyocyte and the change of the phosphorylation level of intracellular cTnI were detected.The specific method was the same as before.Results1.Experimental study on the establishment of isolated cardiomyocytes H/R model（1）Reliability measurement of H/R model establishmentAccording to the results of monitoring the intracellular oxygen concentration by the oxygen concentration measuring probe and t intracellular oxygen content determination kit,it was confirmed that the actual oxygen concentration of the intracellular oxygen concentration reached the expected standard in the model construction.（2）Isolated primary cardiomyocytes identificationLaser scanning confocal fluorescence miroscopy showed that the cells labeled with nucleus dye 4’,6-diamidino-2-phenylindole（DAPI）were blue,and most of the corresponding cells were strong yellow-green fluorescence,suggesting that the cell purity was high.The purity of rat cardiomyocytes was 98.35±0.67%.（3）Cardiomyocytes viability measurementTrypan Blue staining showed that after 30 minutes of hypoxia,the cardiomyocytes viabilityin CTL groupand H/R group were:68.37±3.01%vs.63.63±2.87%（P=0.024）.After120 minutes of reoxygenation,the cardiomyocytes viability in CTL group and H/Rgroup were:62.35±3.16%vs.51.84±3.92%（P=0.020）.The result suggested that hypoxia could cause cell damage and reduce the viability,while reoxygenation could be more harmful over time because of the H/R injury.（4）Morphological observation of isolated rat primary cardiomyocytesThe isolated primary rat cardiomyocytes showed short columnar shape,complete morphology,strong stereoscopic sense,good refraction and clear cross-striation.The ratio of length to width was 4-6:1.Without the electrical stimulation,1/3 of the cardiomyocytes contracted at a low frequency（0.2Hz）.Unlike human cardiomyocytes,about 80%of the rat cardiomyocytes were binuclear cells while 20%were monocytes.（5）Changes of contractile function in response to H/RCompared with CTL group,there was no significant change in sarcomere resting length（SRL）during hypoxia（P=0.916）and reoxygenation（P=0.976）in H/R group.But the sarcomere shortening length（SSL）significantly decreased after 30min of hypoxia（P<0.01）,while the trend exacerbated after 120min of reoxygenation（P=0.017）,which suggesting that myocardial contractility was significantly weakened.In terms of shortening and relaxing velocity,both of them in H/R group were significantly slower than CTL group（P<0.01,P<0.01）after 30 minutes of hypoxia.After 120 minutes of reoxygenation,the shortening and relaxing velocity of H/R group show a further slowdown（P<0.01,P<0.01）which indicates that the H/R injury will decrease the myocardial contractile and diastolic function.（6）Changes of[Ca²⁺]_cytyt and Ca²⁺sensitivity in response to H/RSummarized[Ca²⁺]_cytyt transient metrics（basal,P=0.034;peak,P=0.040）in response to stimulation in H/R group were significantly higher than in CTL group after 30min of hypoxia.This trend exacerbated after 120min of reoxygenation（basal,P<0.01;peak,P<0.01）after 120min of reperfusion which indicating the Ca²⁺overload in the cardiomyocytes.Compared to CTL,H/R phase-loops are shifted rightward during H/R.The rightward shift of the phase-loop indicates that Ca²⁺sensitivity of the myofilament was reduced during exposure to H/R.（7）Changes of cardiac TnI phosphorylation in response to H/RImmunofluorescence assay showed that p-cTnI/total-cTnI in H/R group increased significantly at 30 min of hypoxia（P<0.01）and 120 min of reoxygenation（P<0.01）compared with the initial phosphorylation level,while at 120 minutes of reoxygenation,the ratio of p-cTnI/total-cTnI was further higher than that at the stage of hypoxia（P=0.041）.2.Study on the effect of H/R on mitochondrial function and dynamics（1）Changes of mitochondria morphology under electron microscopyIt showed that ER were surrounded with mitochondria,and the structure of mitochondrial membrane and cristae were clear and complete in CTL group using electron microscopy,few mitochondria were in fission.In H/R group,mitochondrial fission can be seen in a large number of mitochondria,the mitochondrial membrane was incomplete,and mitochondrial cristae disappeared.In TEMPO group,more than 80%of mitochondria were intact,only about 10%were in fission state,suggesting that It can effectively maintain the stability of mitochondrial structure.