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Mononuclear Diploidy at the Heart of Cardiomyocyte Proliferation
Cell Stem Cell
Previews Mononuclear Diploidy at the Heart of Cardiomyocyte Proliferation Paula A. da Costa Martins1,2,* 1Department of Cardiology, CARIM School for Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands 2Department of Physiology and Cardiothoracic Surgery, Faculty of Medicine, University of Porto, Porto, Portugal *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2017.09.012
Reporting in Nature Genetics, Patterson et al. (2017) show that adult mammalian hearts possess an innate capacity to regenerate, which depends on a small population of mononuclear diploid cardiomyocytes. These cells undergo karyokinesis and cytokinesis, raising the possibility that endogenous cardiac muscle cells can be stimulated to proliferate for myocardial repair. Upon a myocardial infarct (MI), approximately 1 billion cardiomyocytes are lost (Laflamme and Murry, 2011) and replaced by fibrotic tissue, resulting in loss of cardiac contractile force and insufficient pumping of blood to the organism. While lower vertebrates have a capacity to undergo cardiomyogenesis following injury, adult mammalian hearts have lost this ability and cannot restore normal cardiac function after injury; this often culminates in chronic heart failure. Over the past decade, continuous work has suggested that the mammalian heart is not a terminally differentiated organ, and that there is a constant cardiomyocyte turnover occurring within adult mammalian hearts throughout life (reviewed in Tzahor and Poss, 2017 and Uygur and Lee, 2016). However, the degree of regenerative potential is controversial, with incongruent rates of cardiomyocyte turnover reported and the source of such cardiomyocyte renewal remaining unclear. Developing strategies for cardiac regeneration, and deciphering mechanisms involved in this process, is a fundamental objective in cardiovascular research. The potential utility of stem and progenitor cells for repairing injured myocardium and enhancing endogenous regenerative mechanisms has received much attention. Patterson et al. (2017) set out to understand the nature of potential proliferative cardiomyocytes that would support heart regeneration after injury and identified mononuclear diploid cardiomyocytes (MNDCMs) as one population of proliferative cardiomyocytes in the adult heart with a vigorous ability to regenerate heart muscle. Although zebrafish, newts, and
newborn mammals, including mice and humans, have large numbers of MNDCMs, these numbers are dramatically decreased in adult mammals. Patterson et al. (2017) performed an exhaustive comparison of adult MNDCMs among 120 inbred mouse strains and found surprisingly high variations (>7-fold) in MNDCM frequency during homeostasis (2.3%–17%). The median frequency (6.1%) for the analyzed strains is consistent with previous studies (Soonpaa et al., 1996); however, the most commonly used strain in cardiac biology studies, C57BL/6, possessed a below-average frequency of MNDCMs, possibly explaining why previous studies have not established MNDCMs as an adult cardiac proliferative cell population. Postnatal cardiac growth in mammals shifts from hyperplasia to hypertrophy. Postnatal DNA synthesis is not matched by a concomitant increase in cardiomyocyte number, but by expansion of cell volume without division. In rodents, a burst of DNA replication and karyokinesis without cytokinesis results in binucleated cardiomyocytes becoming the predominant cell type in the heart. In contrast, this degree of binucleation is not observed in zebrafish or in newts, where >95% of the cardiomyocytes are mononucleated. In fact, studies in newts suggest that mononucleated cardiomyocytes undergo cytokinesis. By studying mouse strains with high and low mononuclear cardiomyocyte populations, Patterson et al. (2017) were able to correlate MNDCM frequency with recovery from MI-induced myocardial injury. High-baseline MNDCM was associated with naturally improved cardiac
function and reduced scar tissue 4 weeks after injury. The authors then assessed de novo DNA synthesis and cell-cycle parameters during the post-infarction period by labeling cells with the thymidine analog 5-ethanyl-20 -deoxyuridine (EdU). They found that most of the EdU+ cardiomyocytes were mononuclear and diploid, strongly supporting the idea that DNA synthesis after cardiac injury goes hand in hand with complete cardiomyocyte karyokinesis and cytokinesis. To understand the differences between strains and to shed light onto the mechanisms driving MNDCM proliferation, Patterson et al. (2017) performed genomewide association studies (GWASs) to identify gene candidates associated with mononuclear cardiomyocyte frequency. They found that expression of the cardiomyocyte-specific kinase Tnni3K negatively correlated with mononuclear cardiomyocyte frequency. TNNI3K protein is highly conserved between human and mouse (91% identity) (Wheeler et al., 2009). Previous studies also showed that overexpression of this gene in the heart does not induce detrimental cardiac phenotypes (Tang et al., 2013) during homeostasis, although increased expression of Tnni3k in zebrafish caused severe cardiac scarring after damage due to failure of cardiomyocyte proliferation. A previously-described T/C polymorphism in the Tnni3K gene is predicted to be recessive, with the C allele causing reduced protein expression. Patterson et al. (2017) found that during homeostasis, neither heart-specific nor global ablation of Tnni3K in mice influenced cardiac muscle morphology or function, suggesting it
