Abstract
Under hypertrophic stimulation, cardiomyocytes enter a hypermetabolic state and accelerate biomass accumulation. Although the molecular pathways that regulate protein levels are well-studied, the functional implications of RNA accumulation and its regulatory mechanisms in cardiomyocytes remain elusive. Here, we have elucidated the quantitative kinetics of RNA in cardiomyocytes through single cell imaging and c-Myc (Myc)-mediated hypermetabolic analytical model using cultured cardiomyocytes. Nascent RNA labeling combined with single cell imaging demonstrated that Myc protein significantly increased the amount of global RNA production per cardiomyocyte. Chromatin immunoprecipitation with high-throughput sequencing clarified that overexpressed Myc bound to a specific set of genes and recruits RNA polymerase II. Among these genes, we identified Btg2 as a novel target of Myc. Btg2 overexpression significantly reduced cardiomyocyte surface area. Conversely, shRNA-mediated knockdown of Btg2 accelerated adrenergic stimulus-induced hypertrophy. Using mass spectrometry analysis, we determined that Btg2 binds a series of proteins that comprise mRNA deadenylation complexes. Intriguingly, Btg2 specifically suppresses cytosolic, but not nuclear, RNA levels. Btg2 knockdown further enhances cytosolic RNA accumulation in cardiomyocytes under adrenergic stimulation, suggesting that Btg2 negatively regulates reactive hypertrophy by negatively regulating RNA accumulation. Our findings provide insight into the functional significance of the mechanisms regulating RNA levels in cardiomyocytes.
Highlights
Cardiac hypertrophy is initially an adaptive response to exogenous stimuli caused by either high blood pressure or neurohumoral factors, and is recognized as the causative mechanism during the progression of heart failure
Ad-MycΔC overexpression did not exhibit the same effect despite the same overall amount of expression levels (Fig. S1C). These results demonstrate that the Myc protein induces an increase in global RNA synthesis in cardiomyocytes, and has a common function to amplify transcription across cell types[15,16,17,18]
We used single cell quantitative imaging combined with nascent RNA labeling to elucidate the functional significance of the mechanisms that regulate RNA levels in cardiomyocytes
Summary
Cardiac hypertrophy is initially an adaptive response to exogenous stimuli caused by either high blood pressure or neurohumoral factors, and is recognized as the causative mechanism during the progression of heart failure. A chemical method to detect RNA synthesis based on the biosynthetic incorporation of the uridine analog into newly transcribed RNA enables the quantitative evaluation of transcription rates in cultured cells[19]. We have used this nascent RNA labeling combined with single cell quantitative imaging to generate an analytical model based on cultured primary rat cardiomyocytes. Using this method, we detected the Myc-mediated hypermetabolic state in cardiomyocytes with accelerated RNA production per nucleus. Based on biochemical and functional analysis using single cell imaging, we found that Btg[2] negatively regulates cardiomyocyte hypertrophy and decreases cytosolic RNA levels by interacting with the Ccr4-Not deadenylation complex
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