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In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.

Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D - Nature (2012)

Bottom Line: Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling.Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function.These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

View Article: PubMed Central - PubMed

Affiliation: 1Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA.

ABSTRACT
The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

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Cellular analysis of the degree of in vivo cardiac reprogramminga, Immunofluorescent (IF) staining for YFP, dsRed, and DAPI on heart sections from tamoxifen “pulse-labeled” reprogrammed hearts injected with GMT plus dsRed (GMTR). Scale bar, 50 μm. b, IF staining for YFP, dsRed, and DAPI on isolated CMs from the infarct/border zone of pulse-labeled reprogrammed hearts. Scale bar: 100 μm. c, Quantification of the percentage of YFP+ cardiomyocytes in the infarct/border zone of pulse-labeled mouse hearts injected with dsRed (control) or GMT compared to sham (**p<0.01, n=3). d–f, IF staining for βGal and DAPI on isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZhearts 4 weeks after dsRed (d) or GMT (e) injection with quantification in (f). n=221 cells from three hearts for dsRed group; n=182 cells from three hearts for GMT group. Scale bar, 200 μm. g–k, Bright-field image of CMs isolated from GMTR-injected Postn-Cre:R26R-lacZ hearts 4 weeks after MI (g). Among these cells, a βGal positive cell is shown (h) that also co-stained with dsRed (j,k). Scale bar, 50 μm. l–o, Immunofluorescent staining for cardiac markers—including α-Actinin, Tropomyosin, cardiac myosin heavy chain (MHC), and cardiac Troponin T (cTnT)—co-labeled with βGal and DAPI, in isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. The images display representative reprogrammed CMs next to endogenous CMs from the same preparation. Quantification of cells with full sarcomere development is shown, with the full spectrum of marker expression, localization, and quantification shown in Suppl. Fig. 8. White boxes in the merged pictures indicate the areas for high magnification images shown in the far right panels. Scale bar, 50 μm for the first three columns, 20 μm for the last column. p–q, Electron microscopy of endogenous CMs or reprogrammed CMs, as identified by the Postn-Cre:R26R-Tomato lineage marker (Tomato+ CM). Asterisk indicates mitochondria and brackets indicate sarcomeric units. Scale bar, 2 μm. r, Heat map of gene expression for a panel of CM- or fibroblast-enriched genes in isolated adult cardiac fibroblasts (CFs), CMs or iCMs based on lineage markers. The complete data set with statistics is provided in Suppl. Fig. 9. Error bars indicate standard error of the mean (SEM).
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Figure 2: Cellular analysis of the degree of in vivo cardiac reprogramminga, Immunofluorescent (IF) staining for YFP, dsRed, and DAPI on heart sections from tamoxifen “pulse-labeled” reprogrammed hearts injected with GMT plus dsRed (GMTR). Scale bar, 50 μm. b, IF staining for YFP, dsRed, and DAPI on isolated CMs from the infarct/border zone of pulse-labeled reprogrammed hearts. Scale bar: 100 μm. c, Quantification of the percentage of YFP+ cardiomyocytes in the infarct/border zone of pulse-labeled mouse hearts injected with dsRed (control) or GMT compared to sham (**p<0.01, n=3). d–f, IF staining for βGal and DAPI on isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZhearts 4 weeks after dsRed (d) or GMT (e) injection with quantification in (f). n=221 cells from three hearts for dsRed group; n=182 cells from three hearts for GMT group. Scale bar, 200 μm. g–k, Bright-field image of CMs isolated from GMTR-injected Postn-Cre:R26R-lacZ hearts 4 weeks after MI (g). Among these cells, a βGal positive cell is shown (h) that also co-stained with dsRed (j,k). Scale bar, 50 μm. l–o, Immunofluorescent staining for cardiac markers—including α-Actinin, Tropomyosin, cardiac myosin heavy chain (MHC), and cardiac Troponin T (cTnT)—co-labeled with βGal and DAPI, in isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. The images display representative reprogrammed CMs next to endogenous CMs from the same preparation. Quantification of cells with full sarcomere development is shown, with the full spectrum of marker expression, localization, and quantification shown in Suppl. Fig. 8. White boxes in the merged pictures indicate the areas for high magnification images shown in the far right panels. Scale bar, 50 μm for the first three columns, 20 μm for the last column. p–q, Electron microscopy of endogenous CMs or reprogrammed CMs, as identified by the Postn-Cre:R26R-Tomato lineage marker (Tomato+ CM). Asterisk indicates mitochondria and brackets indicate sarcomeric units. Scale bar, 2 μm. r, Heat map of gene expression for a panel of CM- or fibroblast-enriched genes in isolated adult cardiac fibroblasts (CFs), CMs or iCMs based on lineage markers. The complete data set with statistics is provided in Suppl. Fig. 9. Error bars indicate standard error of the mean (SEM).

