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Here, we sought to identify microRNAs (miRNAs) that regulate the human cardiac AP and asked whether their manipulation allows for therapeutic modulation of AP abnormalities. Quantitative analysis of the miRNA targetomes in human cardiac myocytes identified miR-365 as a primary miRNA to regulate repolarizing ion channels. AP recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) showed that elevation of miR-365 level significantly prolonged AP duration in hiPSC-CMs derived from a Short-QT syndrome (SQTS) patient, whereas specific inhibition of miR-365 normalized pathologically prolonged AP in Long-QT syndrome (LQTS) iPSC-CMs. Transcriptome analyses in human iPSC-CMs at bulk and single-cell level corroborated the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional AP-regulating genes by this miRNA. Whole-cell patch clamp experiments revealed miR-365-based regulation of repolarizing ionic currents. Finally, refractory period measurements in human myocardial slices substantiated the prolonging effect of miR-365 on AP duration also in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac AP duration by targeting key factors of cardiac repolarization. Overall design: hiPSC-CMs from a healthy individual were assigned to 5 treatment groups: untreated, mimic-Ctrl, mimic-365, antimiR-Ctrl and antimiR-365, and hiPSC-CMs from a short QT syndrome type 1 patient were treated with mimic-Ctrl or mimic-365 (n=3 replicates per each condition).ENALQT2, congenital myasthenia, 9430057C20Rik, HH, l(3)hh, Fibroblast-Derived IPS Cell, Myosin regulatory light chain interacting protein, Induced Pluripotent Stem Cell, IPS, 6.3.2.-, hg., bar, RNA-seq, Fibroblast Derived IPS Cells, hiPSC, ERG1, IDOL, IPS Cells, SQT1, Cell, CG4637, Hh, CG12249, AW228700, human iPS cell line cell, anon-WO0134654.19, CMS, DmelCG4637, Fibroblast Derived Induced Pluripotent Stem Cells, ERG-1, bar3, Whole Transcriptome Shotgun Sequencing, DmelCG12249, Mir, MIR, H-ERG, IPS Cell, congenital MG, Miranda, Fibroblast-Derived IPS Cells, familial limb-girdle myasthenia, l(3)neo56, l(3)neo57, MONDOA, Mrt, Mira, Fibroblast-Derived IPS, HERG, myasthenia gravis congenital, Inducible degrader of the LDL-receptor, mir, Fibroblast-Derived, Human Induced Pluripotent Stem Cells, Myosin regulatory light chain-interacting protein, HERG1, anon-WO0182946.19, bar-3, Cells, Fibroblast-Derived Induced Pluripotent Stem Cells, MIRA, bHLHe36, myasthenia gravis pseudoparalytica, Human Induced Pluripotent Stem Cell, erb-Goldflam syndrome, Idol, Kv11.1LQT2, congenital myasthenia, 9430057C20Rik, HH, l(3)hh, Fibroblast-Derived IPS Cell, Myosin regulatory light chain interacting protein, Induced Pluripotent Stem Cell, IPS, 6.3.2.-, hg., bar, RNA-seq, Fibroblast Derived IPS Cells, hiPSC, ERG1, IDOL, IPS Cells, SQT1, Cell, CG4637, Hh, CG12249, AW228700, human iPS cell line cell, anon-WO0134654.19, CMS, DmelCG4637, Fibroblast Derived Induced Pluripotent Stem Cells, ERG-1, bar3, Whole Transcriptome Shotgun Sequencing, DmelCG12249, Mir, MIR, H-ERG, IPS Cell, congenital MG, Miranda, Fibroblast-Derived IPS Cells, familial limb-girdle myasthenia, l(3)neo56, l(3)neo57, MONDOA, Mrt, Mira, Fibroblast-Derived IPS, HERG, myasthenia gravis congenital, Inducible degrader of the LDL-receptor, mir, Fibroblast-Derived, Human Induced Pluripotent Stem Cells, Myosin regulatory light chain-interacting protein, HERG1, anon-WO0182946.19, bar-3, Cells, Fibroblast-Derived Induced Pluripotent Stem Cells, MIRA, bHLHe36, myasthenia gravis pseudoparalytica, Human Induced Pluripotent Stem Cell, erb-Goldflam syndrome, Idol, Kv11.1falseRNA-seq in healthy and SQT1 hiPSC-CMs upon manipulation of miR-365RNA-seq in healthy and SQT1 hiPSC-CMs upon manipulation of miR-3652025-09-242021-12-04PRJNA770438GSE185687960635017523