title: Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.
abstract: Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.
authors.affiliation: Department of Cardiothoracic and Vascular Sciences-Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Laboratory of Experimental Cardiology for Cell and Molecular Therapy, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Pisano
authors.forename: Claudia
authors.initials: C
authors.lastname: Altomare
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Cervio
authors.forename: Lucio
authors.initials: L
authors.lastname: Barile
authors.forename: Marcella
authors.initials: M
authors.lastname: Rocchetti
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Ciuffreda
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Malpasso
authors.forename: Francesco
authors.initials: F
authors.lastname: Copes
authors.forename: Manuela
authors.initials: M
authors.lastname: Mura
authors.forename: Patrizia
authors.initials: P
authors.lastname: Danieli
authors.forename: Gianluca
authors.initials: G
authors.lastname: Viarengo
authors.forename: Antonio
authors.initials: A
authors.lastname: Zaza
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Gnecchi
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000008:administration & dosage / Q000096:biosynthesis*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D038081:Organogenesis / Q000187:drug effects / Q000502:physiology
chemical_list: C573190:MIRN499 microRNA, mouse; D035683:MicroRNAs; C517732:Mirn133 microRNA, mouse
keywords: Cardiac differentiation; Electrophysiology; MicroRNA; P19 cells; Stem cells