Fig. 8

Impairment autophagy and physiology response in PLEKHM2 mutated DCM-iPSCs-CMs. Cells were treated with 0.1% DMSO or 20 μM Cloroquine (CQ) and 500 nM Rapamycin (RP) for 20 h. RP + CQ treated samples were related to DMSO treatment for each cell line to compare the autophagic response independent of varying basal expression levels among cell lines. Western blot analysis with representative immunoblots (performed on all clones) (A), quantification of LC3B-II normalized to Ponceau S as a loading control (performed on all clones, n = 2–3 of biological replicates) (B), quantification of LC3B-II/ LC3B-I ratio (performed on all clones, n = 2–3 of biological replicates) (C). FACS analysis using CytoID kit (D). Immunostaining of DCM-iPSCs-CM-C and DCM-iPSCs-CM-R (E) Immunostaining of cardiac troponin T (cTnT) and LC3B at day 28 of differentiation after treatment with Rapamycin (500 nM) and Chloroquine (20 μM). Single DCM iPSC – derived cardiomyocytes exhibited a cTNT (green) double stained with LC3B (red) and DAPI (blue) distribution pattern. Scale bars, 50 µm. Spontaneous beat/min rate (F) and mechanical contraction (G, H) were measured following treatment with DMSO or RP and CQ. One clone from each patient was analyzed using MuscleMotion software (DCM-iPSCs-CM clones C1, DCM-iPSCs-CMs clone R7 and DCM-iPSCs-CMs O1) and the results (G, H) are represented as the percentage of the change accrued following RP + CQ treatment. Asterisks represent significance in compared to clone C. G Contraction amplitude represents an average of the maximum peak of contraction during spontaneous beatings. H Half width represents the time duration of 50% raise to 50% decline of the contraction-relaxation signal. Intracellular calcium transients. Asterisks denote significant difference by 1-way ANOVA, *p < 0.05, **p < 0.01 ***p < 0.001 and ****p < 0.0001