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

Vivekkumar Patel, Vivek P. Singh, Jaya Pratap Pinnamaneni, Deepthi Sanagasetti, Jacqueline Olive, Megumi Mathison, Austin Cooney, Elsa R. Flores, Ronald Crystal, Jianchang Yang, Todd K. Rosengart

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish
JournalJournal of Thoracic and Cardiovascular Surgery
DOIs
Publication statusAccepted/In press - 1 Jan 2018

Fingerprint

Cardiac Myocytes
Troponin T
Fibroblasts
Heart Failure
Actinin
p53 Genes
MicroRNAs
Small Interfering RNA
Flow Cytometry
Transcription Factors
Therapeutics
Down-Regulation
Calcium
Gene Expression

Keywords

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

ASJC Scopus subject areas

  • Surgery
  • Pulmonary and Respiratory Medicine
  • Cardiology and Cardiovascular Medicine

Cite this

Patel, V., Singh, V. P., Pinnamaneni, J. P., Sanagasetti, D., Olive, J., Mathison, M., ... Rosengart, T. K. (Accepted/In press). p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells. Journal of Thoracic and Cardiovascular Surgery. https://doi.org/10.1016/j.jtcvs.2018.03.162

p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells. / Patel, Vivekkumar; Singh, Vivek P.; Pinnamaneni, Jaya Pratap; Sanagasetti, Deepthi; Olive, Jacqueline; Mathison, Megumi; Cooney, Austin; Flores, Elsa R.; Crystal, Ronald; Yang, Jianchang; Rosengart, Todd K.

In: Journal of Thoracic and Cardiovascular Surgery, 01.01.2018.

Research output: Contribution to journalArticle

Patel, V, Singh, VP, Pinnamaneni, JP, Sanagasetti, D, Olive, J, Mathison, M, Cooney, A, Flores, ER, Crystal, R, Yang, J & Rosengart, TK 2018, 'p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells', Journal of Thoracic and Cardiovascular Surgery. https://doi.org/10.1016/j.jtcvs.2018.03.162
Patel, Vivekkumar ; Singh, Vivek P. ; Pinnamaneni, Jaya Pratap ; Sanagasetti, Deepthi ; Olive, Jacqueline ; Mathison, Megumi ; Cooney, Austin ; Flores, Elsa R. ; Crystal, Ronald ; Yang, Jianchang ; Rosengart, Todd K. / p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells. In: Journal of Thoracic and Cardiovascular Surgery. 2018.
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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.",
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AU - Singh, Vivek P.

AU - Pinnamaneni, Jaya Pratap

AU - Sanagasetti, Deepthi

AU - Olive, Jacqueline

AU - Mathison, Megumi

AU - Cooney, Austin

AU - Flores, Elsa R.

AU - Crystal, Ronald

AU - Yang, Jianchang

AU - Rosengart, Todd K.

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N2 - 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.

AB - 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.

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