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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

Bioenergetic Profiling of hESC-Derived Cardiomyocytes(A) Contraction frequency in cardiomyocyte monolayers, treated for 5 days with the factors shown, prior to Seahorse measurement.(B) Respiration rates in cardiomyocyte monolayers treated with the factors shown, normalized to cell protein. Basal, endogenous rate; oligomycin, ATP synthase-inhibited rate; FCCP, maximum uncoupled rate; Rot + Ant A, non-mitochondrial respiratory rate.(C) Glycolytic rate measured in parallel with respiration, normalized to cell protein.(D) Theoretical basal ATP production rates from oxidative phosphorylation and anaerobic glycolysis calculated from measurements in (B) and (C).(E) Real-time respiration measurements of vehicle-only and TID-treated cells and response to injection of vehicle-only or nifedipine + blebbistatin, then oligomycin, and finally rotenone and antimycin A.(F and G) Real-time respiration measurements of (F) vehicle-only or (G) TID-treated cells and response to injection of vehicle-only or 40 μM etomoxir or 5 μM UK5099, then oligomycin, FCCP, and finally rotenone and antimycin A.(H) Sensitivity of basal and FCCP-stimulated mitochondrial respiration to etomoxir and UK5099 in vehicle-only or TID-treated cells.Bar data are mean ± SEM from three independent experiments, each comprising four to five measurement wells per condition. Real-time respiration plots show data of a typical experiment mean ± SD of individual wells. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction for (B)–(D). ∗p < 0.05. See also Figure S3.
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fig2: Bioenergetic Profiling of hESC-Derived Cardiomyocytes(A) Contraction frequency in cardiomyocyte monolayers, treated for 5 days with the factors shown, prior to Seahorse measurement.(B) Respiration rates in cardiomyocyte monolayers treated with the factors shown, normalized to cell protein. Basal, endogenous rate; oligomycin, ATP synthase-inhibited rate; FCCP, maximum uncoupled rate; Rot + Ant A, non-mitochondrial respiratory rate.(C) Glycolytic rate measured in parallel with respiration, normalized to cell protein.(D) Theoretical basal ATP production rates from oxidative phosphorylation and anaerobic glycolysis calculated from measurements in (B) and (C).(E) Real-time respiration measurements of vehicle-only and TID-treated cells and response to injection of vehicle-only or nifedipine + blebbistatin, then oligomycin, and finally rotenone and antimycin A.(F and G) Real-time respiration measurements of (F) vehicle-only or (G) TID-treated cells and response to injection of vehicle-only or 40 μM etomoxir or 5 μM UK5099, then oligomycin, FCCP, and finally rotenone and antimycin A.(H) Sensitivity of basal and FCCP-stimulated mitochondrial respiration to etomoxir and UK5099 in vehicle-only or TID-treated cells.Bar data are mean ± SEM from three independent experiments, each comprising four to five measurement wells per condition. Real-time respiration plots show data of a typical experiment mean ± SD of individual wells. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction for (B)–(D). ∗p < 0.05. See also Figure S3.

Mentions: The response to IGF-1 and Dex in terms of cardiomyocyte growth and ΔΨp suggests a possible synergistic role in metabolic stimulation. To test this, bioenergetic profiling of contracting cardiomyocyte monolayers was performed using the Seahorse XF Analyzer on cells treated during the same experimental time course. Contraction rates, measured prior to analysis, were not significantly different between groups (Figure 2A). Under standard conditions (15 mM glucose, 0.5 mM sodium pyruvate) normalized to total cell protein, T3 mildly increased the oligomycin-sensitive respiration rate as previously reported (Yang et al., 2014), indicating increased mitochondrial ATP turnover (Figures 2B and 2D). Neither IGF-1 nor Dex stimulated this further, but the combination of TID did have a further stimulatory effect (TID: 213 ± 4 versus vehicle: 124 ± 6 pmol O2/min/10 μg cell protein; p < 0.05). The anaerobic glycolytic rate (calculated as previously described by Mookerjee et al., 2015) was also significantly increased by TID (TID: 68 ± 4 versus vehicle: 34 ± 4 pmol H+/min/10 μg cell protein; p < 0.05) (Figure 2C). A conversion of these values to ATP production rates (see Experimental Procedures) showed a large increase (1.7-fold) in combined ATP turnover with TID (Figure 2D). The oligomycin-inhibited rates, the maximum uncoupled respiration rates induced by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and the non-mitochondrial rates were not significantly different between conditions (Figure 2B). These results support a synergistic effect for the actions of IGF-1 and Dex on basal cell activity. Inhibition of both excitation and contraction by co-injection of nifedipine and blebbistatin decreased the mitochondrial respiration rate by 39.3% ± 4.2% in the vehicle-only condition and 43.9% ± 9.0% in TID (Figure 1E) after subtraction of a minor buffer-only injection effect. As the starting respiration rate was higher with TID, this showed that the activity of these processes had increased at least proportionally by TID.


