Study of the BRAF-T599R hiPSC line, a new model for impaired cardiac relaxation associated with hypertrophic cardiomyopathy?

Abstract

Introduction

Mutations in the BRAF gene are associated with an upregulation of the MAPK pathway and often leads to hypertrophic cardiomyopathies (HCM). HCM is characterised by an increase in cardiomyocyte size, resulting in left ventricular hypertrophy and impaired myocardial relaxation in patients. Specifically, the BRAF T599R mutation is known to contribute to the development of RASopathies in patients; however, the effects of this mutation on cardiac relaxation have not been thoroughly investigated. Additionally, we have previously identified hsa-miR-548v as a positive regulator of relaxation velocity accompanied by a large decrease of ANKRD1 expression.

Objective

We aim to characterise human BRAF T599R cardiomyocytes. Additionally, we will assess changes in ANRKD1 expression and evaluate the contraction/relaxation cycles in the mutant cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs).

Method

The BRAF T599R hiPSC line was produced using CRISPR/Cas9 genome editing. We characterised the hiPSC-CMs using Western blot analysis on both total and fractionated cells, confocal immunofluorescence, and video microscopy on micro-engineered cardiac tissues.

Results

The mutant hiPSC-CMs an increased activation of the MAPK pathway, along with elevated expression levels of ANKRD1. The mutant hiPSC-CMs displayed increased cell size and a higher density of filamentous actin and titin networks. Following cell fractionation, we observed that ANKRD1 was overexpressed both in the nucleus (indicating transcription factor activity) and in the cytoplasm (as a sarcomeric protein) of the mutant cells. Additionally, the mutant engineered-cardiac tissues demonstrated impaired amplitude, as well as decreased contraction and relaxation velocities when compared to isogenic control tissues. After transfecting with hsa-miR-548v, the amplitude, contraction, and relaxation velocities of the mutant-engineered cardiac tissues significantly improved. Moreover, ANKRD1 expression was notably decreased in both the nucleus and the cytoplasm of the mutant hiPSC-CMs.

Conclusion

The BRAF T599R hiPSC-CMs exhibit hypertrophic cell characteristics and altered contraction and relaxation cycles compared to their isogenic control hiPSC-CMs. Additionally, these cells show higher levels of both nuclear and cytoplasmic ANKRD1. However, transfection with hsa-miR-548v reverses this increase in ANKRD1 expression. This reduction in ANKRD1 is associated with improved contraction and relaxation velocities in engineered cardiac tissue.