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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 non-cardiomyocytes toward a cardiac fate in vivo by GHMTa. Heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. GFP-infected myocardium showed only β-gal+ non-cardiomyocytes, GHMT-infected myocardium showed extensive β-gal+ non-cardiomyocytes and cardiomyocytes. Black boxes in top panels are enlarged in lower panels. Scale bar, 40 μm. b. cTnT immunostaining and X-gal staining of heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. The same cell is marked with the same number. Several β-gal+ cells in GHMT-infected injured hearts expressed cTnT and displayed organized sarcomere structure. White boxes are enlarged in insets. Sections of injured hearts were taken at the border zone. Scale bar, 40 μm. c. Quantification of β-gal+ cardiomyocytes in border zones and infarct zones from GFP-infected (168 sections, n=3) and GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice (20 sections, n=2) post-MI. Sections were taken at 4 levels with an interval of 250 μm below LAD ligation site. Data are presented as mean ± std. d. Staining of border zone from GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice post-MI by X-gal (blue), anti-cTnT (red) and anti-Cx43 (green). Gap junctions (green) were observed between β-gal+ and β-gal−cardiomyocytes (A and B) and between β-gal+ cardiomyocytes (C and D). The same cell is marked with the same letter. Scale bar, 20 um. e. Contractility and Ca2+ transients of β-gal+- and β-gal− -cardiomyocytes. β-gal+-cardiomyocytes (iCLMs) were labeled with a fluorogenic β-gal substrate C12FDG (green). Traces of sarcomere shortening were recorded from field-stimulatedβ-gal−-cardiomyocytes (n=15) and iCLMs (n=7) with clear striated morphology. 71.4% of iCLMs displayed a similar pattern of contractility and Ca2+ transients to β-gal−-cardiomyocytes. 28.6% of iCLMs demonstrated immature contractility.
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Figure 2: Reprogramming non-cardiomyocytes toward a cardiac fate in vivo by GHMTa. Heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. GFP-infected myocardium showed only β-gal+ non-cardiomyocytes, GHMT-infected myocardium showed extensive β-gal+ non-cardiomyocytes and cardiomyocytes. Black boxes in top panels are enlarged in lower panels. Scale bar, 40 μm. b. cTnT immunostaining and X-gal staining of heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. The same cell is marked with the same number. Several β-gal+ cells in GHMT-infected injured hearts expressed cTnT and displayed organized sarcomere structure. White boxes are enlarged in insets. Sections of injured hearts were taken at the border zone. Scale bar, 40 μm. c. Quantification of β-gal+ cardiomyocytes in border zones and infarct zones from GFP-infected (168 sections, n=3) and GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice (20 sections, n=2) post-MI. Sections were taken at 4 levels with an interval of 250 μm below LAD ligation site. Data are presented as mean ± std. d. Staining of border zone from GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice post-MI by X-gal (blue), anti-cTnT (red) and anti-Cx43 (green). Gap junctions (green) were observed between β-gal+ and β-gal−cardiomyocytes (A and B) and between β-gal+ cardiomyocytes (C and D). The same cell is marked with the same letter. Scale bar, 20 um. e. Contractility and Ca2+ transients of β-gal+- and β-gal− -cardiomyocytes. β-gal+-cardiomyocytes (iCLMs) were labeled with a fluorogenic β-gal substrate C12FDG (green). Traces of sarcomere shortening were recorded from field-stimulatedβ-gal−-cardiomyocytes (n=15) and iCLMs (n=7) with clear striated morphology. 71.4% of iCLMs displayed a similar pattern of contractility and Ca2+ transients to β-gal−-cardiomyocytes. 28.6% of iCLMs demonstrated immature contractility.

