The Role And Mechanism Of ERK1/2 Signaling Pathway For Ischemic Injuries In The Aged Heart

Objective:Extracellular signal-regulated kinase 1/2（ERK1/2）signaling pathway is a key branch of the mitogen-activated protein kinase（MAPK）pathway,and it regulated the progresses of cell survival,apoptosis,inflammation,and oxidative stress.However,the regulatory effects and mechanisms of ERK1/2 signaling pathway during the myocardial ischemia in the senescent heart are still uncertain.In the present study,we sought to observe the changes of ERK1/2 pathway in young and old mice exposed myocardial infarction（MI）model,and analyze the relationships between the ERK1/2 signaling pathway and myocardial ischemic injuries.Meanwhile,we aimed to explore the key role of ERK1/2signaling pathway on myocardial ischemic injuries in the ageing heart using constitutively active MEK1 gene（CaMEK）intervention and the specific inhibitor treatment,and to investigate whether ERK1/2 signaling pathway regulated the mitochondrial fusion and fission of cardiomyocytes to exert its effects.Methods:This study was performed in mice and primary cardiomyocytes,and included three parts.Part Ⅰ:Young male C57BL/6J mice aged 12 to 16-week and old male mice with age of16-month were selected.The mice were exposed MI model via left coronary artery ligation.The cardiac functions,infract area,biomarkers of cardiac injury including lactate dehydrogenase（LDH）and creatine kinase（CK）as well as the key proteins expressions of ERK1/2 signaling pathway were tested using appropriate methods or assay kits.Meanwhile,neonatal rat cardiomyocytes were isolated and treated with d-galactose（20g/L）for 48 hours to established senescence model.The senescence-associatedβ-galactosidase staining and senescent biomarkers were used to identify cardiomyocyte senescence.Non-senescent and senescent cardiomyocytes were exposed to hypoxia treatment using the hypoxia incubator chamber.The cell vitality,LDH level,and the key proteins expressions of ERK1/2 signaling pathway were measured by CCK-8,LDH assay kit,and Western blot method,respectively.Part Ⅱ:Old male C57BL/6J mice were divided into sham group,MI blank group,MI+GFP group and MI+CaMEK group.Mice in MI+GFP group and MI+CaMEK group were injected with AAV9-CMV-GFP and AAV9-CMV-CaMEK vector with the dose of 1×10¹¹vg per mouse via the tail vein,respectively.Five weeks after the viral transduction,MI model same as Part I was performed.Cardiac ultrasound,serum LDH,CK concentrations and ATP level were tested according to the manufacturer’s instructions in all mice.Furthermore,DHE staining,TUNEL assay kit and Western blot assay were used to detect the level of reactive oxygen species（ROS）,cell apoptosis and the protein expressions,respectively.Part Ⅲ:Senescent cardiomyocytes induced by d-galactose treatment were divided into control group,hypoxia blank group,hypoxia+GFP group,hypoxia+CaMEK group,hypoxia+DMSO group and hypoxia+PD98059 group.The treatments of hypoxia same as Part I were performed.AAV9-CMV-GFP and AAV9-CMV-CaMEK vector with the dose of 1×10⁷vg per cell were used to infect the cardiomyocytes in hypoxia+GFP group and hypoxia+CaMEK group,respectively,and then the hypoxia was performed.PD98059 at a dose of 50μM was added to inhibit the ERK1/2 signaling pathway before hypoxia.Cell vitality,LDH level,cell apoptosis and cell toxicity were measured using commercial assay kits.Mitochondrial ROS,morphology of mitochondria and mitochondrial membrane potential were detected by Mito SOX,Mito Tracker,and JC-1probes,respectively.Additionally,the proteins related to ERK1/2 signaling pathway,mitochondrial dynamics and cell apoptosis were measured using Western blot method.Results:Part Ⅰ:（1）Cardiac ultrasound results showed that both young and old mice had lower fractional shortening（FS）and left ventricular ejection fraction（LVEF）at day 3 and day28 post-MI compared to their sham groups（all P<0.05）.Compared to young mice exposed to MI model,old mice had lower FS at day 3 post-MI,and lower FS and LVEF at day 28 post-MI（all P<0.05）.（2）Infarct area measurement showed that old mice had a larger infarct area compared with young mice at day 28 post-MI（all P<0.05）.（3）After MI model exposure,both young and old mice had higher serum levels of LDH and CK compared with their sham groups（all P<0.05）,and old mice exposed to MI model had the highest levels of LDH and CK among the groups（all P<0.05）.