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Existing Cardiomyocytes Generate Cardiomyocytes at a Low Rate After Birth in Mice

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Existing Cardiomyocytes Generate Cardiomyocytes at a Low Rate After Birth in Mice Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice Shah R. Alia,1,2, Simon Hippenmeyerb,c,3, Lily V. Saadata, Liqun Luob,c, Irving L. Weissmana,1, and Reza Ardehalid,e,1 aDepartments of Pathology and Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Biology and cHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305; and dDepartment of Internal Medicine, Division of Cardiology, and eBroad Stem Cell Research Center, University of California at Los Angeles School of Medicine, Los Angeles, CA, 90095 Contributed by Irving L. Weissman, May 5, 2014 (sent for review January 17, 2014) The mammalian heart has long been considered a postmitotic (2), but a number of questions about postnatal cardiomyo- organ, implying that the total number of cardiomyocytes is set at genesis remains open. For example, it remains unclear whether birth. Analysis of cell division in the mammalian heart is compli- the daughter cells of cardiomyocytes can also divide (i.e., whether cated by cardiomyocyte binucleation shortly after birth, which daughter cells can behave as transit-amplifying cells). It is also makes it challenging to interpret traditional assays of cell turnover unknown whether the cell-of-origin of postnatal cardiomyo- [Laflamme MA, Murray CE (2011) Nature 473(7347):326–335; genesis can generate other cardiovascular cell types at the time Bergmann O, et al. (2009) Science 324(5923):98–102]. An elegant of division. Because the majority of studies in this field use in- multi-isotope imaging-mass spectrometry technique recently cal- direct assays of cell division that rely on analysis of nuclear culated the low, discrete rate of cardiomyocyte generation in mice division rather than cell division, direct observation of cardio- [Senyo SE, et al. (2013) Nature 493(7432):433–436], yet our cellular- myocyte generation at the single-cell level has remained elusive. level understanding of postnatal cardiomyogenesis remains lim- An understanding of postnatal cardiomyocyte generation at the ited. Herein, we provide a new line of evidence for the differenti- cell level could answer some of the open questions about this ated α-myosin heavy chain-expressing cardiomyocyte as the cell of controversial phenomenon. origin of postnatal cardiomyogenesis using the “mosaic analysis Clonal analysis by lineage tracing is a useful method in cancer with double markers” mouse model. We show limited, life-long, biology to trace precursor-progeny relationships of tumorigenic symmetric division of cardiomyocytes as a rare event that is evi- cells (13) and has also revealed the existence of two develop- dent in utero but significantly diminishes after the first month of mental heart fields (14). Because this technique effectively extends life in mice; daughter cardiomyocytes divide very seldom, which fate-mapping to the single-cell level, we sought to determine this study is the first to demonstrate, to our knowledge. Further- whether differentiated cardiomyocytes could generate cardio- more, ligation of the left anterior descending coronary artery, myocytes postnatally in mammals using clonal analysis, which which causes a myocardial infarction in the mosaic analysis with could strengthen existing observations on this phenomenon as double-marker mice, did not increase the rate of cardiomyocyte well as potentially reveal mechanistic details of this property. The division above the basal level for up to 4 wk after the injury. results of this study could also inform development of cell The clonal analysis described here provides direct evidence of therapy for cardiovascular disease. postnatal mammalian cardiomyogenesis. Results heart development | cardiovascular progenitors | aging | regeneration Mosaic Analysis of Double Markers Transgenesis Unambiguously Labels Progeny Cells. To test our hypothesis, we used the “mosaic lthough it was widely believed that the adult heart is a qui- analysis with double markers” (MADM) model, in which the two Aescent organ, in the past several years reports have argued daughter cells of a dividing cell are indelibly and uniquely “sin- + + in favor of the generation of new cardiomyocytes in the mouse gle-labeled” either GFP or RFP because of interchromosomal and human hearts after birth. The strongest evidence to first Cre-loxP recombination after S phase (15, 16) (Fig. 1A and incontrovertibly demonstrate this phenomenon “date-stamped” 14 autopsied human hearts by correlating levels of C in car- Significance diomyocyte nuclei with atmospheric 14C levels in different years, and revealed that a small percentage of cardiomyocytes is born The nature of postnatal cardiomyogenesis in mammals remains during adulthood (1). Despite this significant finding, which in- in dispute. Here, we provide cell-level evidence for the birth of directly correlated nuclear division with cell division, the