A Repair “Kit” for the Infarcted Heart

Abstract

Transplanted, c-Kit expressing marrow-derived progenitors can enhance the function of an infarcted heart, but the mechanism remains unclear. In this issue of Cell Stem Cell, Loffredo et al. (2011) provide evidence that hematopoietic precursors do not differentiate into new cardiomyocytes, but rather, stimulate production of new cardiomyocytes from endogenous progenitors.

The stem cell biology field is no stranger to paradigm shifts, in particular when reevaluating presumed terminally differentiated tissue. As has been the case in several adult tissues, much interest over the past decade has been directed to the possibility that regenerative progenitor cells exist in the mature heart. The hearts of amphibians and teleost fish retain the ability to regenerate throughout life, and recent work in zebrafish has demonstrated this repair process occurs principally through division of pre-existing cardiomyocytes (Kikuchi et al., 2010). Interestingly, the mouse heart retains the ability to regenerate for a few days after birth, again through cardiomyocyte division, but this replication competence is lost within the first week of postnatal life (Porrello et al. 2011). While mature mammalian hearts clearly lack a robust regenerative response, mounting evidence points to some capacity for cardiomyocyte renewal. In 2007, Richard Lee’s group performed a pulse-chase experiment using inducible Cre recombinase for lineage tracing to elegantly demonstrate that, indeed, the young adult mouse heart does have the capacity to generate new cardiomyocytes post myocardial infarction (Hsieh et al., 2007). Using this system, they fluorescently labeled ~80% of the pre-existing cardiomyocytes and then demonstrated that, eight weeks post myocardial infarction, the percentage of fluorescently labeled mature cardiomyocytes had fallen to roughly 65%, with 15% new cardiomyocytes likely arising from a progenitor population. Since this study, Frisen’s group demonstrated that the adult human heart has the potential to regenerate cardiomyocytes at a low rate throughout adulthood (0.4–1%/year; Bergmann et al., 2009). Clearly, the therapeutic implications of a non-cardiomyocyte regenerative population make these cells a prime target for further characterization.

In the current issue of Cell Stem Cell, Loffredo et al. (2011) re-use this pulse-chase system to assess how bone marrow progenitors expressing c-Kit improve function of the infarcted mouse heart. This question represents a controversial area of research, with initial studies suggesting that hematopoietic c-Kit⁺ progenitors transdifferentiate into cardiomyocytes (Orlic et al., 2001), and subsequent studies demonstrating that these cells mature into inflammatory cells in the heart without becoming cardiomyocytes (Murry et al., 2004, Balsam et al., 2004). Loffredo et al. demonstrate that marrow-derived c-Kit⁺ cells can have a significant impact on heart regeneration, not as direct progenitors but as regulators of an endogenous cardiac stem or progenitor cell. Their genetic pulse-chase system uses the Z/EG reporter mouse line. This mouse is engineered to switch from constitutive expression of β-galactosidase (β-gal; under the β-actin promoter) to constitutive expression of EGFP whenever cells express Cre recombinase. They crossed the Z/EG line with a mouse expressing the tamoxifen-inducible form of Cre recombinase (MerCreMer) only in cardiomyocytes via the α-myosin heavy chain (Myh6) promoter. The strategy, therefore, is to label pre-existing cardiomyocytes with EGFP, and then assess dilution of this EGFP⁺ pool after injury, which would indicate repopulation from an EGFP-negative progenitor pool (Figure 1). Toward that end, six week old mice were given daily tamoxifen injections for two weeks, achieving EGFP labeling of approximately 80% of all mature cardiomyocytes, while in 20% recombination did not occur and the cells continued to express β-gal. The labeled hearts were then infarcted, and 6×10⁵ lineage⁻/c-kit⁺ bone marrow cells or marrow-derived mesenchymal stem cells (MSCs) from wild type animals were administered in a divided dose to the medial and lateral infarct borders. Animals were allowed to recover for eight weeks and were then assessed by immunohistochemistry for the frequency of EGFP- and β-gal-expressing cardiomyocytes in the infarct border zone as well as distal to the infarct site.

Figure 1. Genetic lineage tracing system for the detection of cardiomyocytes regeneration.

