Research On The Molecular Basis And The Role Of Pinch2 In Overwork-induced Heart Injury

Background Overwork is a common phenomenon in today’s society,and epidemiological studies have shown that overwork is one of the risk factors for cardiovascular diseases(CVDs).Studies based on biochemical tests of blood and physical examination suggest that activation of the neuroendocrine system,coagulation,and inflammation are the pathophysiological mechanisms of overwork-induced CVDs.However,the current research on molecular etiology and in-depth molecular mechanism is limited.PINCH2 is an adaptor protein containing five LIM domains and is involved in biological processes such as cell and extracellular matrix adhesion,cell migration,proliferation and survival.PINCH2 plays an important role in the growth,maturation,remodeling and function of the heart,but the role of PINCH2 in overwork-induced myocardial injury or arrhythmia is unclear.Therefore,this study aimed to explore the molecular basis and the role of Pinch2 in overwork-induced heart injury in mouse models.Methods(1)The overwork-induced mouse model was established,and the cardiac function,electrical conduction and pathological structure were evaluated by echocardiography,electrocardiogram,and HE staining of cardiac tissue sections,respectively.(2)The mRNA-and miRNA-sequencing profiles from stress-treated mice and normal mice were performed.Differentially expressed genes(DEGs)and miRNAs(DEmi Rs)were identified.Then,based on the functional enrichment analysis and protein-protein interaction(PPI)network,as well as miRNA-mRNA interactome,the core DEGs and DEmi Rs associated with stress-induced heart injury were marked and validated by q PCR,and the DEmi R targets were validated in vitro.(3)Pinch2 knockout mice(Pinch2-/-)were used to construct overwork models.Cardiac function,electrical conduction,pathological structure,ultrastructure,apoptosis and oxidative stress were compared between Pinch2-/-and wild-type mice.(4)The mRNA sequencing was performed on Pinch2-/-mouse models.Compared with wild-type mice,DEGs were identified,and KEGG and GO enrichment analysis and PPI network were performed to explore the molecular functions and interacitons.Results(1)No serious functional and structural abnormalities were found in echocardiography examination on the overwork-induced mouse model,but the electrocardiogram showed a slight increase in voltage and mild edema of cardiomyocytes in tissue sections,suggesting an early stage of the disease.(2)A total of 293 genes and 29 miRNAs were identified as DEGs and DEmi Rs respectively,and Alb,Stat1,Usp18 were hub genes in the PPI network.The enrichment pathways were related to inflammation and immune,mitochondrial activity,vascular development,cell cycle and extracellular matrix(ECM),which mediated the biological injury processes or reflect the results of damage.The target DEGs of DEmi Rs were clustered in angiogenesis,immune system process and ECM.After the validation in vitro,we found that mi R-29b-3p mimics can down-regulate the expression of its predicted targets,Pxdn and Col15a1.(3)Compared with the wild-type overwork models,Pinch2-/-mouse models showed decreased cardiac function,obvious arrhythmia,aggravated damage to pathological structure in tissue sections and to ultrastructure under electron microscope,and increased cell apoptosis in heart.(4)857 DEGs between the hearts of Pinch2-/-and wild-type mouse models were identified,of which 366 genes were up-regulated and 491 were down-regulated.Functional enrichment showed that the DEGs were involved in biological processes mainly related to immune and inflammatory responses,cell adhesion and lipid metabolism.Stat1 and Irf7 were hub genes in the PPI networkConclusions:(1)In the early stage of overwork-induced heart injury in mice,before severe cardiac dysfunction or structural damage occurs,the expression of a large number of mRNAs and miRNAs has changed significantly.Immunity and inflammation,mitochondrial activity,hypercoagulability,angiogenesis dysfunction,and ECM impairment are potential biological processes involved in overwork-induced heart injury.miRNAs participate in angiogenesis,immune regulation and other activities by regulating target genes.The regulation of target genes Pxdn and Col15a1 by mi R-29b-3 may be a potential way to interfere with overwork-induced disorder.(2)Pinch2 deletion aggravates overwork-induced myocardial structural damage,promotes cardiac dysfunction,arrhythmia and cell apoptosis.At the molecular level,Pinch2 deletion resulted in the differential expression of numerous genes involved in biological processes related to immunity and inflammation,cell adhesion,and lipid metabolism.Pinch2 is essential for maintaining normal cardiac function and structure in overworked states.