Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5

Abstract

Rationale: Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown.

Objective: To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT).

Methods and results: We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes.

Conclusions: Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

Figure 1. Lentiviral-mediated overexpression of Gata4/MEF2C/Tbx5 factors in CFs and TTFs

(A) A diagram of CF and TTF isolation from αMHC-Cre/ROSA26^mTmG and Nkx2.5-Cre/ROSA26^mTmG mice. (B) Schematic representation of the effect of Cre recombinase excision on the expression of dTomato and membrane tethered eGFP. (C) Whole mount fluorescence imaging of heart of 3 week old αMHC-Cre/ROSA26^mTmG mouse (left) and Nkx2.5-Cre/ROSA26^mTmG day 9.5 embryo (right). Note the specific expression of eGFP in the heart of embryonic mice. (D) Fluorescence microscopy (top) and flow cytometric analysis (bottom) of αMHC-Cre/ROSA26^mTmG TTFs with and without infection with GMT lentiviruses and treatment with doxycycline for 3 weeks. (E) Nkx2.5-Cre/ROSA26^mTmG TTFs were assayed as in (D). (F) αMHC-Cre/ROSA26^mTmG CFs were assayed as in (D). (G) Nkx2.5-Cre/ROSA26^mTmG CFs were assayed as in (D). (H) Cardiac genes expression in uninfected (−) and GMT infected (+) TTFs at 3 weeks post-infection compared with cells from E10.5 hearts (H). Mean±SEM from three independent experiments is shown for each gene. Symbols above brackets denote statistically significant differencesin gene expression(* p<0.05, # p <0.01, ns = not significant). (I) CFs were assayed as in [H]. All micrographs were acquired at 10x magnification. CF cardiac fibroblasts. TTF tail tip fibroblasts.

Figure 2. Gene expression and functional analysis of GMT infected fibroblasts

(A) Flow cytometric analysis of infected and uninfected cTnT-Cre/ROSA26^mTmG TTFs. (B) Fluorescence microscopy of infected and uninfected cTnT-Cre/ROSA26^mTmG TTFs. Images obtained at 10x magnification. (C) Pacing evoked potentials from cardiomyocytes derived from in vitro differentiated ES cells (left), uninfected TTFs (middle) and GMT overexpressing TTFs (right). Note the passive exponential decay (τ= 112ms) of uninfected TTFs and the transient depolarization in GMT-infected fibroblasts. (D) Graded voltage response of GMT overexpressing TTFs to increasing pacing amplitudes (0.5, 1, and 2 nA of 5 ms duration from resting membrane potential). (E) Pacing evoked potentials of GMT infected TTFs in the presence (red) or absence (black) of 5 μM nifedipine. In all traces the dotted line represents resting membrane potential.

Figure 3. Survival and reprogramming of GMT infected fibroblasts in an experimental model of MI

(A) Schematic diagram of experimental procedure to transplant GMT reprogrammed luciferase+eGFP+CFs into the injured hearts of SCID female mice. Transplanted mice were subjected to bioluminescent imaging for 8 days before eGFP+ cells wererecovered for single cell PCR array analysis. (B) Representative bioluminescent imaging of mice injected with GMT overexpressing luciferase+eGFP+ CFs. (C) Quantitative analysis of cell survival (i.e. luciferase activity) in transplanted hearts. (D) Single cell PCR array analysis of FACS-purified eGFP+ cells after in vivo engraftment for 8 days.