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-β 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.

Significance: Maximum heart rate (mHR) declines with age, contributing to the reduced aerobic capacity of the elderly. A parallel reduction in "intrinsic heart rate" (which is measured during autonomic blockade) underlies the decline in mHR, and indicates that pacemaker function of the sinoatrial node is compromised during aging. In this study we demonstrate that the slower heart rates in the elderly result from depressed spontaneous activity of individual sinoatrial node myocytes (SAMs). Patch-clamp electrophysiology revealed that aged SAMs have slowed AP firing rates, altered AP waveforms, and changed properties of Ca ²⁺ currents and the cardiac "funny current,"I _f . Our findings demonstrate that age-dependent changes ion channel activity in SAMs are a major cause of the decline in mHR during aging.

The “funny” (pacemaker) current has unusual characteristics, including activation on hyperpolarization, permeability to K⁺ and Na⁺, modulation by internal cAMP, and a tiny, single-channel conductance. In cardiac cells and neurons, pacemaker channels control repetitive activity and excitability. The recent cloning of HCN subunits provides new insight into the molecular basis for the funny channel properties.