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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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Electrophysiological properties of induced cardiomyocytesa–b, Immunofluorescent staining for N-Cadherin (a) or Connexin 43 (b), co-labeled with βGal and DAPI in isolated CMs from Postn-Cre:R26R-lacZ hearts 4 weeks after injury. Boxed areas are shown in higher magnification with the percent of cells having the indicated morphology. Green cells represent endogenous CMs, and red/orange cells are iCMs. (c) Immunohistochemistry for Connexin 43 on sections from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. Scale bar: 50 μm in the 1st and 3rd columns of (a,b) and all of (c); 20 μm in the 2nd and 4th columns of (a,b). d, Representative images of two CMs in contact with one another, including an iCM (red, cell #1) and an endogenous CM (non-red, cell #2) loaded with large (dextran) or small (calcein) dye. The large blue dextrandye loaded in the iCM (cell #1) by whole-cell patch-clamp method did not travel to the CM (cell #2), but the smaller, gap junction–permeable dye calcein did cross the cell border (n=5). Scale bar: 50 μm. e, Video frames captured from a group of myocytes, including endogenous CMs (non-red, #1&3) and an iCM (red, #2) imaged for Fluo-4 fluorescence transients corresponding to sarcoplasmic reticulum Ca2+ releases. Video frames 100 ms apart show that the Ca2+ release has spread throughout the myocyte group, including the iCM (n=6). Scale bar: 50 μm. f, Intracellular electrical recording of in vivo–derived YFP+ iCMs and endogenous YFP− CMs from the same preparation. g, Table of action potential parameters measured for CMs and iCMs, including maximum upstroke velocity (dV/dtMax) and minimum diastolic potential (MDP) measured immediately preceding stimulation, overshoot potential (OSP), and the action potential durations (APD) at 90, 70, and 50% repolarization. h, Characteristic single field–stimulated [Ca2+] transients recorded from endogenous (left panel) or induced (right panel) CMs. Lower panels show the simultaneously recorded percent cell shortening responses triggered by the Ca2+ transients, in the same two cells. Quantifications from 6 iCMs and 4 endogenous CMs are shown in the right four panels. For experiments performed in d–h, Cells were isolated from Postn-Cre:Rosa-YFP mice 8 weeks post-MI and virus transduction. Error bars indicate standard error of the mean (SEM).
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Figure 3: Electrophysiological properties of induced cardiomyocytesa–b, Immunofluorescent staining for N-Cadherin (a) or Connexin 43 (b), co-labeled with βGal and DAPI in isolated CMs from Postn-Cre:R26R-lacZ hearts 4 weeks after injury. Boxed areas are shown in higher magnification with the percent of cells having the indicated morphology. Green cells represent endogenous CMs, and red/orange cells are iCMs. (c) Immunohistochemistry for Connexin 43 on sections from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. Scale bar: 50 μm in the 1st and 3rd columns of (a,b) and all of (c); 20 μm in the 2nd and 4th columns of (a,b). d, Representative images of two CMs in contact with one another, including an iCM (red, cell #1) and an endogenous CM (non-red, cell #2) loaded with large (dextran) or small (calcein) dye. The large blue dextrandye loaded in the iCM (cell #1) by whole-cell patch-clamp method did not travel to the CM (cell #2), but the smaller, gap junction–permeable dye calcein did cross the cell border (n=5). Scale bar: 50 μm. e, Video frames captured from a group of myocytes, including endogenous CMs (non-red, #1&3) and an iCM (red, #2) imaged for Fluo-4 fluorescence transients corresponding to sarcoplasmic reticulum Ca2+ releases. Video frames 100 ms apart show that the Ca2+ release has spread throughout the myocyte group, including the iCM (n=6). Scale bar: 50 μm. f, Intracellular electrical recording of in vivo–derived YFP+ iCMs and endogenous YFP− CMs from the same preparation. g, Table of action potential parameters measured for CMs and iCMs, including maximum upstroke velocity (dV/dtMax) and minimum diastolic potential (MDP) measured immediately preceding stimulation, overshoot potential (OSP), and the action potential durations (APD) at 90, 70, and 50% repolarization. h, Characteristic single field–stimulated [Ca2+] transients recorded from endogenous (left panel) or induced (right panel) CMs. Lower panels show the simultaneously recorded percent cell shortening responses triggered by the Ca2+ transients, in the same two cells. Quantifications from 6 iCMs and 4 endogenous CMs are shown in the right four panels. For experiments performed in d–h, Cells were isolated from Postn-Cre:Rosa-YFP mice 8 weeks post-MI and virus transduction. Error bars indicate standard error of the mean (SEM).

Mentions: To determine whether iCMs expressed proteins involved in cell-cell communication similar to endogenous CMs, we examined the expression pattern of N-Cadherin, a cell-surface Ca2+-dependent adhesion molecule normally found in intercalated disks within the myocardium26. We found that over 90% of iCMs expressed N-Cadherin, with 60% of cells localizing N-Cadherin appropriately at the cell border (Fig. 3a). Similarly, about 90% of iCMs expressed Connexin 43 (Cx43), the major gap junction protein in the heart that promotes electrical coupling and synchronized contraction of myocytes27. Half of the iCMs expressed Cx43 at high levels with good localization relative to endogenous CMs (Fig. 3b), and in 4% of these cells, the Cx43 localization pattern was almost indistinguishable from endogenous CMs (Fig. 3b). Immunohistochemistry also revealed good cell-border localization of Cx43 in iCMs (Fig. 3c).


