Mammalian Heart Renewal by Preexisting Cardiomyocytes

Although recent studies have revealed that heart cells are generated in adult mammals, the frequency and source of new heart cells is unclear. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes¹. Other studies suggest that new cardiomyocytes are born at a very low rate^2,3,4, and that they may be derived from division of pre-existing cardiomyocytes. Thus, the origin of cardiomyocytes in adult mammals remains unknown. Here we combined two different pulse-chase approaches -- genetic fate-mapping with stable isotope labeling and Multi-isotope Imaging Mass Spectrometry (MIMS). We show that genesis of cardiomyocytes occurs at a low rate by division of pre-existing cardiomyocytes during normal aging, a process that increases by four-fold adjacent to areas of myocardial injury. Cell cycle activity during normal aging and after injury led to polyploidy and multinucleation, but also to new diploid, mononucleated cardiomyocytes. These data reveal pre-existing cardiomyocytes as the dominant source of cardiomyocyte replacement in normal mammalian myocardial homeostasis as well as after myocardial injury.

Despite intensive research, fundamental aspects of the mammalian heart’s capacity for self-renewal are actively debated^5,6. Estimates of cardiomyocyte turnover range from less than 1% per year^2,3,4 to more than 40% per year⁷. Turnover has been reported to either decrease³ or increase with age⁷, while the source of new cardiomyocytes has been attributed to both division of existing myocytes⁸ and to progenitors residing within the heart⁹ or in exogenous niches such as bone marrow¹⁰. Controversy persists regarding the plasticity of the adult heart in part due to methodological challenges associated with studying slowly replenished tissues. Toxicity attributed to radiolabeled thymidine¹¹ and halogenated nucleotide analogues¹² limits the duration of labeling and may produce direct biological effects. Tissue autofluorescence can reduce the sensitivity and specificity of immunofluorescent methods of detecting cell cycle activity^5,13, including as cell cycle markers or incorporation of halogenated nucleotide analogues. The challenge of measuring cardiomyocyte turnover is further compounded by the faster rate of turnover of cardiac stromal cells relative to cardiomyocytes¹⁴.

We used Multi-isotope Imaging Mass Spectrometry (MIMS) to study cardiomyocyte turnover and to determine whether new cardiomyocytes are derived from preexisting myocytes or from a progenitor pool (Fig 1a). MIMS uses ion microscopy and mass spectrometry to generate high resolution quantitative mass images and localize stable isotope reporters in domains smaller than one micron cubed^15,16,17. MIMS generates ¹⁴N quantitative mass images by measuring the atomic composition of the sample surface with a lateral resolution of under 50nm and a depth resolution of a few atomic layers. Cardiomyocyte cell borders and intracellular organelles were easily resolved (Fig 1b). Regions of interest can be analyzed at higher resolution, demonstrating cardiomyocyte-specific subcellular ultrastructure, including sarcomeres (Fig 1c, Supplemental Fig 1a). In all subsequent analyses, cardiomyocyte nuclei were identified by their location within sarcomere-containing cells, distinguishing them from adjacent stromal cells.

An immense advantage of MIMS is the detection of nonradioactive stable isotope tracers. As an integral part of animate and inanimate matter, they do not alter biochemical reactions and are not harmful to the organism¹⁸. MIMS localizes stable isotope tracers by simultaneously quantifying multiple masses from each analyzed domain; this enables the generation of a quantitative ratio image of two stable isotopes of the same element¹⁵. The incorporation of a tracer tagged with the rare stable isotope of nitrogen (¹⁵N) is detectable with high precision by an increase in ¹⁵N:¹⁴N above the natural ratio (0.37%). Nuclear incorporation of ¹⁵N-thymidine is evident in cells having divided during a one-week labeling period, as observed in the small intestinal epithelium, which turns over completely in 3–5 days¹⁶ (Fig 1d); in contrast, ¹⁵N-thymidine labeled cells are rarely observed in the heart (Fig 1e) after 1 week of labeling. In subsequent studies, small intestine was used as a positive control to confirm label delivery.

