| Background:Heart failure is caused by systolic or diastolic dysfunction of the heart,and is the end stage of various malignant heart diseases.In the development of heart failure,there are often pathological changes such as myocardial hypertrophy,myocardial fibrosis,mitochondrial dysfunction and abnormal mitochondrial autophagy.Mitochondria are the most important organelles that determine the survival or death of cardiomyocytes.Changes in the morphology and function of mitochondria can induce oxidative stress and cause mitochondrial homeostasis imbalance.Objective:This study aims to explore the role and mechanism of Ndufb10 in regulating mitochondrial homeostasis and improving pathological myocardial hypertrophy during 8-week swimming exercise through in vivo and in vitro experiments,and provide new ideas for the treatment of pathological myocardial hypertrophy.Research methods:In vivo experiment:1.42 SPF grade female 7-week-old SD rats were randomly divided into quiet control group(CN,n=8),SHAM operation group(SHAM,n=8),operation group(SC,n=12),and operation exercise group(SE,n=14).The pathological myocardial hypertrophy model was established in the SC group by abdominal aortic coarctation(TAC),and the SHAM group was not ligation after thoracotomy.SE group received swimming exercise intervention after TAC for 8 weeks.2.After 8 weeks of swimming exercise,echocardiography was performed to examine the cardiac morphology and function of rats in each group.3.Weighing:left ventricular weight,body weight,heart weight,calculate total heart mass index,left heart mass index,etc.4.HE,Masson and TUNEL staining were used to observe myocardial morphology,structure,fibrosis and apoptosis in each group.5.The myocardial ultrastructure,mitochondrial morphology and autophagy of each group were observed by TEM.6.mRNA expression levels of markers related to pathological myocardial hypertrophy,autophagy and apoptosis were detected by RT-qPCR.7.Changes in autophagy,apoptosis and mitochondrial dynamic related protein expression were detected by Western blotting.8.Complete transcriptome differential genes of SC group and SE group were analyzed by RNA-seq sequencing.In vitro experiments:1.There were four groups:blank group(AngⅡ 0h group,n=6),model group(AngⅡintervention for 48h,n=6),knockdown control group(shContorl+AngⅡ intervention for 48h,n=6),knockdown group(shNdufb10+AngⅡ intervention for 48h,n=6).2.The degree of mitochondrial membrane potential was detected by JC-1 method,the degree of MPTP openness was detected by calcein acetyl methyl ester fluorescence probe,ROS level was detected by DCFH-DA fluorescence probe,and the level of NADH in cells was detected by colorimetric method.3.Myocardial cell apoptosis in each group was observed by TUNEL staining.4.Western blotting was used to detect the expressions of autophagy,apoptosis and mitochondrial dynamic related proteins in each group.Research results:In vivo experiment:1.Related indexes of pathological myocardial hypertrophy:compared with CN and SHAM groups,HW/BW and LV/BW in SC group were significantly increased(P<0.05),LVPWd,LVPWs,LVIDd and LVIDs were significantly increased(P<0.05),and left ventricular hypertrophy occurred in SC group.EF and FS were significantly decreased(P<0.05),and cardiac function in SC group was decreased.Myocardial cell diameter was significantly increased(P<0.05),myocardial collagen fiber volume percentage(CVF%)was significantly increased(P<0.05),pathological markers(ANP,BNP,β-MHC)were significantly increased(P<0.05);Compared with SC group,LV/BW in SE group was significantly decreased(P<0.05),LVSd,LVPWd,LVPWs,LVIDd and LVIDs were significantly decreased(P<0.05),EF and FS were significantly increased(P<0.05),and myocardial cell diameter was significantly decreased(P<0.05).Myocardial collagen fiber volume percentage(CVF%)was significantly decreased(P<0.05),pathological markers(ANP,BNP,β-MHC)were significantly decreased(P<0.05).2.Transmission electron microscopy(TEM)results:myocardial fibers and mitochondria in CN and SHAM groups were neatly arranged,and mitochondrial morphology was intact.In the SC group,myofibril was dissolved,transverse tube was compressed and deformed,mitochondrial arrangement was disordered,mitochondria were enlarged,serious mitochondrial edema and obvious vacuolation were observed,mitochondrial crest damage appeared fission,autophagic lysosome envelope components were degraded,and apoptotic bodies were increased.Compared with SC group,SE group had reduced mitochondrial fission,relatively regular ridge structure,reduced edema,vacuolation and apoptotic bodies,and no degraded autopolysosomes were found.3.Apoptosis related indexes:Compared with CN group,there were no significant changes in apoptosis related indexes of SHAM group(p>0.05).Compared with CN and SHAM groups,the apoptosis of myocardial cells in SC group was significantly increased(P<0.05),the ratio of bax/bcl-2 was significantly up-regulated(P<0.05),the protein expression of Bax was significantly up-regulated(P<0.05),and the protein expression of Bcl-2 was significantly down-regulated(P<0.05).The mRNA expressions of Caspase3 and Caspase9 related to mitochondrial apoptosis were significantly increased(P<0.05),while the protein expressions of Caspase3,Caspase9 and Cytochrome C were significantly increased(P<0.05).Compared with SC group,the apoptosis rate of myocardial cells in SE group was significantly decreased(P<0.05),and the bax/bcl-2 ratio was significantly