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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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In vivo delivery of cardiac reprogramming factors improves cardiac function after myocardial infarctiona, Ejection fraction (EF), stroke volume (SV), and cardiac output (CO) of the left ventricle were quantified by magnetic resonance imaging (MRI) 12 weeks after MI (n=9 for each group, *p<0.05). Left four panels show representative transverse images of the thorax, containing hearts at the end of diastole (relaxation) or systole (contraction) from dsRed- or GMT-injected mice, compared to sham-operated age- and strain-matched controls. b, qPCR of atrial natriuretic factor (ANF), brain natriuretic peptide(BNP) and Tenascin (TnC) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. c, qPCR of collagen type I alpha 1 (Col1a1), Col1a2, Col3a1 and elastin (Eln) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. Data in (b) and (c) are relative to dsRed-injected sham-operated mice, indicated by the dashed line. n=3 for each genotype with technical quadruplicates. *p<0.05. d, Masson-Trichrome staining on heart sections 8 weeks post-MI injected with dsRed or GMT with quantification of scar size. Scale bars: 500 μm. (dsRed, n=8; GMT, n=9; *p<0.05). e, Masson-Trichrome (left panel) and immunofluorescent staining for α-Actinin and/or βGal (right panels) in GMT injected Postn-Cre:R26R-lacZ mouse heart 4 weeks post-surgery. Scale bars: 500 μm in the left panel, 50 μm in the right two panels. Error bars indicate standard error of the mean (SEM).
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Figure 4: In vivo delivery of cardiac reprogramming factors improves cardiac function after myocardial infarctiona, Ejection fraction (EF), stroke volume (SV), and cardiac output (CO) of the left ventricle were quantified by magnetic resonance imaging (MRI) 12 weeks after MI (n=9 for each group, *p<0.05). Left four panels show representative transverse images of the thorax, containing hearts at the end of diastole (relaxation) or systole (contraction) from dsRed- or GMT-injected mice, compared to sham-operated age- and strain-matched controls. b, qPCR of atrial natriuretic factor (ANF), brain natriuretic peptide(BNP) and Tenascin (TnC) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. c, qPCR of collagen type I alpha 1 (Col1a1), Col1a2, Col3a1 and elastin (Eln) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. Data in (b) and (c) are relative to dsRed-injected sham-operated mice, indicated by the dashed line. n=3 for each genotype with technical quadruplicates. *p<0.05. d, Masson-Trichrome staining on heart sections 8 weeks post-MI injected with dsRed or GMT with quantification of scar size. Scale bars: 500 μm. (dsRed, n=8; GMT, n=9; *p<0.05). e, Masson-Trichrome (left panel) and immunofluorescent staining for α-Actinin and/or βGal (right panels) in GMT injected Postn-Cre:R26R-lacZ mouse heart 4 weeks post-surgery. Scale bars: 500 μm in the left panel, 50 μm in the right two panels. Error bars indicate standard error of the mean (SEM).

Mentions: Since in vivo reprogrammed iCMs had contractile potential and electrically coupled with viable endogenous CMs (and other iCMs), we asked whether converting endogenous non-myocytes into new myocytes translates into partial restoration of heart function after MI. All studies were performed in a blinded fashion, including the retroviral injections, and were de-coded only after completion of the measurements. By Evans blue/TTC double staining, the area-at-risk (AAR) and the myocardium infarct size were similar in GMT-or dsRed-injected mice 48 hours after coronary ligation (Suppl. Fig. 12a). Three months post-MI, cardiac function was examined by magnetic resonance imaging (MRI). The fraction of blood ejected with each ventricular contraction (ejection fraction), the volume of blood ejected (stroke volume), and the total cardiac output per minute were significantly improved in GMT-infected mice, particularly the stroke volume and cardiac output, possibly due to cardiac enlargement (Fig. 4a). To determine the time course of these improvements, other mice underwent serial high-resolution two-dimensional echocardiography 1 day before MI, and 3 days,1-, 4-, 8-, and 12-weeks post-MI (Supp. Fig. 11b-d). All mice showed a comparable reduction in left ventricular function after coronary artery ligation (Supp. Fig. 11c). Although different imaging approaches yield different absolute value norms, the overarching trends observed by echocardiography were similar to our MRI findings, in that functional improvements for all parameters were statistically significant 8 and 12 weeks post-injection (Supp. Fig. 11c).


