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c-kit+ cells minimally contribute cardiomyocytes to the heart

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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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Figure 1: Kit-Cre lineage tracing.
Figure 2: Analysis of cardiac cells from Kit+/Cre × R-GFP mice.
Figure 3: Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formation.
Figure 4: Assessment of fusion versus de novo cardiomyocyte formation in the heart.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (to J.H.v.B., E.M. and J.D.M.). J.D.M. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

Authors

Contributions

J.D.M., J.H.v.B., O.K., M.M., S.-C.J.L. and R.J.V. designed the experiments. S.-C.J.L. designed the Kit allele targeting construct and targeted mice. J.H.v.B. and O.K. designed the breeding, performed histological experiments and animal procedures. R.J.V. performed the qPCR assays. M.M. performed immunohistochemistry. J.K. performed cell culture experiments. E.M. and R.C.M. designed and conducted the independent verification immunohistochemistry with blinded samples. J.D.M. wrote the manuscript.

Corresponding author

Correspondence to Jeffery D. Molkentin.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Assessing the fidelity and specificity of the Kit-Cre knock-in allele.

a, Representative histological sections from the indicated tissues of Kit+/Cre × R-GFP mice at 4 weeks of age. Blue is nuclei and green is eGFP. The data show eGFP expression in regions of each tissue that is often characteristic of endogenous c-kit protein expression (n = 3 mice). b, Immunohistochemistry for endogenous c-kit expression (red) in the mouse ileum at 4 weeks of age from Kit+/Cre mice that contain the IRES-eGFPnls cassette (but without the × R-GFP reporter allele) so that eGFP expression can be monitored in real time. The inset box and arrows show the co-staining with c-kit antibody and eGFP (n = 3 mice). c, Immunohistochemistry for endogenous c-kit expression (red) in quadriceps muscle of Kit+/Cre mice at 4 weeks of age versus nuclear eGFP (green) from the Kit+/Cre allele (n = 2 mice). Although lineage tracing in Kit+/Cre × R-GFP mice, which is cumulative, showed abundant endothelial cells throughout the skeletal muscle (a), instantaneous c-kit-expressing cells are rare in skeletal muscle, and when identified, are always mononuclear (inset box). d, FACS quantification of bone marrow from Kit+/Cre × R-GFP mice at 4 weeks of age sorted for eGFP expression, of which 94% are positive for the ‘lineage’ cocktail of differentiation-specific antibodies (averages from n = 3 mice, error bars represent s.e.m.). Hence the Kit-Cre allele is properly expressed in bone marrow and traces lineages that arise from c-kit+ progenitors. e, Immunohistochemistry in the hearts of Kit+/Cre × R-GFP mice for endogenous c-kit expression (red) versus all the cells that underwent recombination throughout development and the first 4 weeks of life, shown in green. Although cells that are actively expressing c-kit protein are very rare in the heart (≈5 per heart section), the arrow shows such a cell that is also eGFP+ for recombination. All of the currently c-kit-expressing cells identified in the heart were eGFP+, further verifying the fidelity of the Kit-Cre allele (n = 3 hearts). f, Same experiment as in e except the testis was examined because of the characteristic pattern of Leydig cells that are known to be actively c-kit-expressing cells (n = 3 mice). The data show that greater than 80% of the currently c-kit-antibody-reactive Leydig cells (red outline, better observed in the right panel) are also eGFP+ (arrows show clusters of these cells).

Extended Data Figure 2 Identification of non-myocytes from the hearts of Kit+/Cre × R-GFP mice.

Kit+/Cre × R-GFP mice were collected at 6 weeks of age (constitutive lineage labelling the entire time), although myocardial infarction was performed at week 4 to induce greater vascular remodelling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, whereas the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit-Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, whereas the yellow arrows show two areas with relatively large cells that are eGFP+ and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies (n = 2 mice). b, c, Spread of cells isolated from hearts of 8-week-old Kit+/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers (n = 3 hearts). The large white arrow in panel b shows an eGFP+ (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP+ non-myocytes, which in panel c, were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Blue staining depicts nuclei.