Mentions: Fsp1 is expressed in non-cardiomyocytes such as fibroblasts and transitioning epithelia24, 26. In mouse and human hearts, expression of Fsp1 primarily colocalizes with markers of CFs and increases following MI26. Non-cardiomyocytes in mice carrying alleles of Fsp1-Cre and Rosa26-LacZ are specifically labeled with β-galactosidase (β-gal), providing a marker for fibroblast lineage tracing24. To examine whether cardiac transcription factors were able to activate cardiac genes in non-cardiomyocytes in vivo, we performed LAD ligation on Fsp1-Cre/Rosa26-LacZ mice and injected concentrated retroviruses encoding GHMT or GFP into the border zone immediately following LAD ligation. We then analyzed β-gal activity in histological sections of hearts at various times thereafter. In uninjured hearts, less than one β-gal+ cardiomyocyte per section was observed. After injury, β-galexpression was readily detected in CFs throughout the infarct zone (Fig. 2a, b). In injured hearts infected with GFP viruses, 0.05±0.13% of cardiomyocytes in the injured area were β-gal+ (Fig. 2c), which may be due to low level ectopic activation or to a basal level of new cardiomyocyte formation from a stem cell pool, as reported previously27. In contrast, abundant clusters of intensely stained β-gal+ cardiomyocytes were observed throughout the infarct and border zone of injured hearts infected with the GHMT retrovirus cocktail (Fig. 2b). We observed that 6.5±1.2% of cardiomyocytes in the injured area displayed β-gal activity (Fig. 2c). Generally, more β-gal+ cardiomyocytes were observed in the border zone adjacent to the infarct region, which may be due to intact vascular structures or higher viral infection in this region. Similar results were obtained upon injection with GHMMsT, whereas the inclusion of Nkx2-5 (GHMMsNT) diminished the efficacy of the other five factors (Supplementary Fig. 15), as seen in vitro. β-gal+ cardiomyocytes expressed cTnT and showed clear striations 3 weeks after viral transduction (Fig. 2b). Through quantification of β-gal+ cardiomyocytes in histological sections of infarcted hearts, we calculated that at least 10,000 new myocytes were generated in the injured area after 3 weeks of GHMT infection. This is likely an underestimate of the number of iCLMs generated in vivo since the reporter is only expressed in a subset of non-myocytes, such that unmarked cells could also be reprogrammed but go undetected.


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 non-cardiomyocytes toward a cardiac fate in vivo by GHMTa. Heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. GFP-infected myocardium showed only β-gal+ non-cardiomyocytes, GHMT-infected myocardium showed extensive β-gal+ non-cardiomyocytes and cardiomyocytes. Black boxes in top panels are enlarged in lower panels. Scale bar, 40 μm. b. cTnT immunostaining and X-gal staining of heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. The same cell is marked with the same number. Several β-gal+ cells in GHMT-infected injured hearts expressed cTnT and displayed organized sarcomere structure. White boxes are enlarged in insets. Sections of injured hearts were taken at the border zone. Scale bar, 40 μm. c. Quantification of β-gal+ cardiomyocytes in border zones and infarct zones from GFP-infected (168 sections, n=3) and GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice (20 sections, n=2) post-MI. Sections were taken at 4 levels with an interval of 250 μm below LAD ligation site. Data are presented as mean ± std. d. Staining of border zone from GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice post-MI by X-gal (blue), anti-cTnT (red) and anti-Cx43 (green). Gap junctions (green) were observed between β-gal+ and β-gal−cardiomyocytes (A and B) and between β-gal+ cardiomyocytes (C and D). The same cell is marked with the same letter. Scale bar, 20 um. e. Contractility and Ca2+ transients of β-gal+- and β-gal− -cardiomyocytes. β-gal+-cardiomyocytes (iCLMs) were labeled with a fluorogenic β-gal substrate C12FDG (green). Traces of sarcomere shortening were recorded from field-stimulatedβ-gal−-cardiomyocytes (n=15) and iCLMs (n=7) with clear striated morphology. 71.4% of iCLMs displayed a similar pattern of contractility and Ca2+ transients to β-gal−-cardiomyocytes. 28.6% of iCLMs demonstrated immature contractility.
