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Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

Birket MJ, Ribeiro MC, Kosmidis G, Ward D, Leitoguinho AR, van de Pol V, Dambrot C, Devalla HD, Davis RP, Mastroberardino PG, Atsma DE, Passier R, Mummery CL - Cell Rep (2015)

Bottom Line: Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease.This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency.Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.

No MeSH data available.


Related in: MedlinePlus

Single-Cell Traction Force Measurements in Cardiomyocytes with MYBPC3 Mutation or MYBPC3 shRNA Knockdown(A) Western blot of cMyBP-C protein from two control (Con1 and Con2) and three MYBPC3 mutation lines (HCM1, HCM2, and HCM3). α-actinin is shown as a loading control for cardiomyocyte input.(B) Relative cMyBP-C levels normalized to α-actinin levels based on densitometry of western blot data (n = 3–6 lysates).(C) Typical examples of single aligned (spontaneously contracting) cardiomyocytes from control (Con1) and MYBPC3 mutation (HCM3) lines, showing a bright-field image of the relaxed form and a heatmap of traction stress applied to the substrate calculated from the mean of the traction stress vectors (corresponding to Movie S2).(D and E) (D) Traction stress and (E) cell area of single spontaneously contracting iPSC-derived cardiomyocytes from two control (Con1 [n = 47] and Con2 [n = 36]) and three MYBPC3 mutation lines (HCM1 [n = 44], HCM2 [n = 54], and HCM3 [n = 43]).(F) Western blot of cMyBP-C protein in NKX2-5eGFP/w hESC-derived cardiomyocytes after transduction with a scrambled (Scr) shRNA or two independent MYBPC3-specific shRNAs. Actin is shown as a loading control.(G and H) (G) Traction stress and (H) cell area of single spontaneously contracting cardiomyocytes non-transduced (n = 15), stably expressing the Scr- (n = 32) or MYBPC3-specific (1, n = 26; 2, n = 16) shRNAs.Boxplots and whisker plots show the median, interquartile range, and 10–90 percentile range. Unless otherwise stated, the n signifies the number of individual cells measured, acquired over three independent experiments. Statistical significance was tested with a one-way ANOVA with Tukey’s multiple comparison test in (D) and (E), comparing either control against the HCM lines independently, and a Dunnett’s correction in (G) and (H). Comparison to both controls in (D) are statistically significant ∗p < 0.05. Scale bar, 10 μm. See also Figure S5.
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fig5: Single-Cell Traction Force Measurements in Cardiomyocytes with MYBPC3 Mutation or MYBPC3 shRNA Knockdown(A) Western blot of cMyBP-C protein from two control (Con1 and Con2) and three MYBPC3 mutation lines (HCM1, HCM2, and HCM3). α-actinin is shown as a loading control for cardiomyocyte input.(B) Relative cMyBP-C levels normalized to α-actinin levels based on densitometry of western blot data (n = 3–6 lysates).(C) Typical examples of single aligned (spontaneously contracting) cardiomyocytes from control (Con1) and MYBPC3 mutation (HCM3) lines, showing a bright-field image of the relaxed form and a heatmap of traction stress applied to the substrate calculated from the mean of the traction stress vectors (corresponding to Movie S2).(D and E) (D) Traction stress and (E) cell area of single spontaneously contracting iPSC-derived cardiomyocytes from two control (Con1 [n = 47] and Con2 [n = 36]) and three MYBPC3 mutation lines (HCM1 [n = 44], HCM2 [n = 54], and HCM3 [n = 43]).(F) Western blot of cMyBP-C protein in NKX2-5eGFP/w hESC-derived cardiomyocytes after transduction with a scrambled (Scr) shRNA or two independent MYBPC3-specific shRNAs. Actin is shown as a loading control.(G and H) (G) Traction stress and (H) cell area of single spontaneously contracting cardiomyocytes non-transduced (n = 15), stably expressing the Scr- (n = 32) or MYBPC3-specific (1, n = 26; 2, n = 16) shRNAs.Boxplots and whisker plots show the median, interquartile range, and 10–90 percentile range. Unless otherwise stated, the n signifies the number of individual cells measured, acquired over three independent experiments. Statistical significance was tested with a one-way ANOVA with Tukey’s multiple comparison test in (D) and (E), comparing either control against the HCM lines independently, and a Dunnett’s correction in (G) and (H). Comparison to both controls in (D) are statistically significant ∗p < 0.05. Scale bar, 10 μm. See also Figure S5.

Mentions: Attempts to measure these hiPSC-CMs in medium without TID proved unsuccessful as even the control cells failed to reliably generate robust bead displacement. However, in medium containing TID, traction force on the polyacrylamide substrate could be measured in all cell populations. Under these conditions, traction stress was significantly decreased in all three mutant lines, HCM1 0.31 ± 0.02, HCM2 0.30 ± 0.03, HCM3 0.29 ± 0.03 mN/mm2 in comparison to both controls, Con1 0.57 ± 0.04 and Con2 0.51 ± 0.03 mN/mm2; p < 0.05 (Figures 5C and 5D). This corresponded to traction forces of 0.43 ± 0.04, 0.46 ± 0.06, 0.44 ± 0.05, 0.81 ± 0.16, and 0.86 ± 0.10 μN for HCM1, HCM2, HCM3, Con1, and Con2, respectively. A difference in cardiomyocyte size as measured by cell area was not evident between the control and the mutant cells, suggesting an overt hypertrophic response had not occurred by this stage of development under these conditions (Figure 5E). Contraction frequencies were not different (Figure S4).


Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

Birket MJ, Ribeiro MC, Kosmidis G, Ward D, Leitoguinho AR, van de Pol V, Dambrot C, Devalla HD, Davis RP, Mastroberardino PG, Atsma DE, Passier R, Mummery CL - Cell Rep (2015)

Single-Cell Traction Force Measurements in Cardiomyocytes with MYBPC3 Mutation or MYBPC3 shRNA Knockdown(A) Western blot of cMyBP-C protein from two control (Con1 and Con2) and three MYBPC3 mutation lines (HCM1, HCM2, and HCM3). α-actinin is shown as a loading control for cardiomyocyte input.(B) Relative cMyBP-C levels normalized to α-actinin levels based on densitometry of western blot data (n = 3–6 lysates).(C) Typical examples of single aligned (spontaneously contracting) cardiomyocytes from control (Con1) and MYBPC3 mutation (HCM3) lines, showing a bright-field image of the relaxed form and a heatmap of traction stress applied to the substrate calculated from the mean of the traction stress vectors (corresponding to Movie S2).(D and E) (D) Traction stress and (E) cell area of single spontaneously contracting iPSC-derived cardiomyocytes from two control (Con1 [n = 47] and Con2 [n = 36]) and three MYBPC3 mutation lines (HCM1 [n = 44], HCM2 [n = 54], and HCM3 [n = 43]).(F) Western blot of cMyBP-C protein in NKX2-5eGFP/w hESC-derived cardiomyocytes after transduction with a scrambled (Scr) shRNA or two independent MYBPC3-specific shRNAs. Actin is shown as a loading control.(G and H) (G) Traction stress and (H) cell area of single spontaneously contracting cardiomyocytes non-transduced (n = 15), stably expressing the Scr- (n = 32) or MYBPC3-specific (1, n = 26; 2, n = 16) shRNAs.Boxplots and whisker plots show the median, interquartile range, and 10–90 percentile range. Unless otherwise stated, the n signifies the number of individual cells measured, acquired over three independent experiments. Statistical significance was tested with a one-way ANOVA with Tukey’s multiple comparison test in (D) and (E), comparing either control against the HCM lines independently, and a Dunnett’s correction in (G) and (H). Comparison to both controls in (D) are statistically significant ∗p < 0.05. Scale bar, 10 μm. See also Figure S5.
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fig5: Single-Cell Traction Force Measurements in Cardiomyocytes with MYBPC3 Mutation or MYBPC3 shRNA Knockdown(A) Western blot of cMyBP-C protein from two control (Con1 and Con2) and three MYBPC3 mutation lines (HCM1, HCM2, and HCM3). α-actinin is shown as a loading control for cardiomyocyte input.(B) Relative cMyBP-C levels normalized to α-actinin levels based on densitometry of western blot data (n = 3–6 lysates).(C) Typical examples of single aligned (spontaneously contracting) cardiomyocytes from control (Con1) and MYBPC3 mutation (HCM3) lines, showing a bright-field image of the relaxed form and a heatmap of traction stress applied to the substrate calculated from the mean of the traction stress vectors (corresponding to Movie S2).(D and E) (D) Traction stress and (E) cell area of single spontaneously contracting iPSC-derived cardiomyocytes from two control (Con1 [n = 47] and Con2 [n = 36]) and three MYBPC3 mutation lines (HCM1 [n = 44], HCM2 [n = 54], and HCM3 [n = 43]).(F) Western blot of cMyBP-C protein in NKX2-5eGFP/w hESC-derived cardiomyocytes after transduction with a scrambled (Scr) shRNA or two independent MYBPC3-specific shRNAs. Actin is shown as a loading control.(G and H) (G) Traction stress and (H) cell area of single spontaneously contracting cardiomyocytes non-transduced (n = 15), stably expressing the Scr- (n = 32) or MYBPC3-specific (1, n = 26; 2, n = 16) shRNAs.Boxplots and whisker plots show the median, interquartile range, and 10–90 percentile range. Unless otherwise stated, the n signifies the number of individual cells measured, acquired over three independent experiments. Statistical significance was tested with a one-way ANOVA with Tukey’s multiple comparison test in (D) and (E), comparing either control against the HCM lines independently, and a Dunnett’s correction in (G) and (H). Comparison to both controls in (D) are statistically significant ∗p < 0.05. Scale bar, 10 μm. See also Figure S5.
Mentions: Attempts to measure these hiPSC-CMs in medium without TID proved unsuccessful as even the control cells failed to reliably generate robust bead displacement. However, in medium containing TID, traction force on the polyacrylamide substrate could be measured in all cell populations. Under these conditions, traction stress was significantly decreased in all three mutant lines, HCM1 0.31 ± 0.02, HCM2 0.30 ± 0.03, HCM3 0.29 ± 0.03 mN/mm2 in comparison to both controls, Con1 0.57 ± 0.04 and Con2 0.51 ± 0.03 mN/mm2; p < 0.05 (Figures 5C and 5D). This corresponded to traction forces of 0.43 ± 0.04, 0.46 ± 0.06, 0.44 ± 0.05, 0.81 ± 0.16, and 0.86 ± 0.10 μN for HCM1, HCM2, HCM3, Con1, and Con2, respectively. A difference in cardiomyocyte size as measured by cell area was not evident between the control and the mutant cells, suggesting an overt hypertrophic response had not occurred by this stage of development under these conditions (Figure 5E). Contraction frequencies were not different (Figure S4).

Bottom Line: Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease.This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency.Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.

No MeSH data available.


Related in: MedlinePlus