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c-kit+ cells minimally contribute cardiomyocytes to the heart.

van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD - Nature (2014)

Bottom Line: Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008.By contrast, c-kit(+) cells amply generated cardiac endothelial cells.Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA [2] Department of Medicine, division of Cardiology, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA [3].

ABSTRACT
If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

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Quantitation of Cre activity and DNA recombination in the hearts of Kit+/MCM × R-GFP micea, Time line for tamoxifen administration in Kit+/MCM × R-GFP mice. b, PCR from DNA generated from the bone marrow (BM), whole heart or semi-purified cardiomyocytes after 6 weeks of tamoxifen treatment in Kit+/MCM × R-GFP mice (n=2). Bone marrow shows most of the DNA as having been recombined by Cre, while whole heart is just barely discernable, and purified cardiomyocytes show essentially no recombination given the sensitivity constraints of this assay. c, qPCR was also run to more sensitively detect and quantify the extent of recombination, which was set relative to the recombination in bone marrow. Semi-purified cardiomyocytes (CM) showed very low rates. Averaged data are shown and error bars are s.e.m. of duplicate technical replicates from n=3 Kit+/MCM × R-GFP mice. d, Schematic of the tamoxifen time course and timing of myocardial infarction (MI) in Kit+/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after MI, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analyzed is shown in the bars. Error bars represent the s.e.m. Both the control and experimental groups showed an equivalent reduction in cardiac function post-MI. f, Images of dissociated cardiomyocytes from hearts of Kit+/MCM × R-GFP mice 4 weeks after MI, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at 2 different magnifications. One eGFP+ cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.
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Figure 10: Quantitation of Cre activity and DNA recombination in the hearts of Kit+/MCM × R-GFP micea, Time line for tamoxifen administration in Kit+/MCM × R-GFP mice. b, PCR from DNA generated from the bone marrow (BM), whole heart or semi-purified cardiomyocytes after 6 weeks of tamoxifen treatment in Kit+/MCM × R-GFP mice (n=2). Bone marrow shows most of the DNA as having been recombined by Cre, while whole heart is just barely discernable, and purified cardiomyocytes show essentially no recombination given the sensitivity constraints of this assay. c, qPCR was also run to more sensitively detect and quantify the extent of recombination, which was set relative to the recombination in bone marrow. Semi-purified cardiomyocytes (CM) showed very low rates. Averaged data are shown and error bars are s.e.m. of duplicate technical replicates from n=3 Kit+/MCM × R-GFP mice. d, Schematic of the tamoxifen time course and timing of myocardial infarction (MI) in Kit+/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after MI, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analyzed is shown in the bars. Error bars represent the s.e.m. Both the control and experimental groups showed an equivalent reduction in cardiac function post-MI. f, Images of dissociated cardiomyocytes from hearts of Kit+/MCM × R-GFP mice 4 weeks after MI, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at 2 different magnifications. One eGFP+ cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.

Mentions: To specifically address the question of new cardiomyocyte formation within the adult heart, we generated a mouse model in which the tamoxifen inducible MerCreMer protein was targeted to the Kit locus (Kit+/MCM), followed by cross breeding with the R-GFP reporter line (Fig. 3a). To verify the fidelity of this system, Kit+/MCM × R-GFP mice were given tamoxifen during postnatal maturation for approximately 4 weeks followed by harvesting of tissues with known sites of c-kit expression (Extended Data Fig. 4a). Kit+/MCM × R-GFP mice showed ≈70% overlap in recombination-dependent eGFP expression and endogenous c-kit protein in Leydig cells of the testis (Extended Data Fig. 4b). Importantly, no eGFP+ cells were observed in the absence of tamoxifen at any age examined or after myocardial infarction (MI) injury, demonstrating that the MerCreMer system does not “leak” (Extended Data Fig. 4c). Kit+/MCM × R-GFP mice were also given tamoxifen from day 1 through 6 months of age for continuous labeling (Fig. 3b), which produced eGFP expression in greater than 60% of bone marrow cells, but again no signal in the absence of tamoxifen (Fig. 3c–e). Histological analysis of the heart after 6 months of labeling showed rare examples of eGFP+ adult cardiomyocytes and a relatively large number of non-myocytes (Fig. 3f, g). Careful analysis of the non-myocyte fraction in these hearts showed fibroblasts (rarely), smooth muscle cells (rarely), endothelial cells and immune cells, with the majority again being CD31+ (Extended Data Fig. 5a–h). MI injury also doubled the number of CD31 cells that were eGFP+ in the adult heart with 8 weeks of prior tamoxifen labeling (Extended Data Fig. 5h). We also conducted c-kit lineage labeling from 6–12 weeks of age, just after the postnatal developmental period (Fig. 3h). Upon disassociation of these hearts we observed 0.0055% eGFP+ adult cardiomyocytes (Fig. 3i, j), confirmed as extremely low by PCR and qPCR for Rosa26 locus recombination (Extended Data Fig. 6a, b, c).