Extended Data Figure 3 Analysis of c-kit lineage labelling in the heart at P0 (birth).

a, Diagram of the timing whereby newborn Kit+/Cre × R-GFP mice were analysed for all subsequent experiments in this figure. b, Histological sections for eGFP fluorescence (green) from the ileum and lung at P0 showing the characteristic c-kit labelling pattern as observed at other time points or in other studies when antibodies were used. Blue shows nuclei c, Histological section for eGFP fluorescence (green) from the heart at P0. Blue shows nuclei and magnification was ×40. d, Immunohistochemical tissue section from the P0 heart of Kit+/Cre × R-GFP mice stained with sarcomeric α-actin (red) to show all underlying cardiomyocytes (right panel) or with eGFP expression in green (left panel) as being c-kit-derived. The green cells noted by the arrows are non-myocytes that do not express sarcomeric α-actin. e, eGFP expression alone (left) or eGFP with co-staining for cardiomyocytes in red (sarcomeric α-actin) from heart sections at P0 of Kit+/Cre × R-GFP mice (n = 3 mice). Blue staining depicts nuclei. The cardiomyocyte that is shown has clear striations in the eGFP staining pattern, whereas the two non-myocytes do not show striated eGFP and also lack sarcomeric α-actin staining. f, eGFP expression alone in green (left) with nuclei in blue or eGFP with sarcomeric α-actin co-staining (red) from heart sections at P0 of Kit+/Cre × R-GFP mice. All eGFP+ cells shown lack striations and are non-myocytes although the two cells in the centre sit directly on top of cardiomyocytes and could be easily misinterpreted. Great care is needed in scoring myocytes in the P0 heart because they are small and often the same size as eGFP+ non-myocytes. g, eGFP expression (green) with nuclei in blue and cardiomyocytes identified in red with sarcomeric α-actin antibody from heart histological sections at P0 of Kit+/Cre × R-GFP mice. Here the data show c-kit-lineage-derived cardiomyocytes that appear in a loose cluster (arrows), presumably from a clonal expansion event earlier in development.

Extended Data Figure 4 Additional examination of the Kit-MerCreMer knock-in allele and its potential leakiness in the absence of tamoxifen.

a, Histological analysis of eGFP fluorescent cells from the indicated tissues of Kit+/MCM× R-GFP mice that were given tamoxifen from 2 to 28 days of age and then collected at day 28. Nuclei are shown in blue and green shows eGFP+cells in the expected patterns for known regions of c-kit protein expression, such as the distinct pattern of melanocytes in the skin and widespread expression in the spleen and lungs. b, Representative immunohistochemical analysis in the testis of Kit+/MCM× R-GFP mice for endogenous c-kit expression (red) versus cells that underwent recombination when tamoxifen was given by intraperitoneal injection (2 mg) for five consecutive days (green) (n = 2 mice). The data show that most of the cells currently expressing c-kit protein in testis (only Leydig cells react, red surface staining) are also eGFP+ (intracellular), indicating that recombination only occurs in c-kit-expressing cells, and in the majority of them. c, Representative histological heart sections from Kit+/MCM× R-GFP mice that were placed on tamoxifen-laden food or vehicle food (n = 6 mice per treatment) beginning at 4 weeks of age and then subjected to myocardial infarction injury 4 weeks later, followed by collection 4 weeks after that. In the presence of tamoxifen, histological sections through the myocardial infarction border zone of the heart show widespread eGFP+ cells (green) from the c-kit lineage (left panel), whereas in the absence of tamoxifen no eGFP+ cells are observed (right panel), indicating the Kit-MerCreMer allele does not leak at baseline or after myocardial infarction injury.

