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P63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

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 ?-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.

SUBMITTER: Patel V

PROVIDER: S-EPMC6050095 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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

Patel Vivekkumar V Singh Vivek P VP Pinnamaneni Jaya Pratap JP Sanagasetti Deepthi D Olive Jacqueline J Mathison Megumi M Cooney Austin A Flores Elsa R ER Crystal Ronald G RG Yang Jianchang J Rosengart Todd K TK

The Journal of thoracic and cardiovascular surgery 20180413 2

<h4>Objective</h4>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.<h4>Methods</h4>p63 Knockout (<sup>-/-</sup>) and knockdown murine embryonic fibroblasts (MEFs), p63<sup>-/-</sup> adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or w ...[more]

PMID: 29716728

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Project description:Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63-transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.

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P63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

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

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