Fig 2: PTPN11N308S/+ iPSCs do not display hallmarks of sarcomeric hypertrophic cardiomyopathy(A and B) Representative images of control and PTPN11N308S/+ iPSC-derived cardiomyocytes (iPSC-CMs) seeded at 15,000, 25,000, or 50,000 cells/cm2 15 days before immunofluorescence staining of cardiac troponin T (cTnT) and plakophilin-2 (PKP2) at day 30 of differentiation (A) and corresponding measurements of mean cell surface area of cTnT+ cells indicated as mean ± SEM (B), p-values from a two-way ANOVA with Tukey's multiple comparisons test. Control #1: N = 2 differentiations, n = 715 cells; #2: N = 2 differentiations, n = 1,039 cells. PTPN11N308S/+ #1: N = 2 differentiations, n = 602 cells; #2: N = 2 differentiations, n = 1,303 cells. Scale bar in (A) = 100 µm.(C and D) Representative images of control and PTPN11N308S/+ iPSC-CMs stained for sarcomeric a-actinin (ACTN2) at day 30 (C) and corresponding percentage of cardiomyocytes displaying fully organized, partially organized, or disorganized myofibrils indicated as mean ± SD (D). Control #1: N = 4 differentiations, n = 879 cells; #2: N = 3 differentiations, n = 929 cells. PTPN11N308S/+ #1: N = 2 differentiations, n = 543 cells; #2: N = 3 differentiations, n = 481 cells. Scale bar in (C) = 50 µm; in insets = 10 µm.(E) qRT-PCR analysis of the ratio of expression of ß-myosin heavy chain (MYH7) to a-myosin heavy chain (MYH6) and natriuretic peptide A (NPPA) to natriuretic peptide B (NPPB) and level of expression of cardiac troponin T (TNNT2) and GATA-binding protein 4 (GATA4) in day 30 control and PTPN11N308S/+ iPSC-CMs, indicated as mean ± SEM, both groups #1: N = 2 differentiations, #2: N = 2 differentiations.(F and G) Representative single-cell calcium traces based on Fluo-4 fluorescence imaging on day 30 control and PTPN11N308S/+ iPSC-CMs under 0.5, 1, and 1.5 Hz pacing conditions (F). Calcium transient duration to 50% decay (TD50, left), duration to 90% decay (TD90, middle) and transient amplitude (right) measured by Fluo-4 fluorescence imaging in day 30 control and PTPN11N308S/+ iPSC-CMs under 0.5, 1 or 1.5 Hz field stimulation; dot plots showing all points, the mean and 95% CI (G). Control #1: N = 2 differentiations, n = 135 Ca2+ transients/pacing frequency in 34 cells; #2: N = 1 differentiation, n = 94 Ca2+ transients/pacing frequency in 23 cells (pooled). PTPN11N308S/+ #1: N = 2 differentiations, n = 117 Ca2+ transients/pacing frequency in 30 cells; #2: N = 1 differentiation, n = 99 transients/pacing frequency in 25 cells (pooled). p-values from a two-way ANOVA with Sidak's multiple comparisons test. See also Figures S11 and S12.

Fig 3: Hypoxia does not induce damage in iPSC-CMs. (A) Schematic representation of experimental setup. (B, C) ACTN1 and cTnT expression of iPSC-CMs assessed by immunofluorescence microscopy (B) and flow cytometry (C) (n = 8). (D) Ki-67 expression of iPSC-CMs assessed by flow cytometry (n = 9). (E) Ethidium homodimer-1 (EthD1)/calcein AM staining of iPSC-CMs cultured in four different media (DMEM-Glu/L, RPMI-Glu/B27, RPMI-B27, RPMI-Lac/L) with indicated nutrient compositions, exposed to indicated O2 concentrations and durations. Arrowheads indicate a representative selection of EthD1-positive cells. (F) Quantification (n = 8) and media compositions of E. (G) Flow cytometry analysis of the viability of iPSC-CMs cultured for 24 hours in 21% O2 or 1% O2 and quantification. (H) (n = 30). Data are collected from 2 to 3 technical replicates (exact amounts specified in separate panels) and 3 biological replicates (cardiac differentiations) of 2 cell lines. H: 3 technical replicates from 10 biological replicates (cardiac differentiations). Data were analyzed using one-way ANOVA and Dunnett’s multiple comparison test. *P < .05. Scale bar = 200 µm. Data are shown as mean ± SEM.

Fig 4: Mild hypoxia is sufficient to induce cell death in MM iPSC-CMs. (A) Schematic representation of experimental setup. (B) Bright-field images of MM iPSC-CMs exposed to indicated hypoxia conditions. (C) Microscopic images of ACTN1 and cTnT immunostainings. Square indicates the area of close-up. (D) Microscopic images of TUNEL staining of MM iPSC-CMs. (E) Quantification of TUNEL staining. (F) Glucose concentration in media after 4- to 24-hour incubation of MM iPSC-CMs at indicated hypoxia conditions. E: 3 technical replicates from 6 biological replicates (cardiac differentiations) (n = 28); F: 2 technical replicates from 3 biological replicates (cardiac differentiations) (n = 5). Data were analyzed using one-way ANOVA and Dunnett’s multiple comparison test. *P < .05; ***P < .001. Scale bar = 200 µm. Data are shown as mean ± SEM.

Fig 5: Hypoxia induces cell death of MM iPSC-CMs. (A) Schematic representation of experimental setup. (B, C) ACTN1 and cTnT expression of MM iPSC-CMs by immunofluorescence microscopy (B) and flow cytometric analysis of ACTN1 expression (C) (n = 8, 2 cell lines, 2 biological replicates, 2 technical replicates). (D) Ki-67 expression of MM iPSC-CMs compared to non-MM iPSC-CMs (n = 20, 2 cell lines, 5 biological replicates, 2 technical replicates). (E, F) EthD1/calcein AM staining (LIVE/DEAD assay) of MM iPSC-CMs exposed to 21% O2 for 24 hours or 1% O2 for 4, 8, 16, or 24 hours and quantification (F) (n = 15, 2 cell lines, 4 biological replicates, 1-2 technical replicates). (G) Lactate dehydrogenase media levels of day 40 matured iPSC-CMs exposed to 1% O2 (n = 5, 2 cell lines, 2 biological replicates, 1-2 technical replicates). (H, I) Flow cytometry analysis of MM iPSC-CMs viability after 24-hour exposure to 21% O2 or 1% O2 and quantification (I) (n = 7, 2 cell lines, 3 biological replicates, 1-2 technical replicates). Data were analyzed using one-way ANOVA and Dunnett’s multiple comparison test. **P < .01; ***P < .001. Scale bar = 200 µm. Data are shown as mean ± SEM.