MicroRNA 17-5p modulation for tuning of the hypercontractile function in Hypertrophic Cardiomyopathy

N. Weber (Hannover)¹, K. S. Menge (Hannover)¹, J. Gruber (Hannover)¹, A. A. Derda (Bielefeld)², M. Fuchs (Hannover)³, A. Gietz (Hannover)¹, A. Pfanne (Hannover)¹, I. Riedel (Hannover)¹, A. Just (Hannover)¹, R. Zweigerdt (Hannover)⁴, T. Kraft (Hannover)⁵, Y. Psaras (Headley Way, Oxford)⁶, C. Toepfer (Headley Way, Oxford)⁶, A. Zeug (Hannover)⁷, K. Xiao (Hannover)¹, C. Bär (Hannover)¹, T. Thum (Hannover)^1
1Medizinische Hochschule Hannover Institut für Molekulare und Translationale Therapiestrategien, OE-8886 Hannover, Deutschland; ²Medizinische Hochschule Hannover Kardiologie und Angiologie Hannover, Deutschland; ³Fraunhofer ITEM Cardiovascular Research Group Hannover, Deutschland; ⁴Medizinische Hochschule Hannover Klinik für Herz-, Thorax-, Transplantations- und Gefäßchirurgie, OE 6217 Hannover, Deutschland; ⁵Medizinische Hochschule Hannover Institut für Molekular- und Zellphysiologie Hannover, Deutschland; ⁶University of Oxford University of Oxford Radcliffe Department of Medicine Headley Way, Oxford, Großbritannien; ⁷Institut für Neurophysiologie Hannover, Deutschland

Background and aim:
Hypertrophic cardiomyopathy (HCM) is the most frequent inherited cardiac disease with hypertrophy of the left ventricle and interventricular septum, fibrosis and myocyte disarray. Patients often develop heart failure and arrhythmias with high risk of sudden cardiac death at young age. Mutations in genes for sarcomere proteins cause the majority of inherited HCM-cases leading to changes in contractile function. MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA expressions. Here we aim to modulate miRNAs expressions to influence the gene expressions and ultimately the contractile function in cardiomyocytes and living myocardial slices as proof-of-concept study and for further translation to novel therapies in 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. Using this approach we identified miRNA 17-5p as being deregulated in HCM tissues and involved into the regulation of the functional gene targets in electromechanical coupling of the cardiomyocytes. MiR-17-5p was predicted to target subunits of the Na⁺/K⁺-ATPase (ATP1A2) and the voltage-gated sodium channel (SCN2B). We could confirm the deregulation of this miRNA in HCM tisue with R723 myosin mutation. In the model of patient-specific stem cell-derived cardiomyocytes (hiPSC-CMs) with HCM-myosin mutation R723G regulation of miRNA-17-5p with specific miRNA mimics and LNA-inhibitor showed an inverse regulation of SCN2B as predicted in data mining approach. Additionally, overexpression of miR-17-5p  reduced expression levels of the pro-hypertrophic marker genes ANP and BNP.
We then used AAV6-ACTN2-eGFP virus targeting z-discs of the sarcomere for fluorescent labeling of myofibrils in living HCM-hiPSC-CMs. Videos of electrically paced contracting cardiomyocytes were acquired at the confocal spinning disk microscope with framerate of 90 fps and single sarcomere contraction and relaxation were tracked and analyzed using published Matlab code SarcTrack. Functional analysis revealed reduced fractional shortening of HCM-hiPSC-CMs after miR-17-5p overexpression as compared to miRNA-control treatment. Knockdown of SCN2B with siRNA reduced fractional shortening of the HCM cardiomyocytes as well, but to a lower degree, indicating that miRNA-17-5p mechanism possibly involves additional regulations. We further modulated the expression of miRNA 17-5p in wild-type porcine living myocardial slices (pLMS) with transfection of the miRNA-17-5p into the pLMS inducing alterations of the contractile force of the pLMS.

Conclusion: In conclusion, we have proven that miRNA modulation can affect contactile funcion of cardiomyocytes and living myocardial slices in general. Specifically, miR-17-5p might be a promising candidate for modulation of contractility in HCM with additional anti-hypertrophic capabilities.