Variable cMyBP-C expression from cell to cell in a MYBPC3c.927–2 A>G hiPSC-CM model recapitulates HCM patient phenotype

Fig. 1. Western Blot analysis of cMyBP-C protein levels in MYBPC3c.927–2 A> G hiPSC-CMs and isogenic control. cMyBP-C levels were normalized to α-actinin. The mean value of all cMyBP-C/α-actinin ratios of each time point was set = 1 to enable across gel comparisons. Data are presented as mean ± SD, analyzed from N = 3 independent differentiations. Differentiations were performed using one isogenic control clone and one heterozygous MYBPC3 mutant clone. Statistical comparisons were analyzed using the unpaired t-test

Fig. 2. Analysis of active transcription sites (aTS) of MYBPC3 in MYBPC3c.927–2 A> G and isogenic control hiPSC-CMs. A Distribution of the numbers of aTS per nucleus in cardiomyocytes for MYBPC3. The percentage of nuclei with 0, 1, 2, > 2 aTS was plotted for single cardiomyocytes from N = 3 differentiations. Differentiations were performed using one isogenic control clone and one heterozygous MYBPC3 mutant clone. n = number of analyzed nuclei. B Average number of aTS per nucleus for MYBPC3 in isogenic control (black circles) and mutant (green triangles) at d35, d56 and d70 + post differentiation. C Representative images of 0 aTS, 1 aTS, and 2 aTS in isogenic control. White arrows indicate aTS within the nucleus. Scale bar 5 μm

Fig. 3. Assessment of variable cMyBP-C protein expression from CM to CM. A Immunofluorescent images of isogenic controls and MYBPC3c.927–2 A> G hiPSC-CMs after 70 + days of cultivation showing variable sarcomeric expression of cMyBP-C protein from CM to CM. Scale bar 100 μm. B Percentage of cMyBP-C-positive CMs in mutant and isogenic controls at d35, d56, and d70+, categorized into three distinct groups: cMyBP-C strong pattern (green), and weak pattern (yellow) stands for weak or inhomogeneous cMyBP-C signal in individual CMs, respectively. cMyBP-C negative CMs indicates lack of cMyBP-C expression (red). Only CMs with clear cell boarders were taken into analysis. n = number of analyzed single cells in N = 2 differentiations. Differentiations were performed using one isogenic control clone and one heterozygous MYBPC3 mutant clone

Fig. 4. Morphological analysis of MYBPC3c.927–2 A> G and isogenic control hiPSC-CMs, cultivated on nanopatterned substrates. Cellular area (A), aspect ratio (B), and myofibrillar alignment score (C) calculated for mutant and isogenic controls hiPSC-CMs using ImageJ. n = number of analyzed single cells in N = 2 differentiations. Differentiations were performed using one isogenic control clone and one heterozygous MYBPC3 mutant clone. Data are presented as mean ± SD. Statistical comparisons were analyzed using Mann-Whitney test

Fig. 5. Scatter plots of twitch kinetics parameters of MYBPC3c.927–2 A> G and isogenic control hiPSC-CMs cultivation days. Time to peak (ttp): the time at which the peak is reached starting from the electrical stimulus or the twitch. Half relaxation time (hrt): time for the transient to return to 50% of the peak during the relaxation phase. Shortening velocity (departure velocity, vdep): characterizes the maximum speed with which the cell contracts. Twitch amplitude (peak h): difference between the peak and baseline values. n = number of analyzed single cells in at least two differentiations (N ≥ 2). Differentiations were performed using one isogenic control clone and two heterozygous MYBPC3 mutant clones. Data are presented as mean ± SD. Statistical comparisons were analyzed by Mann-Whitney test

Fig. 6. Scatter plots of Ca2+-transient parameters of MYBPC3c.927–2 A> G and isogenic control hiPSC-CMs. Ca2+ time to peak (Ca2+-ttp): the time at which the Ca2+ peak is reached starting from the electrical stimulus for the twitch. Ca2+ half decay time (Ca2+-hdt): time for Ca2+ transient to reach to 50% of the peak during the Ca2+ reuptake phase. Ca2+ amplitude: difference between the peak and baseline values (340/380 nm ratio). n = number of analyzed single cells in three differentiations (N = 3). Differentiations were performed using one isogenic control clone and two heterozygous MYBPC3 mutant clones. Statistical comparisons were analyzed using Mann-Whitney test. D, E Representative twitch (D) and Ca2+ transient (E) recordings from d70 + of cultivation for isogenic control and mutant MYBPC3c.927–2 A> G hiPSC-CM

Fig. 7. Top 10 of dysregulated pathways and proteins associated with the MYBPC3c.927–2 A> G mutation. A, B The HCM-associated pathways in MYBPC3c.927–2 A> G hiPSC-CMs identified through enrichment analysis (KEGG database) at day 35 (A) and day 70+ (B). Each bar represents a specific pathway, with its length corresponding to the degree of statistical significance (log₁₀(p-value)). C, D Bar chart showing log₂ fold change (log₂FC) values of dysregulated genes in mutant hiPSC-CMs compared to isogenic controls. Dysregulated genes are grouped into two categories: sarcomeric genes (C) and calcium handling genes (D). Green bars represent genes at day 35; orange bars represent genes at day 70+. Bars with a line pattern indicate changes that were not statistically significant (p > 0.05)