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Heart repair by reprogramming non-myocytes with cardiac transcription factors.

Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, Hill JA, Bassel-Duby R, Olson EN - Nature (2012)

Bottom Line: Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias.Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction.Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390-9148, USA.

ABSTRACT
The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

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Reprogramming fibroblasts toward a cardiac phenotype in vitro by GHMTa. and b. Immunofluorescent staining for cardiac markers, α-MHC-GFP and α-actinin. Adult CFs isolated from α-MHC-GFP reporter mice were transduced by retroviruses carrying GHMT or empty vector alone. Immunocytochemistry was performed at (a) 14 days or (b) 30 days following transduction. Three types of iCLMs were categorized based on cardiac gene expression and morphology. Type A iCLMs only express α-MHC-GFP (top panels). Type B iCLMs express both α-MHC-GFP and α-actinin, but do not display sarcomeric structures (middle panels). Type C iCLMs express both α-MHC-GFP and α-actinin, and display sarcomeric structures (bottom panels). Sarcomeres were more organized at day 30 than at day 14 post-transduction. White boxes are enlarged in insets. Scale bar, 20 μm. c. Quantification of Type A, B, and C iCLMs in adult CFs and TTFs 14 days after transduction with GHMT. Ten fields were randomly chosen from each experiment. Three independent experiments were performed. Data are presented as mean ± std. d. Representative calcium transient traces from the indicated cell types depicted as Fura-2 ratios (340/380 nm). iCLMs were derived from adult CFs and displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes. Adult CFs do not display calcium transients.
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Figure 1: Reprogramming fibroblasts toward a cardiac phenotype in vitro by GHMTa. and b. Immunofluorescent staining for cardiac markers, α-MHC-GFP and α-actinin. Adult CFs isolated from α-MHC-GFP reporter mice were transduced by retroviruses carrying GHMT or empty vector alone. Immunocytochemistry was performed at (a) 14 days or (b) 30 days following transduction. Three types of iCLMs were categorized based on cardiac gene expression and morphology. Type A iCLMs only express α-MHC-GFP (top panels). Type B iCLMs express both α-MHC-GFP and α-actinin, but do not display sarcomeric structures (middle panels). Type C iCLMs express both α-MHC-GFP and α-actinin, and display sarcomeric structures (bottom panels). Sarcomeres were more organized at day 30 than at day 14 post-transduction. White boxes are enlarged in insets. Scale bar, 20 μm. c. Quantification of Type A, B, and C iCLMs in adult CFs and TTFs 14 days after transduction with GHMT. Ten fields were randomly chosen from each experiment. Three independent experiments were performed. Data are presented as mean ± std. d. Representative calcium transient traces from the indicated cell types depicted as Fura-2 ratios (340/380 nm). iCLMs were derived from adult CFs and displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes. Adult CFs do not display calcium transients.

Mentions: Cardiac fibroblasts (CFs) are the most prevalent interstitial cell type in adult mammalian hearts. We transduced adult CFs with GHMT, GMT or empty viruses and analyzed expression of cardiac markers by flow cytometry one week later. GHMT induced 6.8% of CFs to become cTnT+/α-MHC-GFP+, compared with 1.4% double-positive cells with GMT (Supplementary Fig. 6a). GHMT induced both cTnT and cTnI (cardiac Troponin I) in 7.5% of cells (Supplementary Fig. 6b). Thus, GHMT represented the most optimal combination of factors for efficient initiation of cardiac gene expression in adult fibroblasts. α-MHC-GFP+ cells derived from adult TTFs and CFs by GHMT transduction showed strong immunostaining of the sarcomeric proteins α-actinin and cTnT (Fig. 1a, b, and Supplementary Fig. 7). More organized sarcomeres were observed in iCLMs at day 30 (Fig. 1b), compared to day 14 (Fig. 1a). In the presence of GHMT, three types of iCLMs, referred to as Types A, B and C, which appear to represent a spectrum of cardiac reprogramming, were induced from adult CFs and TTFs. Type A iCLMs only expressed α-MHC-GFP. Type B iCLMs expressed both α-MHC-GFP and α-actinin. Type C iCLMs not only expressed α-MHC-GFP and α-actinin, but also displayed sarcomere-like structures (Fig. 1a). GHMT induced ~15% of adult CFs and ~18% of adult TTFs to become α-MHC-GFP positive cells, and ~30% and ~10% of these became Type C iCLMs, respectively (Fig. 1c).


