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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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Lineage tracing of GHMT-induced iCLMs in vivoa. Border zone of heart sections isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice that were subjected to LAD ligation followed by injection of GFP or GHMT retroviruses. Three weeks post-MI, hearts were fixed, sectioned and stained for cTnT (green) and visualized for tomato (red). Upper panels show GFP-injected heart in which tomato is seen only in fibroblasts. Lower panels show GHMT-injected heart in which tomato is seen in both fibroblasts and cardiomyocytes. Three types of iCLMs (positive for cTnT and tomato) were observed in injured hearts injected with GHMT-virus: (cell A) small cells without sarcomeres; cell B displays sarcomeres and expresses equivalent cTnT to neighboring tomato-negative cardiomyocytes; cell C displays sarcomeres and expresses less cTnT than neighboring tomato-negative cardiomyocytes. Scale bars, 20 μm. b. Tomato+ iCLMs isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice one month post-MI followed by injection of GHMT retroviruses. Scale bar, 20 μm. c. Quantification of tomato+ cardiomyocytes isolated from uninjured hearts (n=2), injured hearts treated with empty vector retroviruses (n=3), or GHMT retroviruses (n=3). Data are presented as mean ± std. d. Recording of action potential of tomato+ iCLMs and endogenous cardiomyocytes by patch-clamping. Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz by whole-cell current patch-clamping. (n=5 for each group).
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Figure 3: Lineage tracing of GHMT-induced iCLMs in vivoa. Border zone of heart sections isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice that were subjected to LAD ligation followed by injection of GFP or GHMT retroviruses. Three weeks post-MI, hearts were fixed, sectioned and stained for cTnT (green) and visualized for tomato (red). Upper panels show GFP-injected heart in which tomato is seen only in fibroblasts. Lower panels show GHMT-injected heart in which tomato is seen in both fibroblasts and cardiomyocytes. Three types of iCLMs (positive for cTnT and tomato) were observed in injured hearts injected with GHMT-virus: (cell A) small cells without sarcomeres; cell B displays sarcomeres and expresses equivalent cTnT to neighboring tomato-negative cardiomyocytes; cell C displays sarcomeres and expresses less cTnT than neighboring tomato-negative cardiomyocytes. Scale bars, 20 μm. b. Tomato+ iCLMs isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice one month post-MI followed by injection of GHMT retroviruses. Scale bar, 20 μm. c. Quantification of tomato+ cardiomyocytes isolated from uninjured hearts (n=2), injured hearts treated with empty vector retroviruses (n=3), or GHMT retroviruses (n=3). Data are presented as mean ± std. d. Recording of action potential of tomato+ iCLMs and endogenous cardiomyocytes by patch-clamping. Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz by whole-cell current patch-clamping. (n=5 for each group).

Mentions: To rule out the possibility that injury or viral infection might somehow activate the Fsp1 reporter in cardiomyocytes, we generated a strain of mice harboring an inducible MerCreMer expression cassette inserted by homologous recombination into the Tcf21 (capsulin/epicardin) locus (Tcf21iCre), which is expressed specifically in non-cardiomyocytes in the heart (Supplementary Fig. 16)30. Intercrossing of these mice with mice bearing the Cre-inducible R26RtdTomato reporter showed specific expression of Tomato predominantly in cardiac fibroblasts with less expression in endothelial cells (Supplementary Fig. 16). However, we only observed one Tomato+ cardiomyocyte in over 40 histological sections of normal hearts. Tcf21iCre/R26RtdT mice were treated with tamoxifen for three days to mark Tcf21-expressing cells. Eight days after the last tamoxifen treatment, LAD ligation was performed and animals were injected with empty vector, GFP or GHMT retroviruses and analyzed 3–4 weeks later. Numerous Tomato+ cardiomyocytes were observed in GHMT-injected hearts compared to GFP-injected ones (Fig. 3a).


