Fig. 2: MiR-365 prolongs action potential duration in human iPSC-derived cardiac myocytes.

a Protocol for differentiation of the patient-specific iPSCs (left, hiPSC) to cardiac myocytes (right, hiPSC-CMs). The scale bars represent 500 μm. b The structural organization of the myofilaments in hiPSC-CMs, assessed by immunofluorescent staining for actinin and myosin filaments (large scale bar = 25 μm, small scale bar = 2 μm). c The proportion of cell types at day 60 of cardiac differentiation. Myocytes were defined as cells staining positive for alpha-actinin (ACTN2). Ventricular-like CMs expressed the ventricular marker myosin light chain 2 (MYL2) in addition to alpha-actinin (Data from 3 independent differentiations). d Double fluorescent sensor for miR-365 activity in living cells. Representative images of hiPSC-CMs infected with AAV6 expressing miR-365 activity sensor with a native or mutated seed sequence. The activity of miR-365 was determined by high content microscopy upon transfection with mimics or antimiRs (scale bar = 100 μm). e Quantification of data from d. Data are from 3–5 independent experiments performed in duplicates presented as mean ± SEM. f Scheme showing optical AP recordings using di-8-ANEPPS upon manipulation of miR-365 levels in hiPSC-CMs, done using MetaFluor software. g Representative action potential traces (F655/F560) from healthy hiPSC-CMs transfected with mimic-365 or control mimic and were loaded 48 h later with di-8-ANEPPS (left). Average trace of 15 APs recorded from a representative cell in each condition (right). h Quantitative analysis of the ratiometric determination of transmembrane potential (median 379 ms in the control group compared to 509 ms in the mimic-365 group). All measurements were performed at 35 °C. Data are represented as Bazett’s corrected action potential duration at 90% of the repolarization (cAPD90). Data are acquired from 4 independent experiments and presented as Box (25% and 75% quartiles) and whisker (min to max) showing the median and individual values. Source data and detailed statistical analyses for c, e and h are provided as a Source Data file.