Skip to main content
. 2021 Dec 9;25(1):103596. doi: 10.1016/j.isci.2021.103596

Figure 3.

Figure 3

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 α-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 β-myosin heavy chain (MYH7) to α-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.