c-kit+ cells minimally contribute cardiomyocytes to the heart.

van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD - Nature (2014)

Quantitation of Cre activity and DNA recombination in the hearts of Kit+/MCM × R-GFP micea, Time line for tamoxifen administration in Kit+/MCM × R-GFP mice. b, PCR from DNA generated from the bone marrow (BM), whole heart or semi-purified cardiomyocytes after 6 weeks of tamoxifen treatment in Kit+/MCM × R-GFP mice (n=2). Bone marrow shows most of the DNA as having been recombined by Cre, while whole heart is just barely discernable, and purified cardiomyocytes show essentially no recombination given the sensitivity constraints of this assay. c, qPCR was also run to more sensitively detect and quantify the extent of recombination, which was set relative to the recombination in bone marrow. Semi-purified cardiomyocytes (CM) showed very low rates. Averaged data are shown and error bars are s.e.m. of duplicate technical replicates from n=3 Kit+/MCM × R-GFP mice. d, Schematic of the tamoxifen time course and timing of myocardial infarction (MI) in Kit+/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after MI, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analyzed is shown in the bars. Error bars represent the s.e.m. Both the control and experimental groups showed an equivalent reduction in cardiac function post-MI. f, Images of dissociated cardiomyocytes from hearts of Kit+/MCM × R-GFP mice 4 weeks after MI, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at 2 different magnifications. One eGFP+ cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.
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Figure 10: Quantitation of Cre activity and DNA recombination in the hearts of Kit+/MCM × R-GFP micea, Time line for tamoxifen administration in Kit+/MCM × R-GFP mice. b, PCR from DNA generated from the bone marrow (BM), whole heart or semi-purified cardiomyocytes after 6 weeks of tamoxifen treatment in Kit+/MCM × R-GFP mice (n=2). Bone marrow shows most of the DNA as having been recombined by Cre, while whole heart is just barely discernable, and purified cardiomyocytes show essentially no recombination given the sensitivity constraints of this assay. c, qPCR was also run to more sensitively detect and quantify the extent of recombination, which was set relative to the recombination in bone marrow. Semi-purified cardiomyocytes (CM) showed very low rates. Averaged data are shown and error bars are s.e.m. of duplicate technical replicates from n=3 Kit+/MCM × R-GFP mice. d, Schematic of the tamoxifen time course and timing of myocardial infarction (MI) in Kit+/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after MI, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analyzed is shown in the bars. Error bars represent the s.e.m. Both the control and experimental groups showed an equivalent reduction in cardiac function post-MI. f, Images of dissociated cardiomyocytes from hearts of Kit+/MCM × R-GFP mice 4 weeks after MI, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at 2 different magnifications. One eGFP+ cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.
Mentions: To specifically address the question of new cardiomyocyte formation within the adult heart, we generated a mouse model in which the tamoxifen inducible MerCreMer protein was targeted to the Kit locus (Kit+/MCM), followed by cross breeding with the R-GFP reporter line (Fig. 3a). To verify the fidelity of this system, Kit+/MCM × R-GFP mice were given tamoxifen during postnatal maturation for approximately 4 weeks followed by harvesting of tissues with known sites of c-kit expression (Extended Data Fig. 4a). Kit+/MCM × R-GFP mice showed ≈70% overlap in recombination-dependent eGFP expression and endogenous c-kit protein in Leydig cells of the testis (Extended Data Fig. 4b). Importantly, no eGFP+ cells were observed in the absence of tamoxifen at any age examined or after myocardial infarction (MI) injury, demonstrating that the MerCreMer system does not “leak” (Extended Data Fig. 4c). Kit+/MCM × R-GFP mice were also given tamoxifen from day 1 through 6 months of age for continuous labeling (Fig. 3b), which produced eGFP expression in greater than 60% of bone marrow cells, but again no signal in the absence of tamoxifen (Fig. 3c–e). Histological analysis of the heart after 6 months of labeling showed rare examples of eGFP+ adult cardiomyocytes and a relatively large number of non-myocytes (Fig. 3f, g). Careful analysis of the non-myocyte fraction in these hearts showed fibroblasts (rarely), smooth muscle cells (rarely), endothelial cells and immune cells, with the majority again being CD31+ (Extended Data Fig. 5a–h). MI injury also doubled the number of CD31 cells that were eGFP+ in the adult heart with 8 weeks of prior tamoxifen labeling (Extended Data Fig. 5h). We also conducted c-kit lineage labeling from 6–12 weeks of age, just after the postnatal developmental period (Fig. 3h). Upon disassociation of these hearts we observed 0.0055% eGFP+ adult cardiomyocytes (Fig. 3i, j), confirmed as extremely low by PCR and qPCR for Rosa26 locus recombination (Extended Data Fig. 6a, b, c).

Bottom Line: Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008.By contrast, c-kit(+) cells amply generated cardiac endothelial cells.Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA [2] Department of Medicine, division of Cardiology, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA [3].

ABSTRACT
If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

Show MeSH
Related in: MedlinePlus