Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice

Abstract

The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellular-level understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated α-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.

Keywords: aging; cardiovascular progenitors; heart development; regeneration.

Fig. 1.

Cardiomyocytes divide postnatally in mice. (A) MADM recombination in a parent cardiomyocyte in Myh6-expressing cells leads to RFP⁺ and GFP⁺ single-labeled daughter cardiomyocytes (arrowheads). (B) Example of an alternate-label sibling pair. (Scale bar, 15 μm.) (C) TM delivery strategy for D–H. (D) Section through P12 heart reveals double-labeled and sparse single-labeled cells. (Scale bar, 100 µm.) (d′) RFP⁺ single-labeled cell along with its partner GFP⁺ single-labeled cell and double-labeled cells. (d′′) GFP⁺ single-labeled cell (with evident sarcomeric elements) and a double-labeled cell. (d′′′) An RFP⁺ single-labeled cell. (Scale bars, 10 µm.) (E) MADM labeling in the atria. (Scale bar, 50 µm.) (F) Example of GFP⁺ cell and double-labeled cell that stain positive for troponin (white). (Scale bar, 10 μm.) (G) α-Actinin staining (white) reveals sarcomeric structures of a RFP⁺ cardiomyocyte. (Scale bar, 5 μm.) (H) GFP⁺ cardiomyocyte forms gap junctions (Connexin43, white) with underlying syncytium. (Scale bar, 5 μm.) (I, Upper) Frequency of single-labeled cells at different time points after TM administration. (n = 3, P values calculated with Student t test. Error bars indicate SEM) (Lower) Analysis of same-color clones for the above mice. n = total number of single-labeled cells observed per group. (J) Histogram of the distribution of distance between clonal sibling cells (n = 5 mice).

Fig. 2.

Postnatal generation of cardiomyocytes in the actinCreER;MADM model. (A) MADM labeling can occur in any cell type to lead to labeled cardiomyocytes. (B and C) Single-labeled sibling cardiomyocytes exhibit intimate end-on contact. (Scale bars, 10 µm.) (D) Single-labeled RFP cells in a large vessel. [Scale bars, 70 µm (Insets, 5 µm)]. (E and F) Cardiomyocytes and smooth muscle cells do not share MADM labeling. (Scale bars, 20 µm.) (G) Sparse c-kit⁺ cells (white cells within inset box) did not demonstrate MADM labeling. [Scale bar, 30 µm (Inset, 7.5 µm)]. (H and I) Sections of 10-d-old (H) and 4.5-wk-old mice (I) analyzed after TM pulse reveal a temporal decrease in single-labeling frequency. [Scale bars, 100 µm (H) and 1 mm (I).] (n = 3,3 mice). (J) TM administration timeline for I–H.

Fig. 3.

Clonal analysis reveals limited proliferation underlying postnatal cardiogenesis. (A) (Upper) Frequency of single-labeled cells at different time points after recent TM administration (n = 3 per group). (Lower) Analysis of individual-colored clone size for each time point above. (B, (Left) Two GFP⁺ cell clones; (Right) two RFP⁺ cell clones with borders outlined by wheat germ agglutinin staining (orange color). (Scale bars, 5 µm.) (C) Frequency of single-labeled cells 3 and 18 mo after P10 TM pulse reveals no significant proliferation of daughter cells. (Lower) Analysis of clone size for each time point above. N = total number of single-labeled cells observed per group. (P values calculated with Student t test. Error bars indicate SEM).

Fig. 4.

Limited birth of new cardiomyocytes after MI in adult mice. (A) Timeline for injury, TM delivery, and analysis of β-actinCreER;MADM^GT/TG transgenic mouse model. (B) Gross images of the heart after LAD ligation reveals fibrosis (dashed circle) in the infarct region (Top, phase) and limited infarct and peri-infarct labeling (Middle and Bottom: GFP and RFP channel, respectively). (Scale bars, 2 mm.) (C) H&E section of a MI heart demonstrates an area of fibrosis (within dashed lines). (Scale bar, 150 µm.) (D) Frequency of single-labeled cells is similar between the sham (n = 3) and LAD mice (n = 5). (P values calculated with Student t test. Error bars indicate SEM) (E–H) There are few single-labeled cells (white arrowheads) in the left ventricle (LV) after an MI or sham operation. (Scale bars, 120 µm.)

Fig. 5.

Limited division of cardiomyocytes after MI in adult mice. (A) Timeline for injury, TM delivery, and analysis of Myh6CreER; MADM^GT/TG transgenic mouse model. (B) Rare single-labeled cells in the sham heart. (Scale bar, 100 µm.) (Inset) Magnified image of dashed box. (Scale bar, 10 µm). (C, Left) Analysis of clone size for each surgery group; (Right) frequency of single-labeled cells is similar between the sham (n = 3) and LAD mice (n = 5). (P values calculated with Student t test. Error bars indicate SEM) (D) There are few single-labeled cells (white arrowheads) in the left ventricle (LV) after an MI. Dashed lines depict the borders of the infarcted area. [Scale bar, 20 μm (Top), 100 μm (Middle), and 10 μm (Bottom).]