Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation

Federica Pisano, Claudia Altomare, Elisabetta Cervio, Lucio Barile, Marcella Rocchetti, Maria Chiara Ciuffreda, Giuseppe Malpasso, Francesco Copes, Manuela Mura, Patrizia Danieli, Gianluca Viarengo, Antonio Zaza, Massimiliano Gnecchi

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)1187-1199
Number of pages13
JournalStem Cells
Volume33
Issue number4
DOIs
Publication statusPublished - Apr 1 2015

Fingerprint

MicroRNAs
Cell Differentiation
Stem Cells
Cell Count
Excitation Contraction Coupling
Ryanodine
Connexin 43
Troponin T
Troponin I
Nifedipine
Dimethyl Sulfoxide
Caffeine
Mesenchymal Stromal Cells
Cardiac Myocytes
Real-Time Polymerase Chain Reaction
Protein Isoforms
Western Blotting
Immunohistochemistry
Maintenance
Pharmacology

Keywords

  • Cardiac differentiation
  • Electrophysiology
  • MicroRNA
  • P19 cells
  • Stem cells

ASJC Scopus subject areas

  • Cell Biology
  • Developmental Biology
  • Molecular Medicine

Cite this

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation. / Pisano, Federica; Altomare, Claudia; Cervio, Elisabetta; Barile, Lucio; Rocchetti, Marcella; Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Copes, Francesco; Mura, Manuela; Danieli, Patrizia; Viarengo, Gianluca; Zaza, Antonio; Gnecchi, Massimiliano.

In: Stem Cells, Vol. 33, No. 4, 01.04.2015, p. 1187-1199.

Research output: Contribution to journalArticle

Pisano, Federica ; Altomare, Claudia ; Cervio, Elisabetta ; Barile, Lucio ; Rocchetti, Marcella ; Ciuffreda, Maria Chiara ; Malpasso, Giuseppe ; Copes, Francesco ; Mura, Manuela ; Danieli, Patrizia ; Viarengo, Gianluca ; Zaza, Antonio ; Gnecchi, Massimiliano. / Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation. In: Stem Cells. 2015 ; Vol. 33, No. 4. pp. 1187-1199.
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AU - Pisano, Federica

AU - Altomare, Claudia

AU - Cervio, Elisabetta

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AU - Rocchetti, Marcella

AU - Ciuffreda, Maria Chiara

AU - Malpasso, Giuseppe

AU - Copes, Francesco

AU - Mura, Manuela

AU - Danieli, Patrizia

AU - Viarengo, Gianluca

AU - Zaza, Antonio

AU - Gnecchi, Massimiliano

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