（2）Mitochondrial fission/fusion associated protein expression changes in response to H/RWestern blot results showed that the expression of Drp1 in cardiomyocytes of H/R group was significantly higher than that of CTL group（P<0.01）,while the expression of Mfn2 was significantly lower（P<0.01）.This trend was significantly improved by adding TEMPO,a scavenger of ROS（P=0.041,P=0.008）.These results suggested that ROS could induce Drp1 activation and decrease Mfn2 expression,thus promoting mitochondrial fission,causing more ROS production and increasing calcium load,finally leading to mitochondrial dysfunction and forming a vicious circle.（3）Changes of succniate concentration and SDH activity in response to H/RSDH kit results showed that the concentration of succinic acid and SDH activity in H/R group were significantly higher than those in CTL group after 30 min hypoxia（P<0.01）and 120 min reperfusion（P<0.01）.（4）Changes of ROS production in response to H/RMitoSOX Red showed that after H/R,mitochondrialROS levels in H/R group increased significantly compared with CTL group（P<0.01）,while ROS decreased significantly after adding SDH inhibitor Malonate（P=0.034）,suggesting that SDH activity is closely related to ROS production,and the increase of SDH activity may be one of the initial causes of mitochondrial dysfunction.（5）Changes of mitochondrial membrane potential in response to H/RQuantitative measurement of TMRM showed that mitochondrial membrane potential of cardiomyocytes in H/R group was significantly lower than that in CTL group（P<0.01）after H/R,and the change could be alleviated by adding TEMPO,a scavenger of ROS（P=0.029）,suggesting that ROS could affect mitochondrial function by increasing mitochondrial membrane potential.（6）Changes of ATP synthesis in response to H/RATP kit showed that ATP level in H/R group was significantly lower than that in CTL group during H/R（P<0.01）,but this change could be alleviated by adding TEMPO（P=0.026）,suggesting that ROS could affect mitochondrial function by lowering ATP level.3.Study on the effect of mitochondrial protection on ER stress and cardiomyocyte function（1）Changes of IRE1α-XBP1 pathway in response to H/RWestern blot showed that phosphorylationIRE1αexpression significantly increased in H/R group compared with CTL（P<0.01）,then affected the downstream of XBP1expression by increasing the splicing XBP1 expression（P<0.01）which suggested that IRE1α-XBP1 pathway was activated during H/R.（2）Changes of PERK-eIF2αpathway in response to H/RThe results of western blot and phos-tag showed that,in response to H/R,total PERK and p-PERK increased significantly in H/R group（P<0.01,P<0.01）.Meanwhile,the expression of phosphorylated eIF2α（p-eIF2α）was also significantly higher（P<0.01）in H/R group,suggesting that the PERK-eIF2αpathway was activated during H/R.Compared with H/R group,TEMPO group could effectively reduce the phosphorylation level of PERK（P<0.01）and its downstream expression of p-eIF2α（P=0.026）.（3）Changes of ATF6 pathway in response to H/RWestern blot results showed that the expression of ATF6 P50 fragment in H/R group was significantly higher than that in CTL group（P<0.01）,suggesting that ATF6 pathway was activated during H/R.Compared with H/R group,TEMPO group could effectively reduce the expression level of active ATF6 P50（P=0.036）.（4）Changes of relaxation-contraction function,[Ca²⁺]_cytyt concentration,calcium sensitivity and cTnI phosphorylation in response to H/RIonOptix results showed that both TEMPO group could effectively enhance the shortening and relaxing function compared with H/R group（P<0.01）,decrease the basic and peak concentration of[Ca²⁺]_cyt（P<0.01,P<0.01）,increase the calcium sensitivity.What’s more,immunofluorescence results showed that both TEMPO group could effectively reduce the intracellular phosphorylation level of cTnI（P<0.01）.Conclusion1.H/R injury can lead to intracellular calcium overload,morever,reduce the relaxation-contraction function and calcium sensitivity in cardiomyocytes.The mechanism may be related to the increase of phosphorylation level of cTnI.2.Ischemic accumulation of succinate in cardiomyocyte during hypoxia can increase of SDH activity,cause excessive increase of ROS level,and lead to disturbance of mitochondrial structure and function.3.During H/R injury,the mechanism of cardiomyocyte function changes is related to mitochondrial dysfunction mediated ERS.ROS scavengers can improve the function of cardiomyocyte by improving mitochondrial function and inhibiting ERS overactivation.