parent cardiomyocytes in newborn and adult mice. Our clonal analysis, cell of postnatal cardiomyogenesis, as well as the extent of di- based on the mosaic analysis with double markers mouse vision in the postnatal mammalian hearts, remains vigorously model, shows that cardiomyocytes are the parent cell of origin debated. Moreover, the effect of injury on the endogenous rate of cardiomyocytes that are generated postnatally. Our findings of mammalian cardiomyocyte generation is unresolved (2–5). confirm that limited, symmetric division of cardiomyocytes is After resection of the ventricular apex, both adult zebrafish a rare phenomenon in the mouse heart after birth. and neonatal mice exhibit robust regeneration, which fate-map- ping studies suggest occurs through a cardiomyocyte intermediate Author contributions: S.R.A., I.L.W., and R.A. designed research; S.R.A., S.H., and L.V.S. (6–8). However, the study of cardiomyocyte generation by di- performed research; S.R.A., S.H., L.L., I.L.W., and R.A. contributed new reagents/analytic tools; S.R.A., S.H., L.L., I.L.W., and R.A. analyzed data; and S.R.A. and R.A. wrote the paper. vision postnatally has been controversial (9, 10) in the mamma- The authors declare no conflict of interest. lian heart because it often relies on indirect assays of cell division, 1To whom correspondence may be addressed. E-mail: [email protected], irv@stanford. which are challenging to interpret in the setting of cardiomyocyte edu, or [email protected]. polyploidy (11, 12) as well as potential DNA repair upon injury. 2Present address: Department of Internal Medicine, University of California San Francisco, Recently, it was shown using a multi-isotope imaging mass San Francisco, CA, 94117. spectrometry (MIMS) technique and concomitant fate-map- 3Present address: Institute of Science and Technology, A-3400 Klosterneuburg, Austria. ping that cardiomyocytes renew cardiomyocytes after birth in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mice (with, at best, minimal contribution from progenitor cells) 1073/pnas.1408233111/-/DCSupplemental. 8850–8855 | PNAS | June 17, 2014 | vol. 111 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1408233111 A Mitosis G2-X Fig. 1. Cardiomyocytes divide postnatally in mice. (A) MADM recombination in a parent car- Single-labeled cells diomyocyte in Myh6-expressing cells leads to RFP+ + B and GFP single-labeled daughter cardiomyocytes I (arrowheads). (B) Example of an alternate-label RFP GFP 0.1 sibling pair. (Scale bar, 15 μm.) (C) TM delivery RFP+ strategy for D–H.(D) Section through P12 heart C GFP+ Birth TM TM TM TM Analyze reveals double-labeled and sparse single-labeled Age 0.05 + cells. (Scale bar, 100 μm.) (d′)RFP single-labeled (days) Fraction of + Labelled cells cell along with its partner GFP single-labeled cell 0 2 4 6 8 12 + 0 and double-labeled cells. (d′′)GFP single-labeled d Week 1 Week 5 D cell (with evident sarcomeric elements) and + d a double-labeled cell. (d′′′) An RFP single-labeled 1 cell. (Scale bars, 10 μm.) (E) MADM labeling in the 2 d + d 3 atria. (Scale bar, 50 μm.) (F) Example of GFP cell N=1251 N=759 Clone size Time at TM pulse and double-labeled cell that stain positive for (in no. of cells) (no chase) troponin (white). (Scale bar, 10 μm.) (G) α-Actinin d d staining (white) reveals sarcomeric structures of a + + J RFP cardiomyocyte. (Scale bar, 5 μm.) (H) GFP E F 5 cardiomyocyte forms gap junctions (Connexin43, 4 white) with underlying syncytium. (Scale bar, 5 μm.) 3 (I, Upper) Frequency of single-labeled cells at dif- 2 = 1 ferent time points after TM administration. (n 3, 0 P values calculated with Student t test. Error bars G H No. of twin-spot pairs indicate SEM) (Lower)Analysisofsame-colorclones for the above mice. n = total number of single- Distance between sibling labeled cells observed per group. (J) Histogram of daughter cells (µm) the distribution of distance between clonal sibling cells (n = 5mice). Fig. S1). MADM labeling allows unambiguous identification recombination to E16.5) is not clear and conclusions about of progeny cells because cytokinesis is needed to generate cardiomyocyte proliferation rates at different time points in + + individual GFP and RFP cells (a binucleated cell would be utero cannot be ascertained from these data. In addition, be- + + double-labeled as GFP RFP , and appear yellow; DNA re- cause Hprt can be expressed in any cell, the identity of the cell pair would not cause labeling) (17). Because MADM single- type that forms a cluster of cardiomyocytes cannot be revealed by labeling can only be achieved by completion of the cell cycle this system. However, this transgenic model (HprtCre;MADM) through cytokinesis, it permits analysis of cell division that is helped to validate the utility of MADM in the heart, and directly related to cytokinesis and uncoupled from karyoki- prompted us to use an inducible cardiomyocyte-specific Cre nesis, unlike many prior reports that have analyzed division transgenic mouse model (described below) to investigate clonal after birth.
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