Excitability of Direct Reprogrammed Murine Tail Fibroblasts: Between Wild-Type Fibroblasts and Cardiomyocytes

Moshe Rav Acha1, Robert Mills1, Jannet Chen1, Sean M Wu2, David Milan1
1 Cardiac Arrhythmia Center, Massachusetts General Hopsital, Cardiovascular Research Center, USA
2 Division of Cardiology, Stanford School of Medicine, Institute of Stem Cell Biology and Regenerative Medicine, USA

Introduction: Limited regenerative capacity of postnatal cardiomyocytes (CM) creates need for alternative regenerative approaches. Cellular rejection, low efficiency differentiation, and tumor formation have presented hurdles for these approaches. Direct reprogramming of fibroblasts into CM using Gata4, Mef2c, Tbx5 (GMT) was previously described to circumvent some of these challenges. We investigated the electrophysiological (EP) changes induced by overexpression of GMT in murine tail fibroblasts (TF).

Methods: Lentiviral overexpression of GMT was induced in TF from multiple lines of transgenic mice carrying different CM lineage reporters. Infected TF expressed a subset of CM specific genes and protein profiles. Whole cell current and voltage clamp studies of wild-type (WT) TF (n=30), GMT infected TF (n=32) and control CM (n=26) were performed.

Results: All isolated CM showed a spontaneous repetitive action potential (AP) activity which did not appear in any of the WT or GMT infected TF. Pacing of CM with variable amplitudes elicited an “all or none” AP response (Fig 1 left), while all WT and majority (78%) of GMT infected TF showed a passive decay of membrane potential according to the cell’s time constant (Fig 1 middle). Nevertheless, a minority (22%) of GMT infected TF demonstrated a stimulus dependent response consisting of rapid up-sloping nifedipine-sensitive potential followed by a variable duration (50-500 ms) plateau, suggestive of Ca-dependent Chloride current (Fig 1 right). Few GMT infected TF showed repetitive low frequency membrane oscillatory potential. Voltage clamp recordings revealed a voltage gated calcium current in GMT infected TF but in contrast to CM, no voltage gated sodium current could be detected.

Conclusion: GMT overexpression in fibroblasts results in induction of voltage dependent Ca and Ca-dependent chloride currents. These currents are responsible for some excitable features in the reprogrammed cells. However, these changes fall short of the essential characteristic EP properties of functional CMs.

Response of CM, wild-type and reprogrammed fibroblasts to depolarizing stimulus