The ventricular action potential is orchestrated by a set of ion channels that enable the heart to respond to physiological as well as pathological stimuli. Expression and function of these ion channels are tightly regulated by different mechanisms, alterations of which lay the ground for malignant arrhythmias and sudden cardiac death. However, current treatment options are invasive and do not resolve the underlying ion channel disorders directly. Here we sought to identify miRNAs that control cardiac ion channels and asked whether miRNA manipulation would allow for modulation of action potential duration (APD) abnormalities.

Among miRNAs that are abundantly expressed in human myocardium, we identified miR-365 targeting the largest number of repolarizing ion channels in cardiac myocytes. Carrying out double-fluorescent reporter assays, we confirmed a direct interaction between miR-365 and its eight 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 long QT syndrome (LQTS) among all hereditary diseases in human.

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 manipulation of miR-365, we performed optical action potential recording using either a genetically encoded voltage sensor (VSFP-CR) or a voltage-sensitive dye, di-8-ANEPPS. In hiPSC-CMs derived from a patient suffering long QT syndrome type 1 (LQT1), the pathologically prolonged APD was restored to normal levels upon inhibition of miR-365 using a specific LNA-antimiR (median 673 ms in the control group compared to 418 ms in antimiR-365 treated cells, P < 0.01). On the other hand, shortened APD in hiPSC-CMs of a short QT patient (SQT1) was ameliorated by overexpression of miR-365 using a synthetic mimic (median 290 ms compared to 431 ms upon mimic-365 treatment, P < 0.001).

Transcriptome analyses in hiPSC-CMs at bulk and single-cell level corroborated the key cardiac repolarizing channels as direct targets of miR-365 and revealed that this miRNA exerts its regulatory role by significant repression of the potassium ion transport machinery and the repolarization phase of the cardiac action potential.

To determine the relevance of miR-365 in a model that incorporates the complexity and function 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 was served as a functional readout. RP measurements revealed that elevation of miR-365 prolonged the RP (31 ± 6 ms increase upon transfection of mimic-365, P<0.05), whereas treatment with antimiR-365 reduced the RP about 60 ms compared to respective controls.

Overall, our results delineate miR-365 to regulate cardiac action potential duration through post-transcriptional repression of key determinants of cardiac repolarization. Therefore, manipulation of miR-365 may be employed to modulate AP abnormalities in various human QT syndromes.