Cell Stem Cell 21, October 5, 2017 ª 2017 Elsevier Inc. 421
Cell Stem Cell
Previews is not required for viability or normal cardiac function. They performed their gene targeting in the C57BL/6 strain (similar to other groups), which contains low numbers of MNDCMs; although they observed a modest increase in proliferating cells following cardiac stress, no differences in cardiac recovery were observed in comparison to the control group. Nevertheless, there was a clear increase in the population of cardiomyocytes displaying DNA synthesis activation that completed cytokinesis (58% in contrast to 15% of control). The existence of endogenous stem cells in the heart has been questioned over the past several years. The work of Patterson et al. (2017) builds on recent work suggesting that cardiomyocytes themselves may be an appropriate target cell for regenerative interventions. The comprehensive determination of cell-cycle parameters including nucleation, ploidy, and division show that mononuclear cardiomyocyte frequency is a complex trait, with several genes and signaling pathways intertwining toward a common phenotype. While Tnni3k seems to be closely involved in regulation of mononuclear cardiomyocyte frequency, the mechanisms through which it acts remain unclear. How does Tnni3k limit cardiomyocyte progression through the cell cycle? Which other genes are involved in determining whether cell-cycle arrest takes place before either karyokinesis or cytokinesis? It will be of great value and interest to understand how Tnni3k expression and function relates to cell-cy-
422 Cell Stem Cell 21, October 5, 2017
cle checkpoints and/or other transcriptional pathways implicated in cardiomyocyte proliferation such as Wnt, NRG, and Hippo signaling (reviewed in Tzahor and Poss, 2017 and Uygur and Lee, 2016). Tnni3k is also implicated in response to oxidative stress (Nakada et al., 2017), an environment that the heart experiences at birth. With recent studies demonstrating pro-proliferative effects of experimental hypoxia on cardiomyocytes in vivo, further research is necessary to clarify whether MNDCMNs have gene expression signatures associated with hypoxia that would favor cell division instead of hypertrophy, and to unveil the precise contribution of hypoxia to the cardiac regenerative response. Environmental factors or extracellular biomechanical cues such as matrix stiffness that affect cytoskeletal integrity and sarcomere organization must also be considered, as well as the contribution of the different cell types that make the heart. Many different factors are induced by cardiac stress and are secreted not only from cardiomyocytes but also other myocardial cell types such as fibroblasts, nerve cells, and vascular and inflammatory cells. Communication via these paracrine signals or through cellcell junctions may contribute to cell-cycle signaling in cardiomyocytes and therefore to the myocardial regenerative response. Consistently, perturbations in the vasculature in zebrafish and newts inhibit cardiac regeneration and recovery after cardiac injury (Uygur and Lee, 2016). Similarly, macrophages show pro-regen-
erative effects during tissue repair (Eming et al., 2017). The work by Patterson et al. (2017) emphasizes that proliferation of pre-existing cardiomyocytes is the underlying mechanism of cardiomyocyte turnover in both neonatal and adult mammals upon which many of these other factors converge. These findings open new frontiers in cardiac regeneration and suggest future therapeutic strategies aimed at exploiting the power of cardiomyocyte proliferation for heart regeneration. REFERENCES Eming, S.A., Wynn, T.A., and Martin, P. (2017). Science 356, 1026–1030. Laflamme, M.A., and Murry, C.E. (2011). Nature 473, 326–335. Nakada, Y., Canseco, D.C., Thet, S., Abdisalaam, S., Asaithamby, A., Santos, C.X., Shah, A.M., Zhang, H., Faber, J.E., Kinter, M.T., et al. (2017). Nature 541, 222–227. Patterson, M., Barske, L., Van Handel, B., Rau, C.D., Gan, P., Sharma, A., Parikh, S., Denholtz, M., Huang, Y., Yamaguchi, Y., et al. (2017). Nat. Genet. 49, 1346–1353. Soonpaa, M.H., Kim, K.K., Pajak, L., Franklin, M., and Field, L.J. (1996). Am. J. Physiol. 271, H2183–H2189. Tang, H., Xiao, K., Mao, L., Rockman, H.A., and Marchuk, D.A. (2013). J. Mol. Cell. Cardiol. 54, 101–111. Tzahor, E., and Poss, K.D. (2017). Science 356, 1035 LP-1039. Uygur, A., and Lee, R.T. (2016). Dev. Cell 36, 362–374. Wheeler, F.C., Tang, H., Marks, O.A., Hadnott, T.N., Chu, P.L., Mao, L., Rockman, H.A., and Marchuk, D.A. (2009). PLoS Genet. 5, e1000647.