Mentions: We formally tested whether retroviral introduction of GMT into non-myocytes could promote cell fusion events in the heart, thereby generating α-Actinin+:β-galactosidase+ cells. We “pulse-labeled” endogenous CMs in transgenic mice with Cre under inducible control of the αMHC promoter (αMHC-MerCreMer)24 crossed with R26R-EYFP mice (Suppl. Fig. 3). Subsequently, hearts were injured and infected retrovirally with GMT and dsRed to mark infected dividing cells. After 4 weeks, we detected no YFP+ cells co-labeled with dsRed in the GMT dsRed-or dsRed-infected hearts (Fig. 2a, b, and Suppl. Fig. 4a). Since pulse-labeling marks only ~80% of endogenous CMs in the uninjured heart and ~60% in the infarct border zone25, we quantified the percentage of YFP+ pulse–labeled endogenous CMs at the border area. GMT introduction resulted in a reduced percentage of YFP+ endogenous CMs compared to total CMs, indicating that the CMs in this region were refreshed by new iCMs (Fig. 2c). These findings suggest that it is unlikely that cell fusion makes a major contribution to the α-Actinin+:β-galactosidase+ cell population, although a minor contribution cannot be ruled out.


In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.

Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D - Nature (2012)

Cellular analysis of the degree of in vivo cardiac reprogramminga, Immunofluorescent (IF) staining for YFP, dsRed, and DAPI on heart sections from tamoxifen “pulse-labeled” reprogrammed hearts injected with GMT plus dsRed (GMTR). Scale bar, 50 μm. b, IF staining for YFP, dsRed, and DAPI on isolated CMs from the infarct/border zone of pulse-labeled reprogrammed hearts. Scale bar: 100 μm. c, Quantification of the percentage of YFP+ cardiomyocytes in the infarct/border zone of pulse-labeled mouse hearts injected with dsRed (control) or GMT compared to sham (**p<0.01, n=3). d–f, IF staining for βGal and DAPI on isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZhearts 4 weeks after dsRed (d) or GMT (e) injection with quantification in (f). n=221 cells from three hearts for dsRed group; n=182 cells from three hearts for GMT group. Scale bar, 200 μm. g–k, Bright-field image of CMs isolated from GMTR-injected Postn-Cre:R26R-lacZ hearts 4 weeks after MI (g). Among these cells, a βGal positive cell is shown (h) that also co-stained with dsRed (j,k). Scale bar, 50 μm. l–o, Immunofluorescent staining for cardiac markers—including α-Actinin, Tropomyosin, cardiac myosin heavy chain (MHC), and cardiac Troponin T (cTnT)—co-labeled with βGal and DAPI, in isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. The images display representative reprogrammed CMs next to endogenous CMs from the same preparation. Quantification of cells with full sarcomere development is shown, with the full spectrum of marker expression, localization, and quantification shown in Suppl. Fig. 8. White boxes in the merged pictures indicate the areas for high magnification images shown in the far right panels. Scale bar, 50 μm for the first three columns, 20 μm for the last column. p–q, Electron microscopy of endogenous CMs or reprogrammed CMs, as identified by the Postn-Cre:R26R-Tomato lineage marker (Tomato+ CM). Asterisk indicates mitochondria and brackets indicate sarcomeric units. Scale bar, 2 μm. r, Heat map of gene expression for a panel of CM- or fibroblast-enriched genes in isolated adult cardiac fibroblasts (CFs), CMs or iCMs based on lineage markers. The complete data set with statistics is provided in Suppl. Fig. 9. Error bars indicate standard error of the mean (SEM).
