Possible mechanisms of age‐dependent decline in cellular function in c‐kit⁺ cardiac progenitor cells

Cardiovascular disease (CVD) represents the most rapidly rising morbidity and mortality worldwide, with costs exceeding 30 billion dollars annually in North America. Loss of myocardial tissue after cardiac injury can result in progressive heart dysfunction despite medical or surgical interventions. Over the past 30 years, scientists and physicians have been searching for effective treatments to prevent the deterioration of heart function after injury. Stem cell-based therapies have emerged as a promising intervention for cardiac repair and regeneration after cardiac injury. Initial preclinical in vivo animal studies and clinical trials of stem cell therapies successfully limited cardiac dysfunction including reduced ventricular dilatation and improved atrial-ventricular coupling and ejection fraction. However, restoring damaged hearts using stem cells can be problematic in aged individuals. Ageing is not only a major predisposing risk factor for CVD, but it can also influence the effectiveness of stem cell-based therapies by affecting the number and quality of stem cells in elderly individuals. While scientists are seeking ways to enhance their responsiveness to stem cell-based treatments, much remains to be understood in the role of ageing on stem cell-based cardiac rejuvenation and regeneration. In a recent study by Castaldi et al., published in The Journal of Physiology, the authors studied c-kit+ cardiac progenitor cells (CPCs) isolated from young (3 month) and old (24 month) C57BL/6 mice to investigate the role of ageing in the functional outcomes of CPCs and its potential impact on autologous stem cell-based therapies (Castaldi et al. 2017). Detailed functional studies including β-galactosidase staining senescence assay, glycolysis, ATP and cell proliferation assay were performed to measure changes in the “stemness” of c-kit+ CPCs associated with ageing. The authors aimed to investigate whether ageing could pose potential problems of stem cell dysfunction and impact future design of autologous stem cell-based therapies. The authors report that aged c-kit+ CPCs are more senescent than young CPCs and aged c-kit+ CPCs showed impaired proliferative capacity with 40% decrease in proliferation compared to young CPCs. Moreover, aged c-kit+ CPCs showed reduced expression of cardiac lineage markers including MEF2C, GATA4, GATA6 and PECAM. Lastly, the authors show aged, but not young, c-kit+ CPCs failed to upregulate mitochondrial phosphorylation and paracrine factors. Overall, Castaldi et al. believe that the ability of c-kit+ CPCs to proliferate and to effectively respond to dexamethasone treatment is significantly impaired in aged mice. The negative impact of ageing on stem cells is known in other stem cell populations such as haematopoietic stem cells where there is decreased proliferative capacity. Although this study provides a convincing initial characterization of age-dependent differences in cardiac progenitor cells, the mechanisms behind these changes are equally important. Elucidating the underlying mechanisms not only provides a deeper understanding, but yields information for potential therapeutic targets. As the authors have investigated changes in aerobic capacity, proliferation and stemness, possible mechanisms and implications are discussed below. Previous studies have shown that c-kit+ cells reside in hypoxic areas of the heart which encourages stem cell quiescence (Sanada et al. 2014). As a result, it follows that in an aged mouse, this prolonged state of quiescence in a hypoxic region has dramatically downregulated and quieted the aerobic metabolism mechanisms and mitochondrial biogenesis of the cell, and upregulated alternative energy sources, including glycolysis. Indeed, this is what Castaldi et al. have found in their work. The authors stimulated their cells with dexamethasone for 1 week to induce activation and differentiation of the CPCs; it would be interesting to investigate if a longer incubation with dexamethasone would bring these oxidative phosphorylation pathways back to similar levels to those in younger cells. It is possible that the 1 week of stimulation was not sufficient to activate these pathways since they have been inactive for so long. Aerobic exercise and physiological cardiac hypertrophy have been shown to improve c-kit+ cardiac progenitor cell numbers in various animal models (Leite et al. 2015). In the future, studies should be conducted to investigate whether aerobic exercise can offset the effects seen with ageing. It is possible that one of the mechanisms behind this beneficial result of exercise is due to an improvement of oxygen perfusion to these hypoxic regions of the heart, leading to maintenance of the aerobic metabolism capabilities and mitochondrial biogenesis. In turn, ageing c-kit+ CPCs would better maintain their “youthful” proliferative abilities, lineage