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)

Electrophysiological properties of induced cardiomyocytesa–b, Immunofluorescent staining for N-Cadherin (a) or Connexin 43 (b), co-labeled with βGal and DAPI in isolated CMs from Postn-Cre:R26R-lacZ hearts 4 weeks after injury. Boxed areas are shown in higher magnification with the percent of cells having the indicated morphology. Green cells represent endogenous CMs, and red/orange cells are iCMs. (c) Immunohistochemistry for Connexin 43 on sections from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. Scale bar: 50 μm in the 1st and 3rd columns of (a,b) and all of (c); 20 μm in the 2nd and 4th columns of (a,b). d, Representative images of two CMs in contact with one another, including an iCM (red, cell #1) and an endogenous CM (non-red, cell #2) loaded with large (dextran) or small (calcein) dye. The large blue dextrandye loaded in the iCM (cell #1) by whole-cell patch-clamp method did not travel to the CM (cell #2), but the smaller, gap junction–permeable dye calcein did cross the cell border (n=5). Scale bar: 50 μm. e, Video frames captured from a group of myocytes, including endogenous CMs (non-red, #1&3) and an iCM (red, #2) imaged for Fluo-4 fluorescence transients corresponding to sarcoplasmic reticulum Ca2+ releases. Video frames 100 ms apart show that the Ca2+ release has spread throughout the myocyte group, including the iCM (n=6). Scale bar: 50 μm. f, Intracellular electrical recording of in vivo–derived YFP+ iCMs and endogenous YFP− CMs from the same preparation. g, Table of action potential parameters measured for CMs and iCMs, including maximum upstroke velocity (dV/dtMax) and minimum diastolic potential (MDP) measured immediately preceding stimulation, overshoot potential (OSP), and the action potential durations (APD) at 90, 70, and 50% repolarization. h, Characteristic single field–stimulated [Ca2+] transients recorded from endogenous (left panel) or induced (right panel) CMs. Lower panels show the simultaneously recorded percent cell shortening responses triggered by the Ca2+ transients, in the same two cells. Quantifications from 6 iCMs and 4 endogenous CMs are shown in the right four panels. For experiments performed in d–h, Cells were isolated from Postn-Cre:Rosa-YFP mice 8 weeks post-MI and virus transduction. Error bars indicate standard error of the mean (SEM).
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Figure 3: Electrophysiological properties of induced cardiomyocytesa–b, Immunofluorescent staining for N-Cadherin (a) or Connexin 43 (b), co-labeled with βGal and DAPI in isolated CMs from Postn-Cre:R26R-lacZ hearts 4 weeks after injury. Boxed areas are shown in higher magnification with the percent of cells having the indicated morphology. Green cells represent endogenous CMs, and red/orange cells are iCMs. (c) Immunohistochemistry for Connexin 43 on sections from the infarct/border zone of Postn-Cre:R26R-lacZ hearts 4 weeks after GMT injection. Scale bar: 50 μm in the 1st and 3rd columns of (a,b) and all of (c); 20 μm in the 2nd and 4th columns of (a,b). d, Representative images of two CMs in contact with one another, including an iCM (red, cell #1) and an endogenous CM (non-red, cell #2) loaded with large (dextran) or small (calcein) dye. The large blue dextrandye loaded in the iCM (cell #1) by whole-cell patch-clamp method did not travel to the CM (cell #2), but the smaller, gap junction–permeable dye calcein did cross the cell border (n=5). Scale bar: 50 μm. e, Video frames captured from a group of myocytes, including endogenous CMs (non-red, #1&3) and an iCM (red, #2) imaged for Fluo-4 fluorescence transients corresponding to sarcoplasmic reticulum Ca2+ releases. Video frames 100 ms apart show that the Ca2+ release has spread throughout the myocyte group, including the iCM (n=6). Scale bar: 50 μm. f, Intracellular electrical recording of in vivo–derived YFP+ iCMs and endogenous YFP− CMs from the same preparation. g, Table of action potential parameters measured for CMs and iCMs, including maximum upstroke velocity (dV/dtMax) and minimum diastolic potential (MDP) measured immediately preceding stimulation, overshoot potential (OSP), and the action potential durations (APD) at 90, 70, and 50% repolarization. h, Characteristic single field–stimulated [Ca2+] transients recorded from endogenous (left panel) or induced (right panel) CMs. Lower panels show the simultaneously recorded percent cell shortening responses triggered by the Ca2+ transients, in the same two cells. Quantifications from 6 iCMs and 4 endogenous CMs are shown in the right four panels. For experiments performed in d–h, Cells were isolated from Postn-Cre:Rosa-YFP mice 8 weeks post-MI and virus transduction. Error bars indicate standard error of the mean (SEM).
Mentions: To determine whether iCMs expressed proteins involved in cell-cell communication similar to endogenous CMs, we examined the expression pattern of N-Cadherin, a cell-surface Ca2+-dependent adhesion molecule normally found in intercalated disks within the myocardium26. We found that over 90% of iCMs expressed N-Cadherin, with 60% of cells localizing N-Cadherin appropriately at the cell border (Fig. 3a). Similarly, about 90% of iCMs expressed Connexin 43 (Cx43), the major gap junction protein in the heart that promotes electrical coupling and synchronized contraction of myocytes27. Half of the iCMs expressed Cx43 at high levels with good localization relative to endogenous CMs (Fig. 3b), and in 4% of these cells, the Cx43 localization pattern was almost indistinguishable from endogenous CMs (Fig. 3b). Immunohistochemistry also revealed good cell-border localization of Cx43 in iCMs (Fig. 3c).

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