Dynamic links between the cardiac intermediate filaments of the cytoskeleton and nucleoskeleton for proper cardiomyocyte differentiation, maturation and reprogramming

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) ESPA 09SYN-21-966 2) ELIDEK 1594 Accumulating evidence suggests that mechanical forces are integral to the commitment and differentiation of cardiac stem cells. Studies have proposed that the cardiac cytoskeleton and specifically the desmin intermediate filament (IF) network and its association with nuclear lamins through the LINC complex, link the contractile apparatus to both the extracellular and nuclear matrix, thus regulating trafficking processes and mechanotransduction during development and adulthood, governing myocyte homeostasis and survival(1-3). Desmin, in addition to being one of the earliest myogenic markers both in heart and somites(4,5), regulates nuclear shape and positioning(6,7), as well as the expression of myogenic (MyoD, Myogenin)(8) and cardiogenic (MEF2, Nkx2.5) transcription regulators, during embryonic stem cell differentiation(4,5). Consistent with the above findings, desmin deficiency has negative impact in the proper regeneration of skeletal muscle cells by hematopoietic stem cells(9). Despite these overwhelming data linking desmin to the establishment and maintenance of myogenesis and cardiogenesis, the precise underlying mechanisms remain elusive. To dissect the exact mechanism of cardiogenic regulation we took advantage of the fibroblast-to-cardiomyocyte direct reprogramming, where fibroblasts trans-differentiate into functional induced cardiomyocytes (iCM) through ectopic expression of three transcription factors, GATA4, Mef2C and Tbx5 (GMT)(10). We hypothesized that desmin could enhance the reprogramming of fibroblasts into cardiomyocytes. Indeed, we have found that expression of desmin together with the GMT factors increases reprogramming by 40-60%. Involvement of a cytoskeletal protein such as desmin in cell differentiation and potentially regeneration, raises intriguing questions and exciting possibilities. To unravel the mechanism of this enhancement we focused on desmin serving as direct mechanotransducer (through the LINC complex) to chromatin. Toward this goal, we investigate the changes of the genetic profile induced by the GMT cocktail during reprogramming in the presence or absence of desmin. RNA sequence analysis has shown that the Notch developmental pathway is regulated by desmin. With the use of Chromosome Conformation Capture, Hi-C and Chip-sequence analysis, we demonstrated the importance of desmin cytoskeleton on the genome 3D organization. In addition, we show that desmin is expressed in most of the cardiac progenitor cells population declaring its role in proper cardiac formation. Moreover, immunofluorescence, calcium influx and electron microscopy studies establish desmin as a major player for cardiac maturity, a critical subject in cardiac regenerative medicine, since its absence substantial reduces expression and proper localization of cardiac specific proteins, delays myofibril formation and impairs function of the induced cardiomyocytes driving most of them to senescence. All the above data will be presented.