Limits...
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.

Show MeSH

Related in: MedlinePlus

Assessment of fusion versus de novo cardiomyocyte formation in the heart. a, Genetic strategy in which Kit+/MCM mice were crossed with Rosa26 targeted mice containing the membrane targeted tdTomato/eGFP (mT/mG) reporter. b,c,d,e,f, Tamoxifen was given to Kit+MCM × mT/mG mice between 8 and 10 weeks, followed 3 days later by MI injury. c, Quantitation across >50 histological sections of all eGFP+ expressing cardiomyocytes before MI (n=4 hearts) and 1 (n=4 hearts), 2 (n=5 hearts) and 4 (n=3 hearts) weeks after MI injury. Error bars represent s.e.m., *P<0.05 vs mT/mG. d, Example of a c-kit lineage derived de novo cardiomyocyte in which membrane-eGFP (green, left) is expressed and tdTomato fluorescence (red, right) is lost. e, Example of eGFP+ cardiomyocyte (green) that still contains endogenous membrane-tdTomato fluorescence (red), indicating fusion. Nuclei are stained blue. f, Quantitation of fusion percentage. *P<0.05 vs mT/mG. g, h, i, Immunohistological images from embryonic (E) day 16.5 mouse hearts that are either Kit+/Cre × R-GFP (het [h]), KitMCM/Cre × R-GFP (, [i]) or KitMCM/Cre (, no reporter, [g]). Red staining is α-actinin and green is eGFP. j, Higher magnification image from h, showing a definitive eGFP+ cardiomyocyte (arrow). k, Higher magnification image from i, which shows only eGFP+ non-myocytes in Kit  hearts. l, m, Histological heart images from E18.5 Kit+/Cre (het) and KitMCM/Cre () embryos containing the mT/mG reporter, again only the heterozygotes show examples of eGFP+ cardiomyocytes (arrow). n, Western blot showing loss of c-kit protein in KitMCM/Cre embryos (s) versus heterozygous controls.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4127035&req=5

Figure 4: Assessment of fusion versus de novo cardiomyocyte formation in the heart. a, Genetic strategy in which Kit+/MCM mice were crossed with Rosa26 targeted mice containing the membrane targeted tdTomato/eGFP (mT/mG) reporter. b,c,d,e,f, Tamoxifen was given to Kit+MCM × mT/mG mice between 8 and 10 weeks, followed 3 days later by MI injury. c, Quantitation across >50 histological sections of all eGFP+ expressing cardiomyocytes before MI (n=4 hearts) and 1 (n=4 hearts), 2 (n=5 hearts) and 4 (n=3 hearts) weeks after MI injury. Error bars represent s.e.m., *P<0.05 vs mT/mG. d, Example of a c-kit lineage derived de novo cardiomyocyte in which membrane-eGFP (green, left) is expressed and tdTomato fluorescence (red, right) is lost. e, Example of eGFP+ cardiomyocyte (green) that still contains endogenous membrane-tdTomato fluorescence (red), indicating fusion. Nuclei are stained blue. f, Quantitation of fusion percentage. *P<0.05 vs mT/mG. g, h, i, Immunohistological images from embryonic (E) day 16.5 mouse hearts that are either Kit+/Cre × R-GFP (het [h]), KitMCM/Cre × R-GFP (, [i]) or KitMCM/Cre (, no reporter, [g]). Red staining is α-actinin and green is eGFP. j, Higher magnification image from h, showing a definitive eGFP+ cardiomyocyte (arrow). k, Higher magnification image from i, which shows only eGFP+ non-myocytes in Kit hearts. l, m, Histological heart images from E18.5 Kit+/Cre (het) and KitMCM/Cre () embryos containing the mT/mG reporter, again only the heterozygotes show examples of eGFP+ cardiomyocytes (arrow). n, Western blot showing loss of c-kit protein in KitMCM/Cre embryos (s) versus heterozygous controls.

Mentions: Hearts from Kit+/MCM × R-GFP mice showed the presence of cells from blood lineages (CD3, CD45, and CD34), which are known to have fusigenic activity with resident parenchymal cells 3,14–18. To examine fusion we employed a genetic strategy that constitutively expresses a membrane targeted fluorescent tdTomato protein from the Rosa26 locus. Upon Cre-mediated recombination, tdTomato fluorescence is lost and a membrane targeted eGFP becomes expressed (abbreviated “mT/mG”) (Fig. 4a). If cells fuse, both signals would be present but a de novo cardiomyocyte from a c-kit+ lineage cell would be only green. Experimentally, Kit+/MCM × mT/mG mice were given tamoxifen for 2 weeks (8–10 weeks of age) then 3 days later MIs, followed by harvesting at 1, 2 and 4 weeks thereafter (Fig. 4b). Control mice were harvested before MI but after tamoxifen (time 0). Percentages of total cardiomyocyte membrane-eGFP labeling, whether from fusion or not, were approximately 0.01% at all three time points after MI (Fig. 4c). While some de novo cardiomyocytes were identified in the heart (eGFP only), the majority (80–88%) retained the membrane-tdTomato label indicating that these cells likely arose by fusion (Fig. 4d, e, f). Thus, c-kit+ lineage cells can generate cardiomyocytes in the heart, although at ≈5-fold lower values than initially predicted.