markers, and thus utility. Therefore, future studies examining potential benefits of aerobic exercise on c-kit+ CPCs would be of great use to regenerative medicine efforts. It will be important to discern the mechanism behind the lower proliferative abilities and stemness in aged c-kit+ cells. In elucidating this mechanism, autologous transplantation of these stem cells can be performed upon genetic manipulation of the affected pathways. For instance, it has been shown that bradykinin receptors could play a role in mediating the proliferative ability of c-kit+ cells. If bradykinin is altered with age, this can be a possible mechanism behind this deterioration. Although the authors reference a paper which analysed the proteome of cardiac progenitor cell secretome, an in-depth analysis of the total proteome of c-kit+ cells will demonstrate age-dependent differences, with and without exercise. As epigenetic changes can contribute to haematopoietic stem cell ageing, the epigenetic profile of c-kit+ CPCs should also be investigated. Positive hits can be followed up with immunoblot or immunofluorescence. Moreover, super-resolution microscopy can also help with this analysis as it can display quantitative changes in the patterns of expression and localization of these newly found proteins. The therapeutic effects of c-kit+ CPCs for cardiac regeneration have been demonstrated in the past. Although clinical practice of stem cell-based therapies favour the use of young, healthy cells, its therapeutic effects remain modest. In the present paper, the authors successfully measured and reported for the first time the fundamental molecular and signalling differences between young and aged isolated c-kit+ CPCs. A recent study demonstrated, however, that c-kit expression is not sufficient to identify CPCs and in fact, cells sorted based on c-kit expression contain a mixture of CPCs and endothelial cells from the myocardium. Vicinanza et al. believe that ∼90% of c-kit+ cardiac cells are committed to blood or endothelial lineages, leaving behind ∼10% of c-kit+ cardiac cells being multipotent CPCs (Vicinanza et al. 2017). This theory may explain the poor efficacy of stem cell therapies for cardiac repair, although 10% of CPCs appear able to produce significant therapeutic effects in patients suffering from cardiac dysfunction. The differences observed between young and aged CPCs in the current study could have been overestimated (or underestimated) given the heterogeneous nature of c-kit+ cardiac cells. Castaldi et al. provide an excellent introductory analysis of c-kit+ age-dependent differences; however, there are other populations of stem cells, such as Sca-1+, Islet-1+ and epicardium-derived cells, within the heart that have not been studied here. Although, c-kit+ cells have been shown to predominantly differentiate into endothelial cells, it is entirely possible that there are age-dependent differences in these cell types that can explain age-induced decline in cardiac repair. Thus, the authors have opened a novel area of research for these different types of cardiac progenitor cells. As pointed out in a recent Perspectives article in The Journal of Physiology (Garikipati & Kishore, 2017), although autologous stem cell-based therapies offer exciting possibilities for cardiac repair in patients suffering from cardiac injury, the potential implications revolve around whether autologous stem cell-based therapies can effectively induce myocardium regeneration in elderly individuals. Regenerative medicine via repairing damaged myocardial tissue using autologous stem cells brings hope for repair of many cardiac defects. However, aged CPCs may have limited capacity to provide therapeutic benefits and thus their efficacy remains modest compared to young CPCs. Though understanding the underlying mechanisms of ageing-induced CPC dysfunction is important, perhaps focusing on maximizing the effectiveness of allogenic stem cell-based therapies could solve some limitations with autologous stem cell-based therapies in aged individuals despite its immunogenic activity. In summary, Castaldi et al. have demonstrated that c-kit+ cardiac progenitor cells have significant age-dependent functional differences. They have shown that aged c-kit+ cells have lower aerobic metabolism potential, decreased mitochondrial biogenesis, and reduction in proliferative capabilities, stemness and upregulation of cardiac lineage markers. These findings shed light on the reduced efficacy of clinical stem cell therapies in aged patients. Further study on the foundation laid by the authors could thus yield promising therapeutic effects to improve cardiac repair in heart failure. None declared. S.H.-L. and S.-H.L. are supported by an Ontario Graduate Scholarship. The authors wish to acknowledge the relevant articles that could not be cited due to space limitations and Mr Neal Ingraham Callaghan for constructive discussion.

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