Researches On Cardiomyocytes Proliferation During Rat Postnatal Development:a Role For MiRNAs In Modulating Cardiomyocytes Proliferation

Rationale:Recent studies demonstrated that the postnatal cardiomyocytes in mammalian retained the regenerative capacity during early life. The neonatal mice can undergo complete cardiac regeneration through the cell cycle reactivation. However, the exact temporal sequence when cardiomyocytes cease dividing and become terminally differentiated is not certain afer birth. The regulatory mechanisms that govern cardiomyocytes proliferation are poorly understood. Non-coding RNAs have emerged as important regulators which repressed posttranscriptional gene expression by binding to target recognition sequences located within the3’untranslated region of target mRNAs. And microRNAs are small (-20-24nucleotides) noncoding RNAs and it is becoming increasingly evident that individual miRNAs often exert their actions by modestly targeting numerous mRNAs with related biological functions. Despite multiples studies suggested the regulatory potential roles of miRNAs in cell cycle control, little is known about the action of miRNAs in postnatal cardiomyocytes maturation, proliferation and terminal differentiation processes. In this study, the proliferation pattern and miRNAs expression profiles of postanatal cardiomyocytes in rats were characterized in detail and the functional roles of several most abundantly and differentially expressed miRNAs were dissected to demonstrate their roles in postnatal cardiomyocytes proliferation.Objectives:The present study is to investigate the proliferation pattern of cardiomyocytes during the early postnatal period, search for miRNAs in cardiomyocytes that were upregulated and involved in cell proliferation during postnatal development and finally revealed the role of the high abundance expressed miRNAs in older rats on cardiomyocytes proliferation.Methods and Results:Part one:Characterize the proliferation pattern and miRNAs and mRNAs expression profiles in Rat postnatal cardiomyocytes1. Cardiomyocytes proliferation Pattern in Postnatal Rats Cardiomyocytes proliferation and mitoses were identified by double immunofluorescence staining for Ki-67or H3P with troponin T, respectively. Numbers of the positive cardiomyocytes were calculated to draw cardiomyocytes proliferation curve in the hearts of postnatal rats. We found the number of Ki-67positive cardiomyocytes was decreased at day10and the total mitotic indices determined by H3P were almost3-fold lower than Ki-67staining. The number of H3P positive cardiomyocytes was significantly decreased at day7. The proliferation and mitotic potentials of cardiomyocytes has gradually disappeared within4weeks in rats after birth.2. Microarray for miRNAs in postnatal cardiomyocytesTo identify miRNAs involved in the postnatal cardiomyocyte proliferation, miRNA profiling of P2versus P4W carciomyocyte from SD rats was performed with Affymetrix microRNA microarray. MiRNAs expression between P2and P4W cardiomyocytes were differentially regulated during this developmental transition. A total of95miRNAs were differentially regulated in P4W group (at least2-fold).30miRNAs displayed increased expression between P2and P4W and65showed decreased expression between P2and P4W.3. Microarray for mRNAs in postnatal cardiomyocytesGlobal changes in transcriptional profile of cardiomyocytes between P2and P4W were also assessed by microarray. Microarray analysis was performed on RNA samples extracted from P2and P4W cardiomyocytes. mRNA expression in P4W was also clustered together and separated from P2group. We identified1353genes that were upregulated in P4W cardiomyocytes and2842genes that were downregulated at the same time point.4. Bioinformatic analysis of the correlation between differentially expressed miRNAs and mRNAs4.1. Functional analysis of the differentially expressed genes—GO analysisGO analysis was applied to analyze the main function of the differential expression genes according to the Gene Ontology which is the key functional classification of NCBI. Main function of the significantly upregulated miRNA associated genes involved several known gene ontologies:response to drug, protein transport, cell cycle, positive regulation of cell proliferation, apoptosis and oxidation reduction. Main function of the significantly downregulated miRNA associated genes were associated with fatty acid metabolic process, fatty acid beta-oxidation, electron transport chain, ion transport and glycolysis.4.2. Correlation between differentially expressed miRNAs and mRNAs profiles—MicroRNA-GO-Network MicroRNA-GO-Network is built according to the relationship of significant GOs and gene and the relationships among microRNAs and genes. We analyzed miRNA and mRNA expression profiles in P2and P4W cardiomyocytes to recognize miRNA and mRNA expression during the postnatal development. Using a biostatistitical approach to integrate the differentially