(A) Schematic pedigree of the DCM family recruited in this study. Filled squares (male) and circles (female) represent individuals carrying the specific heterozygous TNNT2 R173W mutation. (B) The R173W point mutation was confirmed to be present on exon 12 of the TNNT2 gene in the DCM patients by PCR and DNA sequencing. CON, control. (C) A representative image of the skin fibroblasts expanded from the skin biopsies. Representative images of an (D) ESC-like and (E) TRA-1-60 positive colony derived from reprogramming the skin fibroblasts with Yamanaka factors. (F) Immunofluorescence and alkaline phosphatase staining of the skin fibroblasts-derived iPSCs. (G) Quantitative bisulphite pyrosequencing analysis of the methylation status at the promoter regions of Oct4 and Nanog in the iPSCs. Both Nanog and Oct4 promoter regions were highly demethylated in the iPSCs derived. (H) Teratomas derived from the iPSCs injected into the kidney capsule of immunodeficient mice showing tissues of all three embryonic germ layers. Bars, 200 μm.

(A) Immunostaining of sarcomeric α-actinin and cTnT at day 30 post differentiation. Single DCM iPSC-CMs exhibited punctate sarcomeric α-actinin distribution pattern suggesting a disorganized myofilament structure. Enlarged views of the boxed areas of the merged micrographs showing detailed α-actinin and cTnT staining pattern in the cells. Bars, 20 μm. (B) Compared to control iPSC-CMs (n=368), a significant higher percentage of DCM iPSC-CMs (n=391) showed punctate sarcomeric α-actinin staining pattern in greater than one fourth of the total cellular area (**p=0.008). (C) No significant difference was observed in cell size between control (n=36) and DCM iPSC-CMs (n=39). (D), TEM images of myofibrillar organization in control and DCM iPSC-CMs. Compared to control CMs (n=11, 2 iPSC lines, one each from 2 individuals), myofibrils in DCM CMs (n=12, 2 iPSC lines, one each from 2 patients) exhibited an increased variability in the degree of organization. ZL, Z-line; ZB, Z-bodies; A, A-band; I, I-band; Mt, mitochondria. Extra TEM images of control and DCM CMs can be found in . Bars, 1μm.

(A) A representative MEA assay recording chronotropic response of both control (n=14) and DCM (n=14) beating EBs overtime with 10 μM NE treatment. Electrical signals were recorded before and after NE treatment. Beating frequencies were normalized to the values before NE treatment. (B) Representative images of sarcomeric α-actinin immunostaining on single control and DCM iPSC-CMs after 7 days of NE treatment. Compared to the controls, long term NE treatment significantly aggravated sarcomeric organization of single DCM CMs. Bar, 20 μm. (C) Percentage of CMs with disorganized sarcomeric α-actinin staining pattern with (control, n=210; DCM, n=255) or without (control, n=261; DCM, n=277) NE treatment. NE treatment markedly increased the number of disorganized CMs in DCM group (**p<0.001), and had a less significant effect on control iPSC-CMs (*p=0.05). (D) TEM images of myofibrillar organization in control and DCM iPSC-CMs after NE treatment. Compared to control (n=6, 2 iPSC lines, one each from 2 individuals) CMs, myofibrils in DCM (n=7, 2 iPSC lines, one each from 2 patients) CMs exhibited an increased scattered distribution of Z-bodies. ZL, Z-line; ZB, Z-bodies. Extra TEM images of NE treated control and DCM CMs can be found in . Bars, 1μm. (E) Relative beating frequencies of CM clusters (n = 10) over time after NE treatment by video imaging. Values are normalized to the beating frequencies of CM clusters at the same time points without NE treatment. Data are presented as mean±s.e.m. (F) Tracking morphological and contractility changes of iPSC-CMs overtime after NE treatment by video imaging. Bar, 200 μm. The respective videos were in ().

