In the present study, we sought to identify regulated microRNAs in mouse CPCs during development and determined their relationship to phenotypic variations occurring in the postnatal heart. A cohort of 45 microRNAs was differentially regulated between adult myocytes and CPC from both neonatal and adult mouse hearts. Of these, only eight microRNAs were uniquely regulated between neonatal and adult CPCs () using a stringent criteria (≥5-fold change, P<0.05) and validated by Q-PCR. We utilize three bioinformatic strategies, by weighting the number of downstream protein targets regulating different cellular processes, to predict cellular phenotypes that may be regulated in CPCs during development. This prediction was independent of the direction or magnitude of changes in microRNAs once the microRNAs had met the threshold criteria. Using the above strategy, we identify a cohort of eight microRNAs that is regulated during the post-natal development of CPCs that may predict the observed differences in their cellular proliferation. The difference in proliferative potential predicted by the microRNA analysis of mouse neonatal and adult CPCs while in vivo is recapitulated in human embryonic and adult CPC while in vivo, and in mouse neonatal and adult CPCs in culture.
To test the functional relationship between the regulated microRNA cohort and the predicted changes in CPC proliferation, we tested the gain-of-function of the miR-17-92 cluster. These results suggest a role of this cluster in regulating the exiting from cell cycle of CPCs in the adult hearts. Furthermore, the expression pattern of Rbl2, a cell cycle regulatory protein, predicted to be regulated by the differentially expressed microRNA cohort () was analyzed in neonatal and adult CPCs. Together, these results show an experimental strategy that identifies cohorts of differentially expressed miRNAs that predict cellular phenotypes of CPCs and functional regulators of these phenotypes.
This work provides an additional approach to determine the putative regulatory functions of microRNA cohorts in CPCs biology. In this study, we focused our analysis in developmental stages. It is anticipated that similar analysis in the future would lead to the discovery of microRNA cohorts in the injured heart that may regulate CPC proliferation, and their manipulation may translate into a greater cardiac regeneration and/or repair.
A hallmark of microRNAs is their tremendous potential to target a large number of genes, often in common pathways. With an estimated 60% of human mRNA having conserved microRNA targets, microRNAs represent one of the key regulators and informative markers of a set of genes controlling different cellular processes rather than single genes [36
]. To comprehensively evaluate the influence of microRNAs on multiple proteins by the integration of multiple downstream genes, the global evaluation of microRNA in the signaling cascade becomes essential [38
]. Similarly, we have utilized the overall assessment of the microRNA profile in establishing the possible roles of the differentially expressed microRNAs in the proliferative phenotype of neonatal and adult CPCs.
The miR-17-92 cluster either individually or synergistically has been shown to promote cellular proliferation in multiple tissues [39
]. However, the functions of the miR-17-92 cluster in the heart are not completely understood. The reduced heart weight and ventricular septal defects in mice deficient in miR-17-92 cluster supports an important role of miR-17-92 cluster in the prenatal developing myocardium [42
]. Individual microRNAs from this cluster target a complex network of proliferative cell cycle proteins and modulate proteins that control the transition from G1 to S phase thereby promoting proliferation [35
]. Our results suggest a role for this microRNA cluster in cell proliferation of CPCs since its re-expression contributes to a partial re-entry into the cell cycle. Future experiments into the re-expression of this microRNA cluster and others after injury may further elucidate the function of microRNAs in regulating regeneration in the adult myocardium.
A large network of interacting proteins including Rbl2 (an anti-proliferative cell cycle protein) and the associated E2F transcription factors orchestrates the regulation of cell proliferation. Rbl2 belongs to the retinoblastoma pocket protein family and has been previously validated as a target of miR-17 and miR-20a using a variety of different reporter gene assays [35
]. Our analysis shows that all the members of the miR-17 cluster are expressed at least 2-fold higher in neonatal CPCs. The transcript levels of Rbl2 remain constant in both neonatal and adult CPCs while the adult CPCs show a two-fold increase in Rbl2 protein when compared to the neonatal CPCs. Taken together, our data suggest that in CPCs, the miR-17 cluster may regulate cell proliferation, at least in part, through its action on one of the known target proteins, Rbl2. Nonetheless, the effect of over-expression of miR-17 cluster on Rbl2 protein level in CPCs was not directly demonstrated in our study due to the limited number of CPCs inherent in our in vivo
gain-of-function study design.
Our study demonstrated differential microRNA profiles in murine CPCs that predicted the differences in proliferative capacities in these cells. However, the analyses of human CPCs were limited to the proliferative phenotype of CPCs due to limited amount of tissues. Nonetheless, previous studies have demonstrated that many core cellular processes and microRNA orthologs are conserved between mouse and human [46
]. Moreover, the functions of mature microRNAs are phylogenetically conserved between species for example, the muscle-specificity of miR-1 in Drosophila
, mouse and humans [26
]. Future experiments are required to further evaluate microRNAs regulation in human CPCs. Moreover, previous studies have documented significant differences in the differentiation capacity between neonatal and adult CPCs [18
]. The possible roles of microRNAs in these differences are not yet known and will require future studies.
Recent identification of CPCs in adult hearts provides an exciting and promising new paradigm for cardiac regenerative therapy. However, with the loss of about one billion cardiomyocytes in a typical myocardial infarction in patients [48
], it is vital to ensure a sufficient number of CPCs for transplantation. It has previously been demonstrated that despite the increase in the CPCs in the failing human hearts of transplant recipients, the repair processes remained limited because patients ultimately required transplantation [49
]. The decline in the number and proliferative capacity of CPCs during development from neonate to adult stages [16
] may limit the use of CPCs for an efficient cell-based therapy. Therefore, insights into the proliferative capacity may provide new strategies to maintain the cardiac organ homeostasis and repair of the adult heart.