decreased(P<0.05).The mRNA expressions of Caspase3 and Caspase9 related to mitochondrial apoptosis were significantly decreased(P<0.05).The proteins of Caspase3,Caspase9 and Cytochrome C were significantly decreased(P<0.05).4.Autophagy related genes and proteins:Compared with CN group,autophagy related genes and proteins in SHAM group had no significant changes(p>0.05);Compared with CN and SHAM groups,P62 and LC3 gene and protein expressions in SC group were significantly upregulated(p<0.05),and mitochondrial autophagy related NIX and BNIP3 gene and protein were significantly up-regulated(p<0.05).Compared with SC group,the gene and protein expressions of P62 and LC3 in SE group were significantly down-regulated(p<0.05),and the mitochondrial autophagy related NIX and BNIP3 gene and protein were significantly downregulated(p<0.05).5.Mitochondrial dynamic related proteins:Compared with CN group,there were no significant changes in the expression of mitochondrial fusion proteins(MFN1,OPA1)and fission proteins(DRP1,FIS1)in SHAM group(p>0.05).Compared with CN and SHAM groups,the expressions of mitochondrial fusion proteins MFN1 and OPA1 in SC group were significantly down-regulated(p<0.05),and the expressions of mitochondrial fission proteins DRP1 and FIS1 were significantly increased(p<0.05).Compared with SC group,the expressions of mitochondrial fusion proteins MFN1 and OPA1 in SE group were significantly up-regulated(p<0.05),while the expressions of mitochondrial fission proteins DRP1 and FIS1 were significantly decreased(p<0.05).6.Ndufb10,a key gene,was screened after RNA-seq sequencing.In vitro experiment:1.The pathological hypertrophy model of myocardial cells simulated by AngⅡ in vitro:mRNA expressions of pathological myocardial hypertrophy markers ANP and BNP were significantly increased in H9C2 cells stimulated by AngⅡ for 48h(P<0.05),and cell diameter was significantly increased(P<0.01).2.Transfection of H9C2 cardiomyocytes with Ndufb0 knockdown adenovirus:The expression of Ndufb10 gene in H9C2 cardiomyocytes was lowered,and the mRNA expression of Ndufb10 was significantly decreased 24h after adenovirus transfection(P<0.05).3.Pathological marker genes:Compared with AngⅡ 0h group,BNP mRNA expression in AngⅡ 48h group and shControl+AngⅡ 48h group was significantly increased(P<0.05).Compared with AngⅡ 48h group and shControl+AngⅡ 48h group,BNP mRNA expression in shNdufb10+AngⅡ 48h group was significantly decreased(P<0.05).4.Indicators related to mitochondrial function:Compared with AngⅡ 0h,NADH level and ROS in AngⅡ 48h group and shControl+AngⅡ 48h group were significantly decreased,mitochondrial membrane potential was significantly decreased,and MPTP openness was significantly increased(P<0.05).Compared with AngⅡ 48h group and shControl+AngⅡ 48h group,NADH level,ROS production,mitochondrial membrane potential and MPTP openness in shNdufb10+AngⅡ 48h group were significantly decreased(P<0.05).5.Mitochondrial power-related proteins:Compared with AngⅡ 0h group,the expressions of mitochondrial fusion proteins MFN1 and OPA1 in AngⅡ 48h group and shControl+AngⅡ 48h group were significantly decreased(P<0.01),while the expressions of mitochondrial fission proteins DRP1 and FIS1 were significantly increased(P<0.01).Compared with AngⅡ 48h group and shControl+AngⅡ 48h group,the expression of mitochondrial fusion protein OPA1 in shNdufb 10+AngⅡ 48h group was significantly decreased(P<0.01),while the expressions of mitochondrial fission proteins DRP1 and FIS1 were significantly increased(P<0.01).6.Autophagy related proteins:Compared with AngⅡ 0h group,the expressions of P62,NIX and BNIP3 in AngⅡ 48h group and shControl+AngⅡ 48h group were significantly increased(P<0.01);Compared with AngⅡ 48h group and shControl+AngⅡ 48h group,the protein expressions of P62,NIX and BNIP3 in shNdufb 10+AngⅡ 48h group were significantly upregulated(P<0.01).7.Apoptosis-related indicators:Compared with AngⅡ 0h group,the apoptosis of cells in AngⅡ 48h group and shControl+AngⅡ 48h group was significantly increased(P<0.01),the pro-apoptotic protein bax was significantly up-regulated(P<0.05),and the anti-apoptotic protein bcl-2 was significantly decreased(P<0.05).The expressions of mitochondrial apoptosis related proteins Caspase3,Caspase9 and Cytochrome C were significantly increased(P<0.01).Compared with AngⅡ 48h group and shControl+AngⅡ 48h group,apoptosis in shNdufb 10+AngⅡ 48h group was significantly increased(P<0.01),,pro-apoptotic protein bax was significantly up-regulated(P<0.05),and anti-apoptotic protein bcl-2 was significantly decreased(P<0.05).The expressions of mitochondrial apoptosis related proteins Caspase3,Caspase9,Cytochrome C were significantly increased(P<0.01).Conclusion:1.8 weeks of swimming exercise effectively improved the pathological myocardial hypertrophy induced by TAC surgery.2.After RNA-seq sequencing,KEGG enrichment analysis showed that the differential genes were significantly enriched in the oxidative phosphorylation pathway.After further analysis,the key gene Ndufb10 with significant changes was screened out,and the later RT-qPCR and Western-blot verification results were consistent with the sequencing results.3.8 weeks of swimming exercise can enhance mitochondrial function by activating the expression of Ndufb10,inhibit excessive autophagy and excessive apoptosis of mitochondria,maintain mitochondrial homeostasis,and thus play an important role in improving pathological myocardial hypertrophy. |