The constitutively active β-actin promoter drives expression of a loxP flanked β-galactosidase gene (β-gal; shown in blue). In the presence of cre-recombinase, the β-gal gene is floxed out, now allowing the β-actin promoter to drive EGFP expression (shown in green). In this double transgenic system, the Cre recombinase is under the transcriptional control of the Myh6 promoter, confining expression to mature cardiomyocytes. The cre-recombinase is also fused to the mutant estrogen receptor “Mer” which constrains the protein to the cytoplasm until tamoxifen is present. By administering a daily dose of tamoxifen to 6 week old mice for two weeks, Loffredo et al. were able to label 80% of mature cardiomyocytes permanently green. Following myocardial infarction, this percentage falls to 60% in the infarct border zone as new cardiomyocytes arise. If, at the time of myocardial infarction, bone marrow derived c-kit⁺ cells are administered to the heart, even more new cardiomyocytes are made and the percentage of green-cardiomyocytes in the infarction border zone falls to only 50% of cardiomyocytes. No increase in new cardiomyocytes is seen over sham control if mesenchymal stem cells (MSCs) or resident cardiac c-Kit+ cells are added.

Similar to their prior study, control infarcted mice had ~20% reduction in the number of EGFP-labeled cardiomyocytes, consistent with the generation of new cardiomyocytes from progenitor cells. Hearts that received MSCs were no different from controls, indicating MSCs neither influence endogenous progenitors nor become cardiomyocytes themselves. The mice that received the c-kit⁺ cells, however, had a 30% reduction in the number of EGFP⁺ cardiomyocytes, suggesting an augmentation of endogenous cardiac progenitor activity. This effect was found only in the region of c-kit injection and not in remote areas (which had ~7% reduction in EGFP⁺ cardiomyocytes). These new cardiomyocytes all expressed β-gal and therefore were not due to differentiation of the transplanted wild type c-kit⁺ cells into mature cardiomyocytes. Further evaluation demonstrated that administration of c-Kit+ cells also increased the number of cells expressing early cardiomyocyte transcription factors Gata4 or Nkx2.5 by approximately three fold, and that these cells were actively synthesizing DNA. This population may represent an expanding subset of nascent cardiomyocytes or their progenitors, although at present the origin of these cells remains unclear (Figure 1).

While the authors conclude that their results support the model that an endogenous cardiac stem cell exists and that its activity can be potentiated by c-Kit⁺ hematopoetic stem cells, Loffredo et al. took pains to assay for other possible explanations including transdifferentiation or cell fusion. They designed genetic systems to test for transdifferentiation, but surprisingly, they found that the transplanted cells did not even persist in the hearts 8 weeks later. Intrigued by this finding, they showed that Y chromosome⁺ graft cells could be detected in the peri-infarct zones up to seven days post transplant, but none remained at four weeks. This result effectively rules out transdifferentiation or fusion as underlying mechanisms responsible for the new cardiomyocytes seen at 8 weeks. One remaining possibility is that the 20% of cardiomyocytes in which the Cre system failed to induce recombination is somehow different from the other 80%, e.g. in this population’s ability to re-enter the cell cycle. The initial paper describing this system found no differences in DNA synthesis rates at selected times post-infarction (Hsieh et al., 2007), but this general caveat is worth keeping in mind.

The chemokine stromal-derived factor-1 (SDF-1, aka CXCL12) is up-regulated in the infarcted heart and mediates homing of marrow-derived c-Kit⁺ cells from the circulation. To assess whether recruitment of circulating c-Kit⁺ stem cells could mimic effects of exogenous c-Kit⁺ cells, a protease-resistant version of SDF-1 was directly administered to the infarcted heart. In previous studies, this factor was demonstrated to increase recruitment of CXCR4⁺/c-Kit⁺ progenitor cells to the site of administration and increase microvascular density (Segers et al., 2007). While SDF-1 administration did recapitulate increased vascularization, no enhancement in derivation of cardiomyocytes from progenitors was seen.

A good experiment often opens up more questions than it answers, and that is the case with this study from Loffredo et al. What is the cardiomyocyte progenitor cell? Their genetic system provides strong evidence for their function, but it cannot identify them. These candidate stem cells are clearly not the transplanted c-Kit⁺ cells from bone marrow, and the authors’ transplantation studies do not favor either resident cardiac c-Kit⁺ cells or marrow-derived MSCs. Perhaps the activity resides within perivascular Sca1⁺ cell, the Hoechst dye-effluxing side population cells, or something beyond the list of usual suspects. Another important question is how do the marrow-derived c-Kit⁺ cells exert their effects, and is this mechanism a normal component of the inflammatory response to infarction that has been uncovered through cell transplantation? If we can identify these paracrine signals, perhaps we can control their responses without resorting to cell therapy. Despite these open questions, the study by Loffredo et al. helps to further elucidate the role of bone marrow-derived c-kit⁺ cells to expand the endogenous cardiomyocyte population, and it demonstrates, excitingly, that this endogenous population can be manipulated and enriched. Thus, we have been moved a step closer to one of the most exciting new therapeutics in the field of cardiovascular medicine.