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Related In: Results  -  Collection

Show All Figures
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Figure 2: Reprogramming non-cardiomyocytes toward a cardiac fate in vivo by GHMTa. Heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. GFP-infected myocardium showed only β-gal+ non-cardiomyocytes, GHMT-infected myocardium showed extensive β-gal+ non-cardiomyocytes and cardiomyocytes. Black boxes in top panels are enlarged in lower panels. Scale bar, 40 μm. b. cTnT immunostaining and X-gal staining of heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. The same cell is marked with the same number. Several β-gal+ cells in GHMT-infected injured hearts expressed cTnT and displayed organized sarcomere structure. White boxes are enlarged in insets. Sections of injured hearts were taken at the border zone. Scale bar, 40 μm. c. Quantification of β-gal+ cardiomyocytes in border zones and infarct zones from GFP-infected (168 sections, n=3) and GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice (20 sections, n=2) post-MI. Sections were taken at 4 levels with an interval of 250 μm below LAD ligation site. Data are presented as mean ± std. d. Staining of border zone from GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice post-MI by X-gal (blue), anti-cTnT (red) and anti-Cx43 (green). Gap junctions (green) were observed between β-gal+ and β-gal−cardiomyocytes (A and B) and between β-gal+ cardiomyocytes (C and D). The same cell is marked with the same letter. Scale bar, 20 um. e. Contractility and Ca2+ transients of β-gal+- and β-gal− -cardiomyocytes. β-gal+-cardiomyocytes (iCLMs) were labeled with a fluorogenic β-gal substrate C12FDG (green). Traces of sarcomere shortening were recorded from field-stimulatedβ-gal−-cardiomyocytes (n=15) and iCLMs (n=7) with clear striated morphology. 71.4% of iCLMs displayed a similar pattern of contractility and Ca2+ transients to β-gal−-cardiomyocytes. 28.6% of iCLMs demonstrated immature contractility.
Mentions: Fsp1 is expressed in non-cardiomyocytes such as fibroblasts and transitioning epithelia24, 26. In mouse and human hearts, expression of Fsp1 primarily colocalizes with markers of CFs and increases following MI26. Non-cardiomyocytes in mice carrying alleles of Fsp1-Cre and Rosa26-LacZ are specifically labeled with β-galactosidase (β-gal), providing a marker for fibroblast lineage tracing24. To examine whether cardiac transcription factors were able to activate cardiac genes in non-cardiomyocytes in vivo, we performed LAD ligation on Fsp1-Cre/Rosa26-LacZ mice and injected concentrated retroviruses encoding GHMT or GFP into the border zone immediately following LAD ligation. We then analyzed β-gal activity in histological sections of hearts at various times thereafter. In uninjured hearts, less than one β-gal+ cardiomyocyte per section was observed. After injury, β-galexpression was readily detected in CFs throughout the infarct zone (Fig. 2a, b). In injured hearts infected with GFP viruses, 0.05±0.13% of cardiomyocytes in the injured area were β-gal+ (Fig. 2c), which may be due to low level ectopic activation or to a basal level of new cardiomyocyte formation from a stem cell pool, as reported previously27. In contrast, abundant clusters of intensely stained β-gal+ cardiomyocytes were observed throughout the infarct and border zone of injured hearts infected with the GHMT retrovirus cocktail (Fig. 2b). We observed that 6.5±1.2% of cardiomyocytes in the injured area displayed β-gal activity (Fig. 2c). Generally, more β-gal+ cardiomyocytes were observed in the border zone adjacent to the infarct region, which may be due to intact vascular structures or higher viral infection in this region. Similar results were obtained upon injection with GHMMsT, whereas the inclusion of Nkx2-5 (GHMMsNT) diminished the efficacy of the other five factors (Supplementary Fig. 15), as seen in vitro. β-gal+ cardiomyocytes expressed cTnT and showed clear striations 3 weeks after viral transduction (Fig. 2b). Through quantification of β-gal+ cardiomyocytes in histological sections of infarcted hearts, we calculated that at least 10,000 new myocytes were generated in the injured area after 3 weeks of GHMT infection. This is likely an underestimate of the number of iCLMs generated in vivo since the reporter is only expressed in a subset of non-myocytes, such that unmarked cells could also be reprogrammed but go undetected.

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