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High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

ABSTRACT: Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-? or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC4579788 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

Zhao Yuanbiao Y Londono Pilar P Cao Yingqiong Y Sharpe Emily J EJ Proenza Catherine C O'Rourke Rebecca R Jones Kenneth L KL Jeong Mark Y MY Walker Lori A LA Buttrick Peter M PM McKinsey Timothy A TA Song Kunhua K

Nature communications 20150910

Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming gro ...[more]

PMID: 26354680

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Project description:BACKGROUND:Scleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality in SSc and an unmet medical challenge, mostly because of our limited understanding of the molecular factors and signalling events that trigger and sustain disease progression. Recent evidence has correlated skin fibrosis in SSc with stabilization of proto-oncogene Ha-Ras secondary to auto-antibody stimulation of reactive oxygen species production. The goal of the present study was to explore the molecular connection between Ha-Ras stabilization and collagen I production, the main read-out of fibrogenesis, in a primary dermal fibroblast culture system that replicates the early stages of disease progression in SSc. RESULTS:Forced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous collagen I proteins. Moreover, normal levels of Tgf? transcripts and active transforming growth factor-beta (TGF?) implied Ha-Ras stimulation of the canonical Smad2/3 signalling pathway independently of TGF? production or activation. Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and collagen I production in cells that constitutively express Ha-Ras. CONCLUSIONS:Our study shows for the first time that constitutively elevated Ha-Ras protein levels can directly stimulate Smad2/3 signalling and collagen I accumulation independently of TGF? neo-synthesis and activation. This finding therefore implicates the Ha-Ras pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic targets in SSc.

Dataset Information

High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

Publications

High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

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