EMBRYONIC STEM CELL-DERIVED SINOATRIAL-LIKE CELLS AS A MODEL TO STUDY THE EXERCISE-DEPENDENT EFFECTS OF MIR-1 AND MIR-423 UPREGULATION ON HEART RATE

The cardiovascular benefits of regular exercise are well established. High-intensity endurance exercise induces electrical, structural, and functional adaptations in the heart in order to sustain the increased cardiac output and metabolic needs. These cardiac changes are called ‘athletes heart’ and include typical morphological changes (eccentric hypertrophy) and a slowing of the heart rate with significant brady- and tachy-arrythmias. The heart rhythm depends on the intrinsic heart rate, generated by the pacemaker cells in the sinus node, and on the autonomic (parasympathetic and sympathetic) tone. Bradycardia in endurance athletes is commonly attributed to an increased vagal tone. Recently, D’Souza and colleagues have instead demonstrated that, in rodent, training-induced bradycardia is due to an intrinsic modification of the heart. In particular, they showed that bradycardia persists (in vivo) after blockade of the autonomous nervous system in mice and (in vitro) in the isolated heart. Furthermore, they demonstrated a reduction of the If pacemaker current in sinoatrial node (SAN) cells of trained mice. The molecular mechanism at the basis of this process is not completely understood but our collaborators in Manchester showed also a training-dependent up-regulation of miR-1, one of the main myomiR, and of miR-423. The aim of this work is to understand the role of miR-1 and miR-423 in sinoatrial cells. Given the difficulty to keep SAN cells in culture, we decided to study the role of these microRNAs in sinoatrial-like cells differentiated from mESC overexpressing them. To do this, we generated three different clones of mouse embryonic stem cells overexpressing miR-1, miR-423 and a control line. The overexpression of these miRNAs affected neither the pluripotency nor the capacity of mESC to differentiate into cardiomyocytes. We selected CD166+ sinoatrial-like cells from differentiating mESC by flow cytometry and found that the proportion of CD166+ SAN-like cells is higher in mESC-miR1 (25.20±2.32% n=26) and in mESC-miR423 (18.56±1.36% n=9) than in control lines (11.09±1.09% n=30). Electrophysiological analysis showed a lower firing rate in miR-1-CD166+ cells than in cells from the control line, while no differences are present between miR-423-CD166+ and empty-CD166+ (miR-1-CD166+ 1.37±0.08Hz n=10; miR-423-CD166+ 2.36±0.30Hz n=7; empty-CD166+ 2.22±0.22Hz n=7). We further demonstrated that the miR-1 overexpression leads to around a 50% reduction of the funny current. We found no differences in the expression level of miR-423 in miR-423-CD166+ and empty-CD166+, despite it resulted overexpressed in mESC-miR-423 respect to mESC- empty. We then studied the effect of miR-423 by transiently overexpressing it in neonatal rat ventricular cardiomyocytes, finding again no differences. These data demonstrated that miR-1 modulates the beating frequency of sinoatrial-like cells, reducing the If current, and thus demonstrating that miR-1 contribute to the establishment of the endurance athletes’ bradycardia. For what concern miR-423, from our data it seems to have no effect in the control of cardiac rate, however, since the overexpression of miR-423 was modest in mESC and we could not control the expression level in transfected neonatal rat ventricular cardiomyocytes further experiments are needed to definitely rule out its contribution to exercise-induced bradycardia.