Distinct Roles of MicroRNA-1 and -499 in Ventricular Specification and Functional Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes

Abstract

BackgroundMicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive.Methods and ResultsWe hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased Ito, IKs and IKr, and decreased If (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca2+ transient amplitude and kinetics. Molecular pathway analyses were performed for further insights.ConclusionWe conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.

A New Level of Complexity

“…97 miR-499 also supports myogenic differentiation in vitro in cardiomyocyte progenitor cells isolated from human embryos, which might at least in part be mediated by Sox6. 98 A similar function was disclosed in differentiating human ES cells, in which lentiviral overexpression of miR-499 promotes differentiation of ES cells to the ventricular cardiomyocyte lineage, including upregulation of Myh7/Myh6 and troponin T. 99 In ischemic heart, miR-499 regulates mitochondrial dynamics after myocardial infarction. Thus, overexpression of miR-499 might inhibit cardiomyocyte apoptosis via targeting calcineurin and dynamin-related protein-1.…”

MicroRNAs as novel regulators of stem cell fate

“…Interestingly, the transplantation of murine ESCs overexpressing miR-1 into the border zone of infarcted mouse hearts prevented ischemia-induced apoptosis [80] . In addition, miR-1 facilitates the electrophysiological maturation of ESCs [81] . Furthermore, when miR-1 was transfected into fibroblast cells, gene expression profiles shifted toward that of muscle-like cells [82] .…”

MicroRNAs and Stem Cells

“…In addition, miR-1 was suggested to control electrophysiological maturation of ESCs. 75 Direct targets of miR-1 that might be involved in the regulation of cardiac differentiation include the transcriptional repressor of muscle gene expression HDAC4, 68 the Notch ligand Delta-like 1, 40 and Hand2, a transcription factor regulating the myocyte expansion. 71 The miR-1 and miR-133 are polycistronically clustered on the same chromosome and therefore are transcribed together.…”

“…67 In addition, the expression of myosin heavy chain genes and the cardiac transcription factors were elevated in mouse and human ESC cultures indicative for an augmentation of cardiac differentiation and the maintenance of the differentiated state in vitro. 66,75 Vice versa, inhibition of miR-499 blocked cardiac differentiation, 67 demonstrating that miR-499 controls cardiac commitment in vitro. In vivo, overexpression of miR-499 in human cardiac stem cells resulted in an enhanced myocyte differentiation when implanted into the border zone of infarcted rat hearts.…”