The ventricular action potential is formed by a harmonized interplay between a panel of ion channels. Abnormalities in the expression and function of these ion channels, as observed in long or short QT syndromes (LQTS and SQTS, respectively), are associated with an increased risk of life-threatening arrhythmias and sudden cardiac death. Here we sought to identify microRNAs (miRNAs) that post-transcriptionally regulate cardiac ion channels and asked whether miRNA manipulation would allow for modulation of action potential duration (APD) irregularities.

Among miRNAs that are abundantly expressed in the human myocardium, we identified miR-365 targeting the largest number of repolarizing ion channels. Carrying out double-fluorescent reporter assays, we confirmed a direct interaction between miR-365 and eight corresponding binding sites within the 3’ untranslated region of KCNQ1, KCNH2, KCNJ2, CACNA1C, KCNC3, KCNA1, and KCNJ3 mRNAs. Interestingly, an independent gene enrichment analysis on 2917 predicted targets of miR-365 primarily associated this miRNA to LQTS among all hereditary diseases in humans.

To resolve the regulatory function of miR-365, we employed patient-specific induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a human disease model. Upon manipulating miR-365, we performed action potential recordings using either optical-based voltage sensors, patch-clamp, or multielectrode array (MEA). In hiPSC-CMs derived from a patient suffering LQTS type 1, the pathologically prolonged APD was restored to normal levels upon inhibition of miR-365 using specific LNA-antimiRs (on average 36.5% significant reduction of APD in antimiR-365 treated cells compared to antimiR-ctrl). In contrast, the abnormally shortened APD in hiPSC-CMs of SQTS type 1 was ameliorated by overexpression of miR-365 using synthetic mimics (49.1% average increase in APD upon mimic-365 treatment).

We performed RNA-sequencing and single-cell RNA-sequencing in hiPSC-CMs upon treatment with mimic- and antimiR-365. Transcriptome analysis confirmed that gene sets contributing to cardiac myocyte membrane repolarization were significantly downregulated by miR-365 and revealed that among all ion channel targets of this miRNA, specific potassium channels, namely KCNQ1, KCNJ2, and KCNJ3, were preferentially and more strongly regulated. Using whole-cell patch-clamp in LQTS and SQTS hiPSC-CMs, we measured the currents corresponding to miR-365 targets after manipulation of this miRNA, confirming the most significant regulatory effect in IKs current density (encoded by KCNQ1).

Finally, to determine the relevance of miR-365 in a model that incorporates the complexity of the adult human heart, we employed human myocardial tissue slices. Slices with 300 µm thickness were prepared and cultivated under electromechanical stimulation to preserve their structure and function for up to 4 months. Repeated recordings of the refractory period (RP) upon manipulation of miR-365 revealed that elevation of miR-365 prolonged the RP (25 ± 7 ms increase upon transfection of mimic-365, P<0.05), whereas treatment with antimiR-365 reduced the RP (50 ± 31.18 ms shortening) compared to respective controls.

Taken together, our results delineate miR-365 to regulate cardiac APD through post-transcriptional repression of key determinants of cardiac repolarization. Therefore, manipulation of miR-365 may be employed to modulate APD abnormalities in various human arrhythmias.