Previews Mononuclear Diploidy at the Heart of Cardiomyocyte Proliferation Paula A. da Costa Martins1,2,* 1Department of Cardiology, CARIM School for Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands 2Department of Physiology and Cardiothoracic Surgery, Faculty of Medicine, University of Porto, Porto, Portugal *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2017.09.012
Reporting in Nature Genetics, Patterson et al. (2017) show that adult mammalian hearts possess an innate capacity to regenerate, which depends on a small population of mononuclear diploid cardiomyocytes. These cells undergo karyokinesis and cytokinesis, raising the possibility that endogenous cardiac muscle cells can be stimulated to proliferate for myocardial repair. Upon a myocardial infarct (MI), approximately 1 billion cardiomyocytes are lost (Laflamme and Murry, 2011) and replaced by fibrotic tissue, resulting in loss of cardiac contractile force and insufficient pumping of blood to the organism. While lower vertebrates have a capacity to undergo cardiomyogenesis following injury, adult mammalian hearts have lost this ability and cannot restore normal cardiac function after injury; this often culminates in chronic heart failure. Over the past decade, continuous work has suggested that the mammalian heart is not a terminally differentiated organ, and that there is a constant cardiomyocyte turnover occurring within adult mammalian hearts throughout life (reviewed in Tzahor and Poss, 2017 and Uygur and Lee, 2016). However, the degree of regenerative potential is controversial, with incongruent rates of cardiomyocyte turnover reported and the source of such cardiomyocyte renewal remaining unclear. Developing strategies for cardiac regeneration, and deciphering mechanisms involved in this process, is a fundamental objective in cardiovascular research. The potential utility of stem and progenitor cells for repairing injured myocardium and enhancing endogenous regenerative mechanisms has received much attention. Patterson et al. (2017) set out to understand the nature of potential proliferative cardiomyocytes that would support heart regeneration after injury and identified mononuclear diploid cardiomyocytes (MNDCMs) as one population of proliferative cardiomyocytes in the adult heart with a vigorous ability to regenerate heart muscle. Although zebrafish, newts, and
newborn mammals, including mice and humans, have large numbers of MNDCMs, these numbers are dramatically decreased in adult mammals. Patterson et al. (2017) performed an exhaustive comparison of adult MNDCMs among 120 inbred mouse strains and found surprisingly high variations (>7-fold) in MNDCM frequency during homeostasis (2.3%–17%). The median frequency (6.1%) for the analyzed strains is consistent with previous studies (Soonpaa et al., 1996); however, the most commonly used strain in cardiac biology studies, C57BL/6, possessed a below-average frequency of MNDCMs, possibly explaining why previous studies have not established MNDCMs as an adult cardiac proliferative cell population. Postnatal cardiac growth in mammals shifts from hyperplasia to hypertrophy. Postnatal DNA synthesis is not matched by a concomitant increase in cardiomyocyte number, but by expansion of cell volume without division. In rodents, a burst of DNA replication and karyokinesis without cytokinesis results in binucleated cardiomyocytes becoming the predominant cell type in the heart. In contrast, this degree of binucleation is not observed in zebrafish or in newts, where >95% of the cardiomyocytes are mononucleated. In fact, studies in newts suggest that mononucleated cardiomyocytes undergo cytokinesis. By studying mouse strains with high and low mononuclear cardiomyocyte populations, Patterson et al. (2017) were able to correlate MNDCM frequency with recovery from MI-induced myocardial injury. High-baseline MNDCM was associated with naturally improved cardiac
function and reduced scar tissue 4 weeks after injury. The authors then assessed de novo DNA synthesis and cell-cycle parameters during the post-infarction period by labeling cells with the thymidine analog 5-ethanyl-20 -deoxyuridine (EdU). They found that most of the EdU+ cardiomyocytes were mononuclear and diploid, strongly supporting the idea that DNA synthesis after cardiac injury goes hand in hand with complete cardiomyocyte karyokinesis and cytokinesis. To understand the differences between strains and to shed light onto the mechanisms driving MNDCM proliferation, Patterson et al. (2017) performed genomewide association studies (GWASs) to identify gene candidates associated with mononuclear cardiomyocyte frequency. They found that expression of the cardiomyocyte-specific kinase Tnni3K negatively correlated with mononuclear cardiomyocyte frequency. TNNI3K protein is highly conserved between human and mouse (91% identity) (Wheeler et al., 2009). Previous studies also showed that overexpression of this gene in the heart does not induce detrimental cardiac phenotypes (Tang et al., 2013) during homeostasis, although increased expression of Tnni3k in zebrafish caused severe cardiac scarring after damage due to failure of cardiomyocyte proliferation. A previously-described T/C polymorphism in the Tnni3K gene is predicted to be recessive, with the C allele causing reduced protein expression. Patterson et al. (2017) found that during homeostasis, neither heart-specific nor global ablation of Tnni3K in mice influenced cardiac muscle morphology or function, suggesting it