Heart repair by reprogramming non-myocytes with cardiac transcription factors.

Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, Hill JA, Bassel-Duby R, Olson EN - Nature (2012)

Reprogramming fibroblasts toward a cardiac phenotype in vitro by GHMTa. and b. Immunofluorescent staining for cardiac markers, α-MHC-GFP and α-actinin. Adult CFs isolated from α-MHC-GFP reporter mice were transduced by retroviruses carrying GHMT or empty vector alone. Immunocytochemistry was performed at (a) 14 days or (b) 30 days following transduction. Three types of iCLMs were categorized based on cardiac gene expression and morphology. Type A iCLMs only express α-MHC-GFP (top panels). Type B iCLMs express both α-MHC-GFP and α-actinin, but do not display sarcomeric structures (middle panels). Type C iCLMs express both α-MHC-GFP and α-actinin, and display sarcomeric structures (bottom panels). Sarcomeres were more organized at day 30 than at day 14 post-transduction. White boxes are enlarged in insets. Scale bar, 20 μm. c. Quantification of Type A, B, and C iCLMs in adult CFs and TTFs 14 days after transduction with GHMT. Ten fields were randomly chosen from each experiment. Three independent experiments were performed. Data are presented as mean ± std. d. Representative calcium transient traces from the indicated cell types depicted as Fura-2 ratios (340/380 nm). iCLMs were derived from adult CFs and displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes. Adult CFs do not display calcium transients.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Reprogramming fibroblasts toward a cardiac phenotype in vitro by GHMTa. and b. Immunofluorescent staining for cardiac markers, α-MHC-GFP and α-actinin. Adult CFs isolated from α-MHC-GFP reporter mice were transduced by retroviruses carrying GHMT or empty vector alone. Immunocytochemistry was performed at (a) 14 days or (b) 30 days following transduction. Three types of iCLMs were categorized based on cardiac gene expression and morphology. Type A iCLMs only express α-MHC-GFP (top panels). Type B iCLMs express both α-MHC-GFP and α-actinin, but do not display sarcomeric structures (middle panels). Type C iCLMs express both α-MHC-GFP and α-actinin, and display sarcomeric structures (bottom panels). Sarcomeres were more organized at day 30 than at day 14 post-transduction. White boxes are enlarged in insets. Scale bar, 20 μm. c. Quantification of Type A, B, and C iCLMs in adult CFs and TTFs 14 days after transduction with GHMT. Ten fields were randomly chosen from each experiment. Three independent experiments were performed. Data are presented as mean ± std. d. Representative calcium transient traces from the indicated cell types depicted as Fura-2 ratios (340/380 nm). iCLMs were derived from adult CFs and displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes. Adult CFs do not display calcium transients.
Mentions: Cardiac fibroblasts (CFs) are the most prevalent interstitial cell type in adult mammalian hearts. We transduced adult CFs with GHMT, GMT or empty viruses and analyzed expression of cardiac markers by flow cytometry one week later. GHMT induced 6.8% of CFs to become cTnT+/α-MHC-GFP+, compared with 1.4% double-positive cells with GMT (Supplementary Fig. 6a). GHMT induced both cTnT and cTnI (cardiac Troponin I) in 7.5% of cells (Supplementary Fig. 6b). Thus, GHMT represented the most optimal combination of factors for efficient initiation of cardiac gene expression in adult fibroblasts. α-MHC-GFP+ cells derived from adult TTFs and CFs by GHMT transduction showed strong immunostaining of the sarcomeric proteins α-actinin and cTnT (Fig. 1a, b, and Supplementary Fig. 7). More organized sarcomeres were observed in iCLMs at day 30 (Fig. 1b), compared to day 14 (Fig. 1a). In the presence of GHMT, three types of iCLMs, referred to as Types A, B and C, which appear to represent a spectrum of cardiac reprogramming, were induced from adult CFs and TTFs. Type A iCLMs only expressed α-MHC-GFP. Type B iCLMs expressed both α-MHC-GFP and α-actinin. Type C iCLMs not only expressed α-MHC-GFP and α-actinin, but also displayed sarcomere-like structures (Fig. 1a). GHMT induced ~15% of adult CFs and ~18% of adult TTFs to become α-MHC-GFP positive cells, and ~30% and ~10% of these became Type C iCLMs, respectively (Fig. 1c).

Bottom Line: Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias.Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction.Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390-9148, USA.

ABSTRACT
The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

Show MeSH
Related in: MedlinePlus