Background: it has been described that miR499, 1 and 133 are involved in cardiac development and stem cell differentiation. In particular, miR499 enhances the differentiation of embryonic and adult stem cells into cardiomyocytes (CMC). However, it is currently unknown if different miR may act synergistically to improve differentiation efficiency. Methods: we used mouse P19 cells as cardiogenic differentiation model. P19 cells were transduced with a viral vector expressing EGFP under the control of a cTnI promoter. miR499 was transiently overexpressed in P19 cells individually or together with miR1 or miR133. Cardiac differentiation was quantified by: 1) count of spontaneous beating areas; 2) qPCR of the cardiac markers Nkx2.5, GATA4, MEF2c, MLC2v, Cx43 and cTnT; 3) Western Blot and ICC of cTnT and Cx43; 4) measure of inducibility of RyR-mediated Ca++ transients in P19 exposed to caffeine; 5) recording of mechanical activity after Ca++ depletion and in the presence of nifedipine or ryanodine. Results: the overexpression of miR499 alone increased the number of beating CMC by 1.7 fold compared with P19 CTRL (p<0.05). The association of miR499+133, but not miR499+1, further increased the number of beating CMC (2.02 fold vs. P19 CTRL, p<0.01). qPCR showed that the combination of miR499+133 increase the expression of GATA4, Nkx2.5, Cx43 and cTnT compared with P19 CTRL (p<0.01) and MEF2c and MLC2v compared with miR499 alone (p<0.05). Western Blot and ICC for Cx43 and cTnT confirmed these findings. Caffeine responsiveness was increased by 2.6 fold by miR499+133 compared with P19 CTRL (32.5 vs. 12.5%, p<0.05) and was directly correlated with the activation of the cTnI promoter. Cyclic contractions were reversibly abolished by extracellular Ca++ depletion and by both nifedipine and ryanodine. Finally, the overexpression of miR499+133 is associated with an earlier activation of cTnI promoter. Conclusions: our results show that concomitant overexpression of miR499 and miR133 significantly enhance cardiac differentiation. Most importantly the functional studies have demonstrated that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling.
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