Abstract Tu144: DIAPH1 regulates stress induced senescence in Human cardiomyocytes

Abstract

Introduction: Stress induced senescence (SIS), defined as the loss of cell physiology/function associated with increased senescence associated secretory phenotypes (SASPs), is a potential risk factor for cardiac dysfunction and failure. Human Diaphanous 1(DIAPH1), a formin molecule is known to regulate actin-cytoskeleton dynamics, trafficking of organelles and signal transduction. Our recent work demonstrated the importance of DIAPH1-MFN2 interactions in regulating actin-cytoskeleton dynamics and the mitochondria-sarcoplasmic reticulum(Mito-SR) distance thereby regulating cardiomyocyte function and pathological responses to ischemia reperfusion injury. We tested the hypothesis that DIAPH1 regulated stress induced senescence in cardiomyocytes under aging and hypoxia/reoxygenation (H/R) conditions. Methods: Human cardiomyocytes (HiPSC-CMs) from two different sources of HiPSCs which are double positive for TNT2 and TNNI3 were used. To understand the role of DIAPH1 in SIS, we have used two different approaches. HiPSC-CMs were either kept for 120days in culture or at day25 were treated with mitomycin(200nM/72hrs). DIAPH1 was silenced using shRNA approach ( shDIAPH1 ). Results: We observed increased DIAPH1 gene expression at 120 days of culture vs day 30. Interestingly, shDIAPH1 HiPSC-CMs, attenuated the number of SA-β-galactosidase positive cells, reduced expression of senescence markers SERPINE, CDKN1A, CDKN2A, TP53 , COL1A2 along with improved mitochondrial function when compared to shScr controls. In experiments where HiPSC-CMs at day 25 were treated with mitomycin to induced senescence, we observed that silencing DIAPH1 attenuated the number of SA-β-galactosidase positive cells, reduced F/G actin ratio and robustly decreased DIAPH1-MFN2 interactions as measured by DUOLINK proximity ligation assay. We probed the RNA sequencing datasets from our recent publication (Yepuri et.al., Nat. Comm. 2023) and observed that senescence markers, SASPs and cytokines including CDKN1A, H2AX, LMNA, IL1A, IL6, SERPINE were significantly down regulated in shDIAPH1 HiPSC-CMs exposed to H/R conditions. Conclusion: Taken together our data demonstrates that DIAPH1 mediates stress induced senescence in cardiomyocytes and is linked to actin-cytoskeleton dynamics and DIAPH1-MFN2 interactions. Importantly, our data indicates a causative role of DIAPH1 in SIS and sets the stage for probing DIAPH1 as a therapeutic target for protection of hearts from stress induced senescence.