Cell Stem Cell 21, October 5, 2017 ª 2017 Elsevier Inc. 421
Cell Stem Cell
Previews is not required for viability or normal cardiac function. They performed their gene targeting in the C57BL/6 strain (similar to other groups), which contains low numbers of MNDCMs; although they observed a modest increase in proliferating cells following cardiac stress, no differences in cardiac recovery were observed in comparison to the control group. Nevertheless, there was a clear increase in the population of cardiomyocytes displaying DNA synthesis activation that completed cytokinesis (58% in contrast to 15% of control). The existence of endogenous stem cells in the heart has been questioned over the past several years. The work of Patterson et al. (2017) builds on recent work suggesting that cardiomyocytes themselves may be an appropriate target cell for regenerative interventions. The comprehensive determination of cell-cycle parameters including nucleation, ploidy, and division show that mononuclear cardiomyocyte frequency is a complex trait, with several genes and signaling pathways intertwining toward a common phenotype. While Tnni3k seems to be closely involved in regulation of mononuclear cardiomyocyte frequency, the mechanisms through which it acts remain unclear. How does Tnni3k limit cardiomyocyte progression through the cell cycle? Which other genes are involved in determining whether cell-cycle arrest takes place before either karyokinesis or cytokinesis? It will be of great value and interest to understand how Tnni3k expression and function relates to cell-cy-
422 Cell Stem Cell 21, October 5, 2017
cle checkpoints and/or other transcriptional pathways implicated in cardiomyocyte proliferation such as Wnt, NRG, and Hippo signaling (reviewed in Tzahor and Poss, 2017 and Uygur and Lee, 2016). Tnni3k is also implicated in response to oxidative stress (Nakada et al., 2017), an environment that the heart experiences at birth. With recent studies demonstrating pro-proliferative effects of experimental hypoxia on cardiomyocytes in vivo, further research is necessary to clarify whether MNDCMNs have gene expression signatures associated with hypoxia that would favor cell division instead of hypertrophy, and to unveil the precise contribution of hypoxia to the cardiac regenerative response. Environmental factors or extracellular biomechanical cues such as matrix stiffness that affect cytoskeletal integrity and sarcomere organization must also be considered, as well as the contribution of the different cell types that make the heart. Many different factors are induced by cardiac stress and are secreted not only from cardiomyocytes but also other myocardial cell types such as fibroblasts, nerve cells, and vascular and inflammatory cells. Communication via these paracrine signals or through cellcell junctions may contribute to cell-cycle signaling in cardiomyocytes and therefore to the myocardial regenerative response. Consistently, perturbations in the vasculature in zebrafish and newts inhibit cardiac regeneration and recovery after cardiac injury (Uygur and Lee, 2016). Similarly, macrophages show pro-regen-
erative effects during tissue repair (Eming et al., 2017). The work by Patterson et al. (2017) emphasizes that proliferation of pre-existing cardiomyocytes is the underlying mechanism of cardiomyocyte turnover in both neonatal and adult mammals upon which many of these other factors converge. These findings open new frontiers in cardiac regeneration and suggest future therapeutic strategies aimed at exploiting the power of cardiomyocyte proliferation for heart regeneration. REFERENCES Eming, S.A., Wynn, T.A., and Martin, P. (2017). Science 356, 1026–1030. Laflamme, M.A., and Murry, C.E. (2011). Nature 473, 326–335. Nakada, Y., Canseco, D.C., Thet, S., Abdisalaam, S., Asaithamby, A., Santos, C.X., Shah, A.M., Zhang, H., Faber, J.E., Kinter, M.T., et al. (2017). Nature 541, 222–227. Patterson, M., Barske, L., Van Handel, B., Rau, C.D., Gan, P., Sharma, A., Parikh, S., Denholtz, M., Huang, Y., Yamaguchi, Y., et al. (2017). Nat. Genet. 49, 1346–1353. Soonpaa, M.H., Kim, K.K., Pajak, L., Franklin, M., and Field, L.J. (1996). Am. J. Physiol. 271, H2183–H2189. Tang, H., Xiao, K., Mao, L., Rockman, H.A., and Marchuk, D.A. (2013). J. Mol. Cell. Cardiol. 54, 101–111. Tzahor, E., and Poss, K.D. (2017). Science 356, 1035 LP-1039. Uygur, A., and Lee, R.T. (2016). Dev. Cell 36, 362–374. Wheeler, F.C., Tang, H., Marks, O.A., Hadnott, T.N., Chu, P.L., Mao, L., Rockman, H.A., and Marchuk, D.A. (2009). PLoS Genet. 5, e1000647.