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 5

Transcriptomics analysis reveals downregulation of ankyrin repeat domain 1 protein in hiPSC-CMs transfected with hsa-miR-548v.

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Transcriptomics analysis reveals downregulation of ankyrin repeat domain...
(A) Schematic overview of RNA-sequencing experiment. (B) t-SNE plot of RNA sequencing data based on the 1000 most variant genes from hiPSC-CM transfected with hsa-miR-548v (orange, n = 4) or negative miRNA negative control (gray, n = 4). (C) Volcano plot of downregulated (green) and upregulated (red) genes in hiPSC-CMs transfected with hsa-miR 548v or miRNA negative control. NPPA: Natriuretic peptide A; NPPB: Natriuretic peptide B; ANKRD1: cardiac ankyrin protein 1; ANKRD2: cardiac ankyrin protein 2; DES: Desmin; TNNI3: Troponin I3, Cardiac; MYH6: alpha cardiac myosin heavy chain; MYH7: beta cardiac myosin heavy chain. (D) Log2-Fold change in expression of sarcomere components, mechano-sensing proteins, intermediate filament components and microtubules. (E) Representative western blot showing ankyrin repeat domain 1 protein (CARP1) expression in control cells before transfection (H0), and 24, 48 and 72 hours after transfection with miRNA negative control or with hsa-miR-548v. (F) Relative level of ankyrin repeat domain 1 protein expression normalized to the basal expression before transfection (n = 4) and expressed as ratio between hsa-miR-548v treated cells/miRNA negative control–treated cells at 24 hours (n = 6), 48 hours (n = 6), and 72 hours (n = 7) after transfection. *P < 0.05, **P < 0.01, ordinary 1-way ANOVA. (G) Representative images of control CM-hiPSCs transfected with miRNA negative control or hsa-miR-548v 72 hours after transfection, stained for troponinT (red) and ankyrin D1 (green) as well as DAPI (blue). Scale bar: 50 μm. (H) Representative images of control CM-hiPSCs transfected with miRNA negative control or hsa-miR-548v 72 hours after -transfection, stained for ankyrin D1 (green) and DAPI (blue). Scale bar: 50 μm. (I) Quantification of raw integrated density of ankydrin D1 protein expression in the nucleus and in the cytoplasm in hiPSC-CMs 72 hours after transfection with hsa-miR-548v or miRNA negative control.