Background and aim: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy with high worldwide prevalence. Hypertrophy of the left ventricle and interventricular septum, fibrosis and myocyte disarray often lead to development of heart failure and arrhythmias with high risk of sudden cardiac death at young age. Mutations in genes for sarcomere proteins account for the majority of inherited HCM cases often leading to contractile dysfunction. Besides alterations in the contractile function, electrophysiological abnormalities and alterations in calcium homeostasis are also detected.

MicroRNAs (miR) are small non-coding RNAs, which regulate mRNA expression levels and have been reported to be deregulated in HCM contributing to pathological phenotype development and possibly to alterations in the processes of excitation-contraction coupling (EC-coupling). Here we aimed to identify and modulate miRNAs involved into regulation of genes involved into EC-coupling processes to find novel potential non-coding RNA therapies for HCM.

Methods and results: Using publicly available miRNA and mRNA-sequencing datasets we performed data mining to identify miRNAs which were differentially expressed in HCM in comparison to healthy donor hearts. We identified miR-17-5p to be significantly downregulated in HCM. MiR-17-5p was predicted to target subunits of the Na⁺/K⁺-ATPase (ATP1A2) and the voltage-gated sodium channel (SCN2B), which were also significantly upregulated in the mRNA-Seq dataset. We confirmed downregulation of miR-17-5p in HCM tissue with the specific myosin mutation R723G, from which we next generated patient-specific human induced pluripotent stem cell derived cardiomyocytes (R723G- hiPSC-CMs). Using qPCR and Luciferase assays we validated that modulation of miR-17-5p expression leads to changes in target gene expression in R723G-hiPSC-CMs. Modulation of miR-17-5p regulated markers of cardiac remodeling such as ANP and BNP. We next generated AAV6-ACTN2-eGFP virus targeting z-discs of the sarcomere for fluorescent labeling of myofibrils in living R723G-hiPSC-CMs. Videos of electrically paced contracting cardiomyocytes were acquired at the confocal spinning disk microscope with framerate of 90fps and single sarcomere contraction and relaxation were tracked and analyzed using recently published Matlab code SarcTrack. First results from high-throughput contractility analysis using SarcTrack Matlab code revealed reduced fractional shortening of R723G- hiPSC-CMs after miR-17-5p overexpression as compared to miR-control treatment. We further investigate molecular effects and mechanisms of miR-17-5p action in electrophysiological recordings and analyses of intracellular calcium transients in R723G-hiPSC-CMs and to next explore the therapeutic potential of miR-17-5p in vivo in HCM mice.

Conclusion: In conclusion, miR-17-5p might be a promising candidate for therapeutic modulation of contractility in HCM with additional anti-hypertrophic capabilities.