Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury

Abstract

An emerging concept is that the mammalian myocardium has the potential to regenerate, but that regeneration might be too inefficient to repair the extensive myocardial injury that is typical of human disease. However, the degree to which stem cells or precursor cells contribute to the renewal of adult mammalian cardiomyocytes remains controversial. Here we report evidence that stem cells or precursor cells contribute to the replacement of adult mammalian cardiomyocytes after injury but do not contribute significantly to cardiomyocyte renewal during normal aging. We generated double-transgenic mice to track the fate of adult cardiomyocytes in a 'pulse-chase' fashion: after a 4-OH-tamoxifen pulse, green fluorescent protein (GFP) expression was induced only in cardiomyocytes, with 82.7% of cardiomyocytes expressing GFP. During normal aging up to one year, the percentage of GFP+ cardiomyocytes remained unchanged, indicating that stem or precursor cells did not refresh uninjured cardiomyocytes at a significant rate during this period of time. By contrast, after myocardial infarction or pressure overload, the percentage of GFP+ cardiomyocytes decreased from 82.8% in heart tissue from sham-treated mice to 67.5% in areas bordering a myocardial infarction, 76.6% in areas away from a myocardial infarction, and 75.7% in hearts subjected to pressure overload, indicating that stem cells or precursor cells had refreshed the cardiomyocytes.

Figure 1

Generation of MerCreMer-ZEG mice for genetic fate mapping of adult mammalian cardiomyocytes. (a) A dual reporter strain ZEG mouse was crossbred with the MerCreMer strain to create MerCreMer-ZEG double heterozygous mice. Upon the pulse of 4-OH-tamoxifen, the reporter switches from β-galactosidase (encoded by lacZ) to GFP in cardiomyocytes only. A reduction in the percentage of GFP⁺ cardiomyocytes during aging or following myocardial infarction (MI) or pressure overload might indicate that new cardiomyocytes have been generated by stem or progenitor cells. (b) Dose-dependent effects of the 4-OH-tamoxifen pulse on GFP labeling in MerCreMer-ZEG mouse myocardium. Representative immunohistochemistry images of β-galactosidase (left) or GFP (center) after different durations of daily 4-OH-tamoxifen, counterstained with hematoxylin. A computer-based color separation technique (right) was used to facilitate the counting of GFP-positive cardiomyocytes; the result is shown in c. (d) High-efficiency cardiomyocyte GFP labeling. We used immunofluorescence costaining to visualize nuclei (DAPI; blue), cardiomyocytes (cardiac troponin T or cardiac MHC; red), and GFP (green). The yellowish cells indicate cardiomyocytes labeled with GFP. (e) Labeling was specific for cardiomyocytes. Immunostaining was used to examine GFP expression in other cell types, including vascular smooth muscle cells (α-smooth muscle actin; red), endothelial cells (isolectin-B4, red), and fibroblasts (vimentin; red). Scale bars, 20 μm. Data shown as mean ± s.e.m.

Figure 2

Adult mammalian cardiomyocytes are not detectably replaced by stem or progenitor cells during normal aging. (a) Immunohistochemistry for GFP during aging 0, 3, 6 and 12 months after the 4-OH-tamoxifen pulse. There were no significant differences in the percentage of GFP⁺ cardiomyocytes. (b) Immunohistochemistry for β-galactosidase (β-gal) during aging 0, 3, 6 and 12 months after the 4-OH-tamoxifen pulse. (c) Leakage of GFP in the absence of 4-OH-tamoxifen pulse was at a very low level, even over 14 months. Shown are representative GFP immunohistochemistry (upper; brown with hematoxylin counterstain) and immunofluorescence (lower; yellow) images showing rare GFP labeling in the absence of 4-OH-tamoxifen (red, troponin T; blue, DAPI). The graph shows leakage of GFP labeling at different mouse ages. Scale bars, 20 μm. Data shown as mean ± s.e.m.

Figure 3

Stem or progenitor cells replenish adult mammalian cardiomyocytes after myocardial injury. (a) The percentage of GFP⁺ cardiomyocytes decreased after myocardial injury. MerCreMer-ZEG mice with 4-OH-tamoxifen pulse labeling received experimental myocardial infarction or left ventricular overload. After 3 months, mouse hearts were fixed and stained with antibodies to GFP (green) or to β-galactosidase (red) (double staining in yellow). Shown are representative images of GFP and β-galactosidase staining (brown) in hearts after sham operation, myocardial infarction (border and remote areas), and pressure overload. (b) Percentage of GFP⁺ cardiomyocytes. (c) Percentage of β-galactosidase⁺ cardiomyocytes. (d) Percentage of GFP⁺ β-galactosidase⁺ cardiomyocytes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars, 20 μm. Data shown as mean ± s.e.m.