Extended Data Figure 5 Analysis of eGFP+ non-myocytes in the hearts of Kit+/MCM × R-GFP mice at baseline or after myocardial infarction injury.

ag, Tamoxifen was given to Kit+/MCM × R-GFP mice for 1 day–6 months of age (a, e, f) or in mice given tamoxifen and myocardial infarction injury (b, c, d, g), followed by collection of the hearts for immunohistochemistry with antibodies for GFP (green), or the indicated antibodies in red: CD45 (a), CD3 (b), α-SMA (c), vimentin (d), CD34 (e), CD31 (f), vWF (g). Nuclei are shown in blue. The white arrows show cells with coincident green and red reactivity for each of the markers, although sometimes the red marker is membrane-localized whereas the green (eGFP) is always cytoplasmic. The most overlapping activity with GFP expression was observed for CD31 (endothelial cells), then CD34, followed by CD45 (haematopoietic cells). n = 2 Kit+/MCM× R-GFP mice for 1 day–6 months of age; n = 4 Kit+/MCM× R-GFP myocardial infarction. h, Averages from FACS plots for the CD31 cellular fraction (antibody-detected) in the heart that are also eGFP+ from Kit+/MCM × R-GFP mice (pre-MI, n = 3) after 8 weeks of tamoxifen in early adulthood at either baseline or 4 weeks after myocardial infarction injury (post-MI, n = 3). The data show about a doubling in the number of CD31 cells that are eGFP+ after myocardial infarction (*P < 0.05 versus pre-MI).

Extended Data Figure 6 Quantification of Cre activity and DNA recombination in the hearts of Kit+/MCM × R-GFP mice.

a, Timeline for tamoxifen administration in Kit+/MCM × R-GFP mice. b, PCR from DNA isolated 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, whereas 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 in Kit+/MCM × R-GFP mice. e, Echocardiography measured cardiac fractional shortening (FS%) was assessed in the mice after myocardial infarction, which shows a reduction in cardiac ventricular performance at 1, 2 and 4 weeks after injury. The number of mice analysed 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-myocardial infarction. f, Images of dissociated cardiomyocytes from hearts of Kit+/MCM × R-GFP mice 4 weeks after myocardial infarction, which were fixed and stained for sarcomeric α-actin antibody (red) and eGFP (green) at two different magnifications. One eGFP+ cardiomyocyte is shown with sarcomeric patterning of the eGFP fluorescence.

Extended Data Figure 7 Analysis of eGFP+ myocytes in the hearts of Kit+/MCM × R-GFP mice after isoproterenol infusion-induced injury.

a, Schematic diagram showing tamoxifen treatment of Kit+/MCM × R-GFP mice between 7 and 14 weeks of age with isoproterenol (ISO) infusion occurring between weeks 10–14. b, c, Quantification and imaging of disassociated cardiomyocytes (separate images shown at two different magnifications) from the hearts of isoproterenol-injured Kit+/MCM × R-GFP mice, which showed rare but definitive cardiomyocyte labelling. *P < 0.05 versus R-GFP, 31 eGFP+ cells of 395,302 counted from two hearts.

Extended Data Figure 8 Verifying the extent of eGFP+ cardiomyocytes by an independent laboratory from blinded histological heart samples.

Unprocessed 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 cryopreserved blocks are shown at ×200 magnification in which the cryosection was processed for eGFP fluorescence (green) and α-actinin antibody (red) to show cardiomyocytes. The data show two 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-labelled cardiomyocytes observed by us. c, Image taken at ×630 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.

Extended Data Figure 9 Assessing cardiomyocyte differentiation markers from total non-myocytes in the heart.

Adult cardiac interstitial cells isolated from a Kit+/Cre × R-GFP mouse were treated with dexamethasone for 1 week. Cells were then fixed and subjected to immunocytochemistry for the indicated antibodies. c-kit-lineage-derived cells were green (eGFP+) and showed fluorescence in the cytosol and nucleus. The data show eGFP cells that express markers of differentiated cardiomyocytes such as α-actinin, troponin T and the transcription factor GATA4 (all in red) but not the fibroblast marker vimentin (white), nuclei were stained blue (right panels). These results indicate that eGFP+ Kit-Cre-expressing cells can generate pre-differentiated cardiomyocytes as well as non-eGFP interstitial cells; hence the cells identified by the Kit-Cre (knock-in) reporter strategy are representative of how endogenous c-kit+-expressing cells truly function.

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van Berlo, J., Kanisicak, O., Maillet, M. et al. c-kit+ cells minimally contribute cardiomyocytes to the heart. Nature 509, 337–341 (2014). https://doi.org/10.1038/nature13309

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