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)

In vivo delivery of cardiac reprogramming factors improves cardiac function after myocardial infarctiona, Ejection fraction (EF), stroke volume (SV), and cardiac output (CO) of the left ventricle were quantified by magnetic resonance imaging (MRI) 12 weeks after MI (n=9 for each group, *p<0.05). Left four panels show representative transverse images of the thorax, containing hearts at the end of diastole (relaxation) or systole (contraction) from dsRed- or GMT-injected mice, compared to sham-operated age- and strain-matched controls. b, qPCR of atrial natriuretic factor (ANF), brain natriuretic peptide(BNP) and Tenascin (TnC) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. c, qPCR of collagen type I alpha 1 (Col1a1), Col1a2, Col3a1 and elastin (Eln) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. Data in (b) and (c) are relative to dsRed-injected sham-operated mice, indicated by the dashed line. n=3 for each genotype with technical quadruplicates. *p<0.05. d, Masson-Trichrome staining on heart sections 8 weeks post-MI injected with dsRed or GMT with quantification of scar size. Scale bars: 500 μm. (dsRed, n=8; GMT, n=9; *p<0.05). e, Masson-Trichrome (left panel) and immunofluorescent staining for α-Actinin and/or βGal (right panels) in GMT injected Postn-Cre:R26R-lacZ mouse heart 4 weeks post-surgery. Scale bars: 500 μm in the left panel, 50 μm in the right two panels. Error bars indicate standard error of the mean (SEM).
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Figure 4: In vivo delivery of cardiac reprogramming factors improves cardiac function after myocardial infarctiona, Ejection fraction (EF), stroke volume (SV), and cardiac output (CO) of the left ventricle were quantified by magnetic resonance imaging (MRI) 12 weeks after MI (n=9 for each group, *p<0.05). Left four panels show representative transverse images of the thorax, containing hearts at the end of diastole (relaxation) or systole (contraction) from dsRed- or GMT-injected mice, compared to sham-operated age- and strain-matched controls. b, qPCR of atrial natriuretic factor (ANF), brain natriuretic peptide(BNP) and Tenascin (TnC) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. c, qPCR of collagen type I alpha 1 (Col1a1), Col1a2, Col3a1 and elastin (Eln) on RNA extracted from the border zone of hearts 4 weeks after MI and injection of dsRed or GMT. Data in (b) and (c) are relative to dsRed-injected sham-operated mice, indicated by the dashed line. n=3 for each genotype with technical quadruplicates. *p<0.05. d, Masson-Trichrome staining on heart sections 8 weeks post-MI injected with dsRed or GMT with quantification of scar size. Scale bars: 500 μm. (dsRed, n=8; GMT, n=9; *p<0.05). e, Masson-Trichrome (left panel) and immunofluorescent staining for α-Actinin and/or βGal (right panels) in GMT injected Postn-Cre:R26R-lacZ mouse heart 4 weeks post-surgery. Scale bars: 500 μm in the left panel, 50 μm in the right two panels. Error bars indicate standard error of the mean (SEM).
Mentions: Since in vivo reprogrammed iCMs had contractile potential and electrically coupled with viable endogenous CMs (and other iCMs), we asked whether converting endogenous non-myocytes into new myocytes translates into partial restoration of heart function after MI. All studies were performed in a blinded fashion, including the retroviral injections, and were de-coded only after completion of the measurements. By Evans blue/TTC double staining, the area-at-risk (AAR) and the myocardium infarct size were similar in GMT-or dsRed-injected mice 48 hours after coronary ligation (Suppl. Fig. 12a). Three months post-MI, cardiac function was examined by magnetic resonance imaging (MRI). The fraction of blood ejected with each ventricular contraction (ejection fraction), the volume of blood ejected (stroke volume), and the total cardiac output per minute were significantly improved in GMT-infected mice, particularly the stroke volume and cardiac output, possibly due to cardiac enlargement (Fig. 4a). To determine the time course of these improvements, other mice underwent serial high-resolution two-dimensional echocardiography 1 day before MI, and 3 days,1-, 4-, 8-, and 12-weeks post-MI (Supp. Fig. 11b-d). All mice showed a comparable reduction in left ventricular function after coronary artery ligation (Supp. Fig. 11c). Although different imaging approaches yield different absolute value norms, the overarching trends observed by echocardiography were similar to our MRI findings, in that functional improvements for all parameters were statistically significant 8 and 12 weeks post-injection (Supp. Fig. 11c).

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