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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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Verifying the extent of eGFP+ cardiomyocytes by an independent laboratory from blinded histological heart samplesUnprocessed cryosections and paraffin sections from the hearts of Kit+/MCM × R-GFP mice after 8 weeks of tamoxifen were blinded and sent to the Marbán laboratory along with negative control sections from hearts that should not have staining. a, b, Two separate images from cryo-preserved blocks are shown at 200x magnification in which the cryo-section was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show 2 regions where a single eGFP+ myocyte is visible in a region with several hundred GFP-negative cardiomyocytes. The single eGFP+ cardiomyocyte is circled and the inset box shows a higher magnification. Sections were also stained for nuclei (blue). In general, approximately 1–2 definitive eGFP+ cardiomyocytes were identified per entire heart section in the Marbán laboratory, a result that is consistent with the approximate numbers of kit lineage-labeled cardiomyocytes observed by us. c, Image taken at 630x magnification from a paraffin embedded and processed histological section in which both an eGFP antibody (green) and α-actinin antibody (red) was used. Nuclei are shown in blue. The arrow shows a single eGFP+ expressing cardiomyocyte and the arrowheads show eGFP+ non-myocytes.
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Figure 12: Verifying the extent of eGFP+ cardiomyocytes by an independent laboratory from blinded histological heart samplesUnprocessed cryosections and paraffin sections from the hearts of Kit+/MCM × R-GFP mice after 8 weeks of tamoxifen were blinded and sent to the Marbán laboratory along with negative control sections from hearts that should not have staining. a, b, Two separate images from cryo-preserved blocks are shown at 200x magnification in which the cryo-section was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show 2 regions where a single eGFP+ myocyte is visible in a region with several hundred GFP-negative cardiomyocytes. The single eGFP+ cardiomyocyte is circled and the inset box shows a higher magnification. Sections were also stained for nuclei (blue). In general, approximately 1–2 definitive eGFP+ cardiomyocytes were identified per entire heart section in the Marbán laboratory, a result that is consistent with the approximate numbers of kit lineage-labeled cardiomyocytes observed by us. c, Image taken at 630x magnification from a paraffin embedded and processed histological section in which both an eGFP antibody (green) and α-actinin antibody (red) was used. Nuclei are shown in blue. The arrow shows a single eGFP+ expressing cardiomyocyte and the arrowheads show eGFP+ non-myocytes.

Mentions: Cardiac injury increases cellular turnover in the heart, hence we subjected Kit+/MCM × R-GFP mice to MI at 10 weeks of age during a 6 week tamoxifen labeling protocol (Fig. 3k and Extended Data Fig. 6d–f). The percentage of eGFP+ cardiomyocytes increased to 0.016% within the heart, with more being localized to the infarct border zone (Fig. 3l, m, n). c-kit+ lineage cells within the heart were also pre-labeled by giving tamoxifen only before MI injury, which again showed a very low percentage of eGFP+ cardiomyocytes (Fig. 3o, p). Percentages of eGFP+ cardiomyocytes in the heart during 4 weeks of isoproterenol infusion-induced injury were 0.007% (Extended Data Fig. 7a–c). These astonishingly low values of cardiomyocyte formation were independently verified using blinded heart histological sections from Kit+/MCM × R-GFP mice sent to an outside academic laboratory (Extended Data Fig. 8a, 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)

Verifying the extent of eGFP+ cardiomyocytes by an independent laboratory from blinded histological heart samplesUnprocessed cryosections and paraffin sections from the hearts of Kit+/MCM × R-GFP mice after 8 weeks of tamoxifen were blinded and sent to the Marbán laboratory along with negative control sections from hearts that should not have staining. a, b, Two separate images from cryo-preserved blocks are shown at 200x magnification in which the cryo-section was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show 2 regions where a single eGFP+ myocyte is visible in a region with several hundred GFP-negative cardiomyocytes. The single eGFP+ cardiomyocyte is circled and the inset box shows a higher magnification. Sections were also stained for nuclei (blue). In general, approximately 1–2 definitive eGFP+ cardiomyocytes were identified per entire heart section in the Marbán laboratory, a result that is consistent with the approximate numbers of kit lineage-labeled cardiomyocytes observed by us. c, Image taken at 630x magnification from a paraffin embedded and processed histological section in which both an eGFP antibody (green) and α-actinin antibody (red) was used. Nuclei are shown in blue. The arrow shows a single eGFP+ expressing cardiomyocyte and the arrowheads show eGFP+ non-myocytes.
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Related In: Results  -  Collection

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Figure 12: Verifying the extent of eGFP+ cardiomyocytes by an independent laboratory from blinded histological heart samplesUnprocessed cryosections and paraffin sections from the hearts of Kit+/MCM × R-GFP mice after 8 weeks of tamoxifen were blinded and sent to the Marbán laboratory along with negative control sections from hearts that should not have staining. a, b, Two separate images from cryo-preserved blocks are shown at 200x magnification in which the cryo-section was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show 2 regions where a single eGFP+ myocyte is visible in a region with several hundred GFP-negative cardiomyocytes. The single eGFP+ cardiomyocyte is circled and the inset box shows a higher magnification. Sections were also stained for nuclei (blue). In general, approximately 1–2 definitive eGFP+ cardiomyocytes were identified per entire heart section in the Marbán laboratory, a result that is consistent with the approximate numbers of kit lineage-labeled cardiomyocytes observed by us. c, Image taken at 630x magnification from a paraffin embedded and processed histological section in which both an eGFP antibody (green) and α-actinin antibody (red) was used. Nuclei are shown in blue. The arrow shows a single eGFP+ expressing cardiomyocyte and the arrowheads show eGFP+ non-myocytes.
Mentions: Cardiac injury increases cellular turnover in the heart, hence we subjected Kit+/MCM × R-GFP mice to MI at 10 weeks of age during a 6 week tamoxifen labeling protocol (Fig. 3k and Extended Data Fig. 6d–f). The percentage of eGFP+ cardiomyocytes increased to 0.016% within the heart, with more being localized to the infarct border zone (Fig. 3l, m, n). c-kit+ lineage cells within the heart were also pre-labeled by giving tamoxifen only before MI injury, which again showed a very low percentage of eGFP+ cardiomyocytes (Fig. 3o, p). Percentages of eGFP+ cardiomyocytes in the heart during 4 weeks of isoproterenol infusion-induced injury were 0.007% (Extended Data Fig. 7a–c). These astonishingly low values of cardiomyocyte formation were independently verified using blinded heart histological sections from Kit+/MCM × R-GFP mice sent to an outside academic laboratory (Extended Data Fig. 8a, 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