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)

Assessment of fusion versus de novo cardiomyocyte formation in the heart. a, Genetic strategy in which Kit+/MCM mice were crossed with Rosa26 targeted mice containing the membrane targeted tdTomato/eGFP (mT/mG) reporter. b,c,d,e,f, Tamoxifen was given to Kit+MCM × mT/mG mice between 8 and 10 weeks, followed 3 days later by MI injury. c, Quantitation across >50 histological sections of all eGFP+ expressing cardiomyocytes before MI (n=4 hearts) and 1 (n=4 hearts), 2 (n=5 hearts) and 4 (n=3 hearts) weeks after MI injury. Error bars represent s.e.m., *P<0.05 vs mT/mG. d, Example of a c-kit lineage derived de novo cardiomyocyte in which membrane-eGFP (green, left) is expressed and tdTomato fluorescence (red, right) is lost. e, Example of eGFP+ cardiomyocyte (green) that still contains endogenous membrane-tdTomato fluorescence (red), indicating fusion. Nuclei are stained blue. f, Quantitation of fusion percentage. *P<0.05 vs mT/mG. g, h, i, Immunohistological images from embryonic (E) day 16.5 mouse hearts that are either Kit+/Cre × R-GFP (het [h]), KitMCM/Cre × R-GFP (, [i]) or KitMCM/Cre (, no reporter, [g]). Red staining is α-actinin and green is eGFP. j, Higher magnification image from h, showing a definitive eGFP+ cardiomyocyte (arrow). k, Higher magnification image from i, which shows only eGFP+ non-myocytes in Kit  hearts. l, m, Histological heart images from E18.5 Kit+/Cre (het) and KitMCM/Cre () embryos containing the mT/mG reporter, again only the heterozygotes show examples of eGFP+ cardiomyocytes (arrow). n, Western blot showing loss of c-kit protein in KitMCM/Cre embryos (s) versus heterozygous controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127035&req=5

Figure 4: Assessment of fusion versus de novo cardiomyocyte formation in the heart. a, Genetic strategy in which Kit+/MCM mice were crossed with Rosa26 targeted mice containing the membrane targeted tdTomato/eGFP (mT/mG) reporter. b,c,d,e,f, Tamoxifen was given to Kit+MCM × mT/mG mice between 8 and 10 weeks, followed 3 days later by MI injury. c, Quantitation across >50 histological sections of all eGFP+ expressing cardiomyocytes before MI (n=4 hearts) and 1 (n=4 hearts), 2 (n=5 hearts) and 4 (n=3 hearts) weeks after MI injury. Error bars represent s.e.m., *P<0.05 vs mT/mG. d, Example of a c-kit lineage derived de novo cardiomyocyte in which membrane-eGFP (green, left) is expressed and tdTomato fluorescence (red, right) is lost. e, Example of eGFP+ cardiomyocyte (green) that still contains endogenous membrane-tdTomato fluorescence (red), indicating fusion. Nuclei are stained blue. f, Quantitation of fusion percentage. *P<0.05 vs mT/mG. g, h, i, Immunohistological images from embryonic (E) day 16.5 mouse hearts that are either Kit+/Cre × R-GFP (het [h]), KitMCM/Cre × R-GFP (, [i]) or KitMCM/Cre (, no reporter, [g]). Red staining is α-actinin and green is eGFP. j, Higher magnification image from h, showing a definitive eGFP+ cardiomyocyte (arrow). k, Higher magnification image from i, which shows only eGFP+ non-myocytes in Kit hearts. l, m, Histological heart images from E18.5 Kit+/Cre (het) and KitMCM/Cre () embryos containing the mT/mG reporter, again only the heterozygotes show examples of eGFP+ cardiomyocytes (arrow). n, Western blot showing loss of c-kit protein in KitMCM/Cre embryos (s) versus heterozygous controls.
Mentions: Hearts from Kit+/MCM × R-GFP mice showed the presence of cells from blood lineages (CD3, CD45, and CD34), which are known to have fusigenic activity with resident parenchymal cells 3,14–18. To examine fusion we employed a genetic strategy that constitutively expresses a membrane targeted fluorescent tdTomato protein from the Rosa26 locus. Upon Cre-mediated recombination, tdTomato fluorescence is lost and a membrane targeted eGFP becomes expressed (abbreviated “mT/mG”) (Fig. 4a). If cells fuse, both signals would be present but a de novo cardiomyocyte from a c-kit+ lineage cell would be only green. Experimentally, Kit+/MCM × mT/mG mice were given tamoxifen for 2 weeks (8–10 weeks of age) then 3 days later MIs, followed by harvesting at 1, 2 and 4 weeks thereafter (Fig. 4b). Control mice were harvested before MI but after tamoxifen (time 0). Percentages of total cardiomyocyte membrane-eGFP labeling, whether from fusion or not, were approximately 0.01% at all three time points after MI (Fig. 4c). While some de novo cardiomyocytes were identified in the heart (eGFP only), the majority (80–88%) retained the membrane-tdTomato label indicating that these cells likely arose by fusion (Fig. 4d, e, f). Thus, c-kit+ lineage cells can generate cardiomyocytes in the heart, although at ≈5-fold lower values than initially predicted.

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