Abstract
Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)+/α-myosin heavy chain (αMHC)-GFP+ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFPhigh iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFPlow cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.
Highlights
Cardiomyocytes (CMs) in the adult heart have limited regenerative capacity [1]
Recent studies have found that mouse [2,3,4,5] and human [6,7,8,9] fibroblasts can be directly reprogrammed into induced CMs, which holds a great promise to develop a new therapeutic approach for heart disease
Since the epigenetic status dynamically fluctuates throughout the cell cycle [21], we investigated if synchronizing a specific cell-cycle phase in GMT-infected fibroblasts could improve induced CMs (iCMs)-reprogramming
Summary
Cardiomyocytes (CMs) in the adult heart have limited regenerative capacity [1]. At the onset of heart disease, lost CMs are typically replaced with fibrotic scar tissue, subsequently leading to chronic heart failure, which remains one of the leading causes of death worldwide. None of the α-Actinin+ iCMs expressed proliferation marker, Ki67 four weeks after reprogramming [20] It is unknown whether cell-cycle exit of reprogrammed iCMs happens right upon reprogramming induction or at a later stage of reprogramming process. Cell-cycle pre-synchronization at the G1-phase could markedly enhance the reprogramming efficiency of induced dopaminergic neurons [24]. These studies suggested that manipulation of cell-cycle progression has a significant impact on epigenetic reprogramming, it is unknown whether a particular phase of cell cycle favors the initiation of iCM- reprogramming and if manipulating the cell cycle (i.e., synchronization) of post-infected fibroblasts influences the progression of reprogramming
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