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J Thorac Cardiovasc Surg. 2018 Aug;156(2):556-565.e1. doi: 10.1016/j.jtcvs.2018.03.162. Epub 2018 Apr 13.

p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

Author information

1
Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Tex.
2
Department of Pediatrics, The University of Texas at Austin, Dell Medical School, Austin, Tex.
3
Department of Molecular Oncology, Moffitt Cancer Center, Tampa, Fla.
4
Department of Genetic Medicine, Weill Cornell Medical College, New York, NY.
5
Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Tex. Electronic address: todd.rosengart@bcm.edu.

Abstract

OBJECTIVE:

Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming.

METHODS:

p63 Knockout (-/-) and knockdown murine embryonic fibroblasts (MEFs), p63-/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2-Myocardin (HM).

RESULTS:

Flow cytometry revealed that a significantly greater number of p63-/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63-/- MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63-/- adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure.

CONCLUSIONS:

Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming.

KEYWORDS:

cellular reprogramming; heart failure; induced-cardiomyocytes (iCMs); myocardial infarction; regenerative medicine; transdifferentiation

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