（4）Compared to non-senescent group,d-galactose treatment for 48 hours increased the expressions of p16and p21 and the numbers of senescence-associatedβ-galactosidase staining positive cell in cardiomyocytes（all P<0.05）.（5）Hypoxia decreased the cell vitality and increased LDH level both in non-senescent and senescent groups compare to their control groups（all P<0.05）,respectively.Senescent cardiomyocytes had lower cell vitality and higher LDH level compared with non-senescent cells after hypoxia exposure（all P<0.05）.（6）Western blot tests showed that,after ischemic or hypoxic injuries,ERK1/2 signaling pathway was activated in young mice hearts and non-senescent cardiomyocytes compared to their controls（all P<0.05）,respectively.However,the activation of ERK1/2 signaling pathway was not found in aged myocardium and senescent cardiomyocytes after ischemic or hypoxic injuries.Part Ⅱ:（1）The MEK expression and p-ERK/t-ERK ratio of myocardium in MI+CaMEK group was highest among all groups（all P<0.05）（2）Compared with mice in MI+GFP group,mice in MI+CaMEK group had higher LVEF and lower serum CK level at day 3 post-MI,and smaller infarct area at the day 28 post-MI（all P<0.05）.（3）DHE staining showed that ROS level of myocardium in MI blank group mice was significantly elevated than those in sham group,and CaMEK gene transduction ameliorated the ROS level（all P<0.05）.（4）Mice in MI blank group had lower Mfn1 and ATP level,as well as increased Drp1 expression and Drp1 Ser616 level in ageing myocardium compared to those in sham group（all P<0.05）.（5）Compared with mice in sham group,mice in MI blank group had lower Bcl-2/Bax ratio and more apoptotic cells（all P<0.05）.Those changes were reversed by CaMEK gene transduction in infarcted myocardium.Part Ⅲ:（1）AAV9-CMV-CaMEK infection significantly increased the MEK expression and p-ERK/t-ERK ratio in senescent cardiomyocytes exposed to hypoxic injury,and PD98059 significantly inhibited ERK1/2 signaling pathway（all P<0.05）.（2）Compared with the hypoxic controls,CaMEK gene transduction increased the cell vitality and reduced the LDH release in senescent cardiomyocytes（all P<0.05）,and PD98059treatment had the opposite effects.（3）The mitochondrial ROS level of senescent cardiomyocytes elevated after hypoxia treatment,and was ameliorated and increased by AAV9-CMV-CaMEK infection and PD98059 treatment,respectively.（4）Compared with cardiomyocytes in control group,cardiomyocytes in hypoxia blank group had lower Mfn1expression,mitochondrial membrane potential and ATP level,and more fragmented mitochondria,Drp1 and Drp1 Ser616 levels（all P<0.05）.Furthermore,AAV9-CMV-CaMEK infection reversed hypoxia induced changes,whereas PD98059treatment aggravated those injuries.（5）Compared with cardiomyocytes of control group,cardiomyocytes in hypoxia blank group had lower Bcl-2/Bax ratio and more apoptotic cells（all P<0.05）.AAV9-CMV-CaMEK reduced the cell apoptosis while PD98059 had the opposite effects on cell apoptosis.Conclusions:（1）The tolerability of ischemic and hypoxic injuries in senescent heart was worse than non-senescent one,and the activation of ERK1/2 signaling pathway was limited.It was indicated that ERK1/2 signaling pathway may play an important role in regulating the injuries of aging hearts.（2）AAV9-CMV-CaMEK used in the present study could effectively activate the ERK1/2 signaling pathway of myocardium,and it was a reliable gene targeting method for hearts.（3）In aged mice heart exposed to MI model,activation of ERK1/2 signaling pathway by AAV9-CMV-CaMEK injection attenuated the oxidative damages,regulated the expressions of proteins related with mitochondrial fusion and fission,ameliorated the energy supply,reduced cell apoptosis.It helps improve the cardiac functions after ischemic injury,implying that ERK1/2 signaling pathway exerts a cardioprotective effect.（4）For senescent cardiomyocytes,activation of ERK1/2 signaling pathway ameliorated mitochondrial ROS production,regulated the protein expressions of Mfn1 and Drp1,inhibited the excessive mitochondrial fission and enhanced mitochondrial fusion,maintained the morphology and functions of mitochondria during hypoxic injury,resulting in increased cell vitality and reduced cell apoptosis.It is suggested that AAV9-CMV-CaMEK may be an effective vector for improving senescent cardiomyocyte injuries.