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)

Lineage tracing of GHMT-induced iCLMs in vivoa. Border zone of heart sections isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice that were subjected to LAD ligation followed by injection of GFP or GHMT retroviruses. Three weeks post-MI, hearts were fixed, sectioned and stained for cTnT (green) and visualized for tomato (red). Upper panels show GFP-injected heart in which tomato is seen only in fibroblasts. Lower panels show GHMT-injected heart in which tomato is seen in both fibroblasts and cardiomyocytes. Three types of iCLMs (positive for cTnT and tomato) were observed in injured hearts injected with GHMT-virus: (cell A) small cells without sarcomeres; cell B displays sarcomeres and expresses equivalent cTnT to neighboring tomato-negative cardiomyocytes; cell C displays sarcomeres and expresses less cTnT than neighboring tomato-negative cardiomyocytes. Scale bars, 20 μm. b. Tomato+ iCLMs isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice one month post-MI followed by injection of GHMT retroviruses. Scale bar, 20 μm. c. Quantification of tomato+ cardiomyocytes isolated from uninjured hearts (n=2), injured hearts treated with empty vector retroviruses (n=3), or GHMT retroviruses (n=3). Data are presented as mean ± std. d. Recording of action potential of tomato+ iCLMs and endogenous cardiomyocytes by patch-clamping. Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz by whole-cell current patch-clamping. (n=5 for each group).
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Related In: Results  -  Collection

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Figure 3: Lineage tracing of GHMT-induced iCLMs in vivoa. Border zone of heart sections isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice that were subjected to LAD ligation followed by injection of GFP or GHMT retroviruses. Three weeks post-MI, hearts were fixed, sectioned and stained for cTnT (green) and visualized for tomato (red). Upper panels show GFP-injected heart in which tomato is seen only in fibroblasts. Lower panels show GHMT-injected heart in which tomato is seen in both fibroblasts and cardiomyocytes. Three types of iCLMs (positive for cTnT and tomato) were observed in injured hearts injected with GHMT-virus: (cell A) small cells without sarcomeres; cell B displays sarcomeres and expresses equivalent cTnT to neighboring tomato-negative cardiomyocytes; cell C displays sarcomeres and expresses less cTnT than neighboring tomato-negative cardiomyocytes. Scale bars, 20 μm. b. Tomato+ iCLMs isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice one month post-MI followed by injection of GHMT retroviruses. Scale bar, 20 μm. c. Quantification of tomato+ cardiomyocytes isolated from uninjured hearts (n=2), injured hearts treated with empty vector retroviruses (n=3), or GHMT retroviruses (n=3). Data are presented as mean ± std. d. Recording of action potential of tomato+ iCLMs and endogenous cardiomyocytes by patch-clamping. Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz by whole-cell current patch-clamping. (n=5 for each group).
Mentions: To rule out the possibility that injury or viral infection might somehow activate the Fsp1 reporter in cardiomyocytes, we generated a strain of mice harboring an inducible MerCreMer expression cassette inserted by homologous recombination into the Tcf21 (capsulin/epicardin) locus (Tcf21iCre), which is expressed specifically in non-cardiomyocytes in the heart (Supplementary Fig. 16)30. Intercrossing of these mice with mice bearing the Cre-inducible R26RtdTomato reporter showed specific expression of Tomato predominantly in cardiac fibroblasts with less expression in endothelial cells (Supplementary Fig. 16). However, we only observed one Tomato+ cardiomyocyte in over 40 histological sections of normal hearts. Tcf21iCre/R26RtdT mice were treated with tamoxifen for three days to mark Tcf21-expressing cells. Eight days after the last tamoxifen treatment, LAD ligation was performed and animals were injected with empty vector, GFP or GHMT retroviruses and analyzed 3–4 weeks later. Numerous Tomato+ cardiomyocytes were observed in GHMT-injected hearts compared to GFP-injected ones (Fig. 3a).

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