hsa-miR-548v controls the viscoelastic properties of human cardiomyocytes and improves their relaxation rates
Eva Vermersch, Salomé Neuvendel, Charlène Jouve, Andrea Ruiz-Velasco, Céline Pereira, Magali Seguret, Marie-Elodie Cattin-Messaoudi, Sofia Lotfi, Thierry Dorval, Pascal Berson, Jean-Sébastien Hulot
Eva Vermersch, Salomé Neuvendel, Charlène Jouve, Andrea Ruiz-Velasco, Céline Pereira, Magali Seguret, Marie-Elodie Cattin-Messaoudi, Sofia Lotfi, Thierry Dorval, Pascal Berson, Jean-Sébastien Hulot
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Research Article Cardiology Stem cells

hsa-miR-548v controls the viscoelastic properties of human cardiomyocytes and improves their relaxation rates

Abstract

The impairment of left ventricular (LV) diastolic function with an inadequate increase in myocardial relaxation velocity directly results in lower LV compliance, increased LV filling pressures, and heart failure symptoms. The development of agents facilitating the relaxation of human cardiomyocytes requires a better understanding of the underlying regulatory mechanisms. We performed a high-content microscopy-based screening in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) using a library of 2,565 human miRNA mimics and measured relaxation kinetics via high-computing analyses of motion movies. We identified hsa-miR-548v, a primate-specific miRNA, as the miRNA producing the largest increase in relaxation velocities. This positive lusitropic effect was reproduced in engineered cardiac tissues generated with healthy and BRAF T599R mutant hiPSC-CMs and was independent of changes in calcium transients. Consistent with improvements in viscoelastic responses to mechanical stretch, RNA-Seq showed that hsa-miR-548v downregulated multiple targets, especially components of the mechanosensing machinery. The exogenous administration of hsa-miR-548v in hiPSC-CMs notably resulted in a significant reduction of ANKRD1/CARP1 expression and localization at the sarcomeric I-band. This study suggests that the sarcomere I-band is a critical control center regulating the ability of cardiomyocytes to relax and is a target for improving relaxation and diastolic dysfunction.

Authors

Eva Vermersch, Salomé Neuvendel, Charlène Jouve, Andrea Ruiz-Velasco, Céline Pereira, Magali Seguret, Marie-Elodie Cattin-Messaoudi, Sofia Lotfi, Thierry Dorval, Pascal Berson, Jean-Sébastien Hulot

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Figure 6

Effect on hsa-miR-548V on BRAF T599R mutated hiPSC-CMs.

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Effect on hsa-miR-548V on BRAF T599R mutated hiPSC-CMs. (A) Sequence ali...
(A) Sequence alignment showing the BRAF 1796C>G nucleotide substitution leading to the nonsense T599R mutation (red square). Of note, the 1794T>G silent variant was introduced for the CRISPR/Cas9 processing. (B) Comparison of hiPSC-CMs between isogenic control (n = 124, 5 independent experiments) and BRAF T599R (n = 127, 6 independent experiments) cell lines. ***P = 0.0004, unpaired t test. (C) Representative Western blot for ERK phosphorylation (pERK) and ankyrin repeat domain 1 protein (CARP1) expression on hiPSC-CMs from BRAF T599R and its isogenic control. Vinculin was used as a loading control. (D) Quantification of total ERK1/2 normalized to vinculin (left) and quantification of pERK relative to ERK1/2 expression level (middle) in BRAF T599R (n = 8) cells and the isogenic control (n = 7). **P = 0.0012, Mann-Whitney U test. Quantification of ankyrin D1 level expression (right) in BRAF T599R (n = 8) and the isogenic control (n = 6) *P < 0.05, Mann-Whitney U test. (E) Western blot for ankyrin repeat domain 1 protein on BRAF T599R cells (n = 3) 72 hours after transfection with miRNA negative control or hsa-miR-548v. Quantification of ANKRD1 relative level expression in BRAF T599R cells 72 hours after transfection, normalized to the basal expression before transfection, and expressed as ratio between hsa-miR-548v treated cells/miRNA negative control–treated cells. P = 0.10, Mann-Whitney U test. (F) Schematic overview of hECT generation and representative bright-field image of tissues obtained using the BRAF mutant hiPSC-CMs. (G–J) Beat rate (G), amplitude (H), maximum contraction speed (I), and maximum relaxation speed (J) of BRAF T599R engineered cardiac tissues at 0, 24, 48, and 72 hours after transfection with miRNA negative control (n = 7) or hsa-miR-548v (n = 10), from 1 differentiation. **P < 0.01, ****P < 0.0001 from Šídák post hoc comparisons between treatment groups, 2-way ANOVA.