expressed miRNAs and mRNAs, we found many interacting pairs between miRNAs and mRNAs. The most highly interconnected modules included several important miRNAs:miR-34a,-139-5p,-185,-30e,30a,-1,-29a,-195,-499,-133a,-133b. Several differentially expressed genes in the P4W cardiomyocytes are involved in response to drugs, response to hypoxia, oxidation reduction, aging, positive regulation of proliferation, which are potential targets of miRNAs. Significant GOs and genes and the relationships among microRNA and genes were put together to build the miRNAs-GO-networks. Several differentially expressed genes and miRNAs in the P4W cardiomyoeytes were involved in cell cycle and proliferation regulation processes. Cell cycle and cell proliferation regulation networks are enriched by miRNA and mRNA expression profiling.Part Two:miR-29a regulates the proliferation of cardiomyocytes Methods and Results:1. Identification of miRNAs upregulated during postnatal heart developmentA subset of miRNAs from the microarray analysis were further comfirmed by real-time PCR analysis in neonatal2to10day,2weeks,3weeks and4weeks cardiomyocytes and heart tissues. Based on their significant upregulated in P4W cardiomyocytes and might involved in cell proliferation regulation, we chose six of the upregulated miRNAs in P4W cardiomyocytes for further validation:miR-1, miR-34a, miR-29a, miR-30e, miR-139and miR-185. One of the most highly upregulated miRNAs in cardiomyocytes and heart tissues between P2and P4W was miR-29a. Obviously, miR-29a expression was also found to be temporally upregulated in cardiomyocytes from postnatal day10and sustained until postnatal4weeks.2. H9c2proliferation analysisMTS analysis was was conducted to evaluate the effect of miRNAs on H9c2proliferation. The overexpression of miR-1, miR-34a, miR-30e, miR-139and miR-185in H9c2could not increase cell proliferation24hours after the transfection compared with H9c2transfected with the control mimics or inhibitors. However, overexpression of miR-29a could significantly suppress the proliferation of H9c2, while inhibition of miR-29a would promote the proliferation of H9c2.3. Effects of miR-29a on cardiomyocytes proliferationTo further investigate whether miR-29a could regulate the proliferation of cardiomyocytes, we transfected the neonatal2day cardiomyocytes with miR-29a and its inhibitor; then we assessed the proliferation and mitosis status of cardiomyocytes by immunofluorescence staining, cell cycle associated regulated genes-cyclin D2(CCND2) and CDK2gene expression determined with qRT-PCR and cell cycle distribution detected with flow cytometry. We found that miR-29a overexpression (P29a) in cardiomyocytes could decrease the number of proliferation and mitosis cardiomyocytes48h after transfection compared with cells transfected with a control mimic, while miR-29a inhibitor transfection would increase the proliferation and mitosis of neonatal cardiomyocytes. MiR-29a inhibitor transfection reduced the amount of cardiomyocytes in G0/G1phase, but increased the fraction of cells resided in S and G2/M-cell cycle phases, in comparison to control inhibitor. MiR-29a inhibition induces the proliferation of cardiomyocytes potentially through the modulation of cell-cycle regulators.4. miR-29a directly inhibits the expression of CCND2via its3’UTRTargetscan was used to predict the candidate targets for miR-29a and found CCND2was among the top predicted targets, with a conserved site for miR-29a. Using the double-luciferase report system, we found that the luciferase activities were inhibited by the overexpression of CCND2at the presence of the wild type3’UTR but not by the mutations. After the loss and gain of function of miR-29a transfection, we found that miR-29a overexpression could significantly suppressed CCND2expression in cardiomyocytes, and miR-29a inhibition up-regulated CCND2expression. Furthermore, the number of CCND2positive cardiomyocytes was obviously downregulated in ventricular cardiomyocytes in P4W rat. Together, these data suggest that CCND2is downregulated during the postnatal development and miR-29a targets CCND2by binding to its3’UTR and negatively regulates cardiomyocytes proliferation.Conclusions:We show that cardiomyocytes proliferative and mitosis activity remained constant during the first several days and decreased thereafter in rat. As a result, the cardiomyocytes gradually lost their capacity to proliferate within the first4weeks of age. Multiple miRNAs and mRNAs were upregulated or downregulated during the postnatal cardiomyocytes switching from proliferation to differentiation. And a subset of the upregulated miRNAs was involved in cell cycle control. We identified miR-29a as one of the most robustly up-regulated miRNAs in P4W cardiomyocytes and found that miR-29a could suppress cardiomyocytes proliferation. CCND2was identified as a miR-29a target which may be involved in the process of cardiomyocyte cell cycle and proliferation regulation.