Therapeutic Role Of MiRNA-19a/19b In Cardiac Regeneration And Protection From Myocardial Infarction

Background:Cardiovascular disease remains the leading cause of mortality and morbidity in developed countries.The primary cause of heart failure is the loss of cardiomyocytes in the diseased adult heart.The adult heart has been viewed as an organ with limited regenerating capability because mature cardiomyocytes exit the cell cycle and stop proliferating.Numerous strategies have been applied to preserve cardiac function after a heart attack with little success.Previously,we reported that the miR-17-92 cluster plays a key role in cardiomyocyte proliferation.Objective:We want to elucidate the over-expression of miR-19a/19b,members of the miR-17-92 cluster,enhanced cardiomyocyte proliferation and stimulates cardiac regeneration in response to myocardial infarction(MI)injury.Methods:We detected the expression level of miR-19a/19b in various cardiovascular disease models via qRT-PCR,and found that miR-19a/19b are involved in cell proliferation and the expression of these miRNAs is altered in the hearts of human patients with cardiovascular diseases.To test the function of miR-19a/19b in the heart,firstly,the left anterior descending(LAD)coronary artery was permanently ligated(LAD-ligation)in adult mice to establish a myocardial infarction(MI)and heart failure model,simultaneously,we delivered miR-19a/19b or control mimic formulated with MaxSuppressor in vivo RNALancerⅡ,a lipid-based delivery reagent via intra-cardiac injection.Secondly,we used an adeno-associated virus 9(AAV9)in which the cardiac specific cTNT promoter has been incorporated to achieve cardiac-specific overexpression of miR-19a and miR-19b(AAV9-miR-19a/19b),intra-cardiac injection meanwhile MI.Thirdly,to answer whether miR-19a/19b could serve as potential therapeutic targets to treat myocardial infarcted hearts,6 h after MI,we delivered miR-19a/19b or control mimic via tail vein injection formulated with MaxSuppressor in vivo RNALancerll and Lipofectamine RNAiMAX respectively.Next,carefully examined the pharmacokinetics of miRNA mimics after intracardiac injection and myocardial infarction via qRT-PCR and immunofluorescence staining.Cardiac function was measured using echocardiography,we also performed histological analysis via sirus red/fast green staining and quantified the infarct size.The expression of molecular marker genes were analyzed via qRT-PCR.In order to determine whether miR-19a/19b mimics in adult mouse hearts are able to activate cardiomyocyte proliferation,we examined EdU incorporation in cardiomyocytes in mouse hearts after miR-19a/19b or control mimic injection and myocardial infarction.We detected increased EdU+signal in cardiomyocytes after miR-19a/19b mimic injection when compared to control via immunofluorescence staining.To verify this observation,we examined the expression of phospho-Histone H3(pH3)and Aurora B in control and miR-19a/19b mimic injected hearts.In addition,the expression levels of the cell cycle-related genes cyclin B1(CCNB1),cyclin D1(CCND1)and cyclin dependent kinase 1(CDK1)were detected via qRT-PCR.Next,adult cardiomyocytes were dissociated from hearts three weeks after miR-19a/19b or control mimic injection and MI,quantification analysis confirmed total number of cardiomyocytes,stained isolated adult cardiomyocytes with connexin 43,α-actinin and DAPI to determine the integrity and nucleation status of cardiomyocytes.Using TUNEL assay and western blot analysis,we assessed apoptosis in infarcted hearts injected with miR-19a/19b or control mimics.Then we performed next generation RNA-sequencing(RNA-seq)in miR-19a/19b mimic and control mimic injected heart samples to obtain an unbiased genome-wide view.Using quantitative RT-PCR(qRT-PCR)analysis and verified down-regulated genes,immunohistochemical staining and western blot analysis were further applied to verified the underlying signaling pathway.Results:We show that intra-cardiac injection of miR-19a/19b mimics into adult mouse hearts.Intriguingly,miR-19a/19b protected the adult heart in two distinctive phases:an early phase immediately after MI,apparently by reducing MI-induced immune response and cell death pathways;and long-term protection,in part by enhancing cardiomyocyte proliferation.Genome-wide transcriptome analysis confirmed that genes related to immune response were repressed by miR-19a/19b in infarcted hearts.Using an adeno-associated virus approach to deliver miR-19a/19b into infarcted hearts,we validated that these miRNAs reduced MI-induced cardiac damage and protected cardiac function.Conclusions:We confirmed that miR-19a/19b promotes cardiomyocyte proliferation and cardiac regeneration post myocardial infarction.And improves cell survival and cardiac remodeling via regulates cell death and inflammation pathways in infarcted hearts.Also confirmed the therapeutic potential of miR-19a/19b in protecting cardiac function by systemically delivering miR-19a/19b into mice post-MI.Our study therefore establishes miR-19a/19b as potential therapeutic targets to treat heart failure.