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)

Bioenergetic Profiling of hESC-Derived Cardiomyocytes(A) Contraction frequency in cardiomyocyte monolayers, treated for 5 days with the factors shown, prior to Seahorse measurement.(B) Respiration rates in cardiomyocyte monolayers treated with the factors shown, normalized to cell protein. Basal, endogenous rate; oligomycin, ATP synthase-inhibited rate; FCCP, maximum uncoupled rate; Rot + Ant A, non-mitochondrial respiratory rate.(C) Glycolytic rate measured in parallel with respiration, normalized to cell protein.(D) Theoretical basal ATP production rates from oxidative phosphorylation and anaerobic glycolysis calculated from measurements in (B) and (C).(E) Real-time respiration measurements of vehicle-only and TID-treated cells and response to injection of vehicle-only or nifedipine + blebbistatin, then oligomycin, and finally rotenone and antimycin A.(F and G) Real-time respiration measurements of (F) vehicle-only or (G) TID-treated cells and response to injection of vehicle-only or 40 μM etomoxir or 5 μM UK5099, then oligomycin, FCCP, and finally rotenone and antimycin A.(H) Sensitivity of basal and FCCP-stimulated mitochondrial respiration to etomoxir and UK5099 in vehicle-only or TID-treated cells.Bar data are mean ± SEM from three independent experiments, each comprising four to five measurement wells per condition. Real-time respiration plots show data of a typical experiment mean ± SD of individual wells. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction for (B)–(D). ∗p < 0.05. See also Figure S3.
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fig2: Bioenergetic Profiling of hESC-Derived Cardiomyocytes(A) Contraction frequency in cardiomyocyte monolayers, treated for 5 days with the factors shown, prior to Seahorse measurement.(B) Respiration rates in cardiomyocyte monolayers treated with the factors shown, normalized to cell protein. Basal, endogenous rate; oligomycin, ATP synthase-inhibited rate; FCCP, maximum uncoupled rate; Rot + Ant A, non-mitochondrial respiratory rate.(C) Glycolytic rate measured in parallel with respiration, normalized to cell protein.(D) Theoretical basal ATP production rates from oxidative phosphorylation and anaerobic glycolysis calculated from measurements in (B) and (C).(E) Real-time respiration measurements of vehicle-only and TID-treated cells and response to injection of vehicle-only or nifedipine + blebbistatin, then oligomycin, and finally rotenone and antimycin A.(F and G) Real-time respiration measurements of (F) vehicle-only or (G) TID-treated cells and response to injection of vehicle-only or 40 μM etomoxir or 5 μM UK5099, then oligomycin, FCCP, and finally rotenone and antimycin A.(H) Sensitivity of basal and FCCP-stimulated mitochondrial respiration to etomoxir and UK5099 in vehicle-only or TID-treated cells.Bar data are mean ± SEM from three independent experiments, each comprising four to five measurement wells per condition. Real-time respiration plots show data of a typical experiment mean ± SD of individual wells. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction for (B)–(D). ∗p < 0.05. See also Figure S3.
Mentions: The response to IGF-1 and Dex in terms of cardiomyocyte growth and ΔΨp suggests a possible synergistic role in metabolic stimulation. To test this, bioenergetic profiling of contracting cardiomyocyte monolayers was performed using the Seahorse XF Analyzer on cells treated during the same experimental time course. Contraction rates, measured prior to analysis, were not significantly different between groups (Figure 