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Figure 2: Cellular analysis of the degree of in vivo cardiac reprogramminga, Immunofluorescent (IF) staining for YFP, dsRed, and DAPI on heart sections from tamoxifen “pulse-labeled” reprogrammed hearts injected with GMT plus dsRed (GMTR). Scale bar, 50 μm. b, IF staining for YFP, dsRed, and DAPI on isolated CMs from the infarct/border zone of pulse-labeled reprogrammed hearts. Scale bar: 100 μm. c, Quantification of the percentage of YFP+ cardiomyocytes in the infarct/border zone of pulse-labeled mouse hearts injected with dsRed (control) or GMT compared to sham (**p<0.01, n=3). d–f, IF staining for βGal and DAPI on isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZhearts 4 weeks after dsRed (d) or GMT (e) injection with quantification in (f). n=221 cells from three hearts for dsRed group; n=182 cells from three hearts for GMT group. Scale bar, 200 μm. g–k, Bright-field image of CMs isolated from GMTR-injected Postn-Cre:R26R-lacZ hearts 4 weeks after MI (g). Among these cells, a βGal positive cell is shown (h) that also co-stained with dsRed (j,k). Scale bar, 50 μm. l–o, Immunofluorescent staining for cardiac markers—including α-Actinin, Tropomyosin, cardiac myosin heavy chain (MHC), and cardiac Troponin T (cTnT)—co-labeled with βGal and DAPI, in isolated CMs from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. The images display representative reprogrammed CMs next to endogenous CMs from the same preparation. Quantification of cells with full sarcomere development is shown, with the full spectrum of marker expression, localization, and quantification shown in Suppl. Fig. 8. White boxes in the merged pictures indicate the areas for high magnification images shown in the far right panels. Scale bar, 50 μm for the first three columns, 20 μm for the last column. p–q, Electron microscopy of endogenous CMs or reprogrammed CMs, as identified by the Postn-Cre:R26R-Tomato lineage marker (Tomato+ CM). Asterisk indicates mitochondria and brackets indicate sarcomeric units. Scale bar, 2 μm. r, Heat map of gene expression for a panel of CM- or fibroblast-enriched genes in isolated adult cardiac fibroblasts (CFs), CMs or iCMs based on lineage markers. The complete data set with statistics is provided in Suppl. Fig. 9. Error bars indicate standard error of the mean (SEM).
Mentions: We formally tested whether retroviral introduction of GMT into non-myocytes could promote cell fusion events in the heart, thereby generating α-Actinin+:β-galactosidase+ cells. We “pulse-labeled” endogenous CMs in transgenic mice with Cre under inducible control of the αMHC promoter (αMHC-MerCreMer)24 crossed with R26R-EYFP mice (Suppl. Fig. 3). Subsequently, hearts were injured and infected retrovirally with GMT and dsRed to mark infected dividing cells. After 4 weeks, we detected no YFP+ cells co-labeled with dsRed in the GMT dsRed-or dsRed-infected hearts (Fig. 2a, b, and Suppl. Fig. 4a). Since pulse-labeling marks only ~80% of endogenous CMs in the uninjured heart and ~60% in the infarct border zone25, we quantified the percentage of YFP+ pulse–labeled endogenous CMs at the border area. GMT introduction resulted in a reduced percentage of YFP+ endogenous CMs compared to total CMs, indicating that the CMs in this region were refreshed by new iCMs (Fig. 2c). These findings suggest that it is unlikely that cell fusion makes a major contribution to the α-Actinin+:β-galactosidase+ cell population, although a minor contribution cannot be ruled out.

Bottom Line: Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling.Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function.These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

View Article: PubMed Central - PubMed

Affiliation: 1Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA.

ABSTRACT
The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

Show MeSH
Related in: MedlinePlus