(A) Three types of spontaneous APs were observed in both control and DCM iPSC-CMs (left, ventricular-like; center, atrial-like; right, nodal-like). An estimated 70–80% cells were ventricular-like CMs, whereas the others were atrial- and/or nodal-like cells. There is no significant difference in cardiac cell fate between control and DCM iPSCs (data not shown). (B) Spontaneous APs in control and DCM ventricular-like myocytes using current-clamp recording. There was no significant difference in the AP durations (C), frequency (D), the peak amplitude (E), or in the resting membrane potential (F) between control and DCM cells at the time of measurements (day 19- day 25 post differentiation) (control, n=18; DCM, n=17). Statistical difference was tested using the two tailed Student’s T-test.

(A) Representative line-scan images and (B) spontaneous Ca²⁺ transients in control (left) and DCM iPSC-CMs (right). (C) Frequency of spontaneous Ca²⁺ transients in control and DCM iPSC-CMs. (D) Integration of [Ca²⁺]_i transients in control and DCM iPSC-CMs showed less Ca²⁺ released in each transient in DCM relative to control cells (control, n=87 cells; DCM, n=40 cells, **P=0.002). There were no significant differences in the irregularity of timing (standard deviation/mean) (E) or irregularity of amplitude (F) of the spontaneous Ca²⁺ transients between control and DCM cells. (G–J) Affected caffeine-evoked Ca²⁺ release in DCM iPSC-CMs. (G) Representative Ca²⁺ transients induced with 20 mM caffeine in Ca²⁺-free condition in control and DCM iPSC-CMs (control, n=12, 2 iPSC lines, one each from 2 individuals; DCM, n=12, 2 iPSC lines, one each from 2 patients). (H) Peak amplitude of caffeine-evoked Ca²⁺ transients in control and DCM iPSC-CMs. The difference in the amplitude between control and DCM was observed but fail to reach statistical significance. (I) Time to peak in caffeine-evoked Ca²⁺ transients in control and DCM iPSC-CMs. (J) Decay time (50% from peak) in caffeine-evoked Ca²⁺ transients in control and DCM iPSC-CMs. Two-tailed Student’s t-test (* P < 0.05, ** P < 0.01).

(A) Dot plots of mean contraction force of single CMs measured by AFM. One-way ANOVA analysis indicated that there was significant difference among the mean of all the groups (**p=0.002). Tukey’s Multiple Comparison Test indicated that both control iPSC-CMs (n=13) (P=0.001) and Ad.Serca2a (n=12) (P=0.005) transduced DCM iPSC-CMs exhibited significantly stronger contraction force than that transduced by Ad.GFP (n=17). Ad.Serca2a transduced DCM iPSC-CMs showed comparable contraction force to that of the control iPSC-CMs (p=0.578). (B) Histograms of contraction forces of all the single iPSC-CMs measured by AFM over 100–400 beats. Over-expression of Serca2a significantly restored the contraction force of DCM iPSC-CMs to a level close to that of the controls. (C) Western blotting of Serca2a expression after adenoviral transduction of cells dissociated from iPSC beating EBs. Serca2a protein level was upregulated in cells transduced with Ad.Serca2a but not in cells transduced with Ad.GFP. (D) A representative image showing the AFM cantilever approaching GFP positive single beating CMs. Bar, 50 μm. (E) Representative spontaneous Ca²⁺ transients in single DCM iPSC-CMs transduced with Ad.GFP and Ad.Serca2a, respectively. (F) DCM iPSC-CMs transduced with Ad.Serca2a (n=22) exhibited increased global Ca²⁺ transients compared to cells transduced with Ad.GFP (n=14). (*p=0.04) (two-tailed Student’s t-test). (G) Half decay time of Ca²⁺ transients in DCM iPSC-CMs transduced with Ad.Serca2a or with Ad.GFP. (H) Percentage of CMs with disorganized sarcomeric α-actinin staining pattern in single DCM iPSC-CMs with Ad.Serca2a (n=40) or Ad.GFP (n=40) over-expression. No significant difference was observed between the two groups (two-tailed Student’s t-test). Data are presented as mean±s.e.m.