To evaluate for an age-related change in cell cycle activity, we administered ¹⁵N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) (Supplemental Fig 2). We then performed MIMS analysis (Fig 2a, b, Supplemental Fig 3). In the neonatal group, 56% (±3% s.e.m., n=3 mice) of cardiomyocytes demonstrated ¹⁵N nuclear labeling, consistent with the well-accepted occurrence of cardiomyocyte DNA synthesis during post-natal development¹⁹. We observed a marked decline in the frequency of ¹⁵N-labeled cardiomyocyte nuclei (¹⁵N⁺CM) in the young adult (neonate= 1.00%¹⁵N⁺CM/day ±0.05 s.e.m. vs young adult=0.015% ¹⁵N⁺CM/day ±0.003 s.e.m., n=3 mice/group, p<0.001) (Fig 2a, c; Supplemental Fig 3). We found a further reduction in cardiomyocyte DNA synthesis in old mice (young adult=0.015%¹⁵N⁺CM/day ±0.003 s.e.m. vs. old adult=0.007 %¹⁵N⁺CM/day ±0.002 s.e.m., n=3/group, p<0.05) (Fig 2c). The observed pattern of nuclear ¹⁵N-labeling in cardiomyocytes is consistent with the known chromatin distribution pattern in cardiomyocytes²⁰ (Supplemental Fig 1b) and was measured at levels that could not be explained by DNA repair (Supplemental Fig 4). Extrapolating DNA synthesis measured in cardiomyocytes over 8 weeks yields a yearly rate of 5.5% in the young adult and 2.6% in the old mice. Given that cardiomyocytes are known to undergo DNA replication without completing the cell cycle^19,21,22, these calculations represent the upper limit of cardiomyocyte generation under normal homeostatic conditions, indicating a low rate of cardiogenesis.

To test whether cell cycle activity occurred in preexisting cardiomyocytes or was dependent on a progenitor pool, we performed ¹⁵N-thymidine labeling of double-transgenic MerCreMer/ZEG mice, previously developed for genetic lineage mapping (Fig 3a)^23,24. MerCreMer/ZEG cardiomyocytes irreversibly express green fluorescent protein (GFP) after treatment with 4OH-tamoxifen, allowing pulse labeling of existing cardiomyocytes with a reproducible efficiency of approximately 80%. Although some have reported rare GFP expression by non-cardiomyocytes with this approach²⁵, we did not detect GFP expression in interstitial cells isolated from MerCreMer/ZEG hearts nor did we detect GFP expression by Sca1 or ckit-expressing progenitors in histological sections (Supplemental Fig 5). Thus, during a chase period, cardiomyocytes generated from progenitors should be GFP⁻, whereas cardiomyocytes arising from preexisting cardiomyocytes should express GFP at a frequency similar to the background rate induced by 4OH-tamoxifen. We administered 4OH-tamoxifen for two weeks to 8 wk-old mice (n=4); during a subsequent 10-week chase, mice received ¹⁵N-thymidine via osmotic minipump (20μg/h). With MIMS analysis, we identified 35 ¹⁵N-labeled cardiomyocyte nuclei (of 4190 analyzed) over 10 weeks, yielding a projected yearly rate of cardiomyocyte DNA replication of 4.4%. Extrapolating from prior reports of high stem cell-dependent cardiomyocyte turnover⁷, we had anticipated detecting >320 cardiomyocytes entering the cell cycle; these results exclude such a high rate of turnover (expected ¹⁵N⁺ cardiomyocytes=321; observed=35; Fisher’s exact<0.0001). Immunofluorescent staining for GFP was performed on adjacent sections and an observer unaware of MIMS analysis results assessed GFP status. Of ¹⁵N⁺ cardiomyocytes, 77% expressed GFP, a frequency essentially identical to that of surrounding ¹⁵N⁻ cardiomyocytes (¹⁵N⁺ CM, 77% vs ¹⁵N⁻ cardiomyocytes, 84%; Fisher’s exact=n.s.) (Table 1). If new cardiomyocytes were derived from progenitors, ¹⁵N⁺ cardiomyocytes would have been GFP⁻ (expected=0/35; observed=27/35, Fisher’s exact<0.0001). These data reveal that ¹⁵N-labeled cardiomyocytes resulted from DNA synthesis by preexisting cardiomyocytes and exclude a substantial contribution from stem cells to cardiomyocyte replacement in the uninjured heart.

Cardiomyocytes can undergo DNA replication without completing the cell cycle. Although multinucleation and polyploidization occur during early post-natal development and in response to myocardial stress^2,19, we considered the possibility these processes could account for ¹⁵N⁺ cardiomyocytes in the uninjured adult mouse. We performed fluorescent in situ hybridization in adjacent sections to assess the ploidy state of each ¹⁵N⁺ cardiomyocyte and surrounding ¹⁵N⁻ cardiomyocytes, and an observer unaware of the results of MIMS analysis identified fluorescently-labeled chromosomes (Supplemental Fig 6). Although we found ¹⁵N⁺ cardiomyocytes that were polyploid (4n or greater), we observed a higher frequency of diploid nuclei in the ¹⁵N⁺ pool compared to surrounding ¹⁵N⁻ cardiomyocytes (¹⁵N⁺ diploid:polyploid = 22:12 vs ¹⁵N⁻ diploid:polyploid = 9:56, Fisher’s exact p<0.0001), consistent with ongoing cell division. We then assessed each cell for multinucleation using serial 0.5μm sections adjacent to the section used for MIMS analysis (Supplemental Fig 6). We observed that 49% of ¹⁵N⁺ cardiomyocytes were mononucleated, in contrast to a frequency of 24% for surrounding ¹⁵N⁻ cardiomyocytes (Fisher’s exact p<0.01), also consistent with cell division. The majority of cardiomyocyte DNA synthesis occurred in polyploid and/or multinucleated cardiomyocytes as might be expected with a physiologic hypertrophic response and thus unlikely to indicate cardiomyocyte division; however, 17% (6 of 35 ¹⁵N⁺ CM) were diploid and mononucleated, consistent with newly generated cardiomyocytes (Supplemental Fig 7). The mononucleated, diploid, ¹⁵N⁺ cardiomyocytes were also predominantly GFP⁺ (5 of 6 = 83% vs. 82% background frequency, p=n.s.), suggesting that they arose from preexisting cardiomyocytes at a slow annual rate of 0.76% (n=6 of 4190 over 10 weeks).

We next used MIMS and genetic fate mapping to study myocardial injury. Cardiomyocyte GFP labeling was induced in MerCreMer/ZEG mice with 4OH-tamoxifen. Mice then underwent experimental myocardial infarction or sham surgery followed by continuous labeling with ¹⁵N-thymidine for 8wks. The frequency of ¹⁵N-labeled cardiomyocytes in sham-operated mice was similar to prior experiments in unoperated mice (yearly projected rates: sham=6.8%; unoperated=4.4%), but increased significantly adjacent to infarcted myocardium (total ¹⁵N⁺ cardiomyocyte nuclei: MI=23.0% vs sham=1.1%, Fig 4a–b, Supplemental Fig 8). We examined GFP expression, nucleation and ploidy status of ¹⁵N-labeled cardiomyocytes and surrounding unlabeled cardiomyocytes. We found a significant dilution of the GFP⁺ cardiomyocyte pool at the border region as previously shown^23,24 (67% vs. 79%, p<0.05, Table 2, Supplemental Fig 9); however, ¹⁵N⁺ myocytes demonstrated a similar frequency of GFP expression compared to unlabeled myocytes (71% vs. 67%, Fisher’s exact=n.s.), suggesting that DNA synthesis was primarily occurring in pre-existing cardiomyocytes. Of ¹⁵N-labeled cardiomyocytes, approximately 14% were mononucleated and diploid consistent with division of pre-existing cardiomyocytes (Supplemental Fig 6, 7). We observed higher DNA content (>2N) in the remaining cardiomyocytes as expected with compensatory hypertrophy after injury. Thus, in the 8wks after myocardial infarction, approximately 3.2% of the cardiomyocytes adjacent to the infarct had unambiguously undergone division (total ¹⁵N⁺ × mononucleated diploid fraction = 23% × 0.14 = 3.2%). The low rate of cardiomyocyte cell cycle completion is further supported by the absence of detectable Aurora B Kinase, a transiently expressed cytokinesis marker, which was detected in rapidly proliferating small intestinal cells but not in cardiomyocytes (Supplemental Fig 10). We also considered the possibility that a subset of ¹⁵N⁺ myocytes that were multinucleated and/or polyploid resulted from division followed by additional rounds of DNA synthesis without division. However, quantitative analysis of the ¹⁵N⁺ population did not identify a subpopulation that had accumulated additional ¹⁵N-label as would be expected in such a scenario (Supplemental Fig 11). Together, these data suggest that adult cardiomyocytes retain some capacity to reenter the cell cycle, but that the majority of DNA synthesis after injury occurs in preexisting cardiomyocytes without completion of cell division.

If dilution of the GFP⁺ cardiomyocyte pool cannot be attributed to division and differentiation of endogenous progenitors, do these data exclude a role for progenitors in the adult mammalian heart? These data could be explained by preferential loss of GFP⁺ cardiomyocytes after injury, a process that we have previously considered but for which we have not found supporting evidence²³. Such an explanation excludes a role for endogenous progenitors in cardiac repair and would be consistent with data emerging from lower vertebrates^8,26 and the neonatal mouse²⁷ in which preexisting cardiomyocytes are the cellular source for cardiomyocyte repletion. A second possibility to explain the dilution of the GFP⁺ cardiomyocyte pool is that injury stimulates progenitor differentiation without division; inevitably, this would lead to exhaustion of the progenitor pool, which if true could explain the limited regenerative potential of the adult mammalian heart.

In summary, this study demonstrates birth of cardiomyocytes from preexisting cardiomyocytes at a projected rate of approximately 0.76%/year (¹⁵N⁺ annual rate × mononucleated diploid fraction = 4.4% × 0.17) in the young adult mouse under normal homeostatic conditions, a rate that declines with age but increases by approximately four-fold after myocardial injury in the border region. This study shows that cardiac progenitors do not play a significant role in myocardial homeostasis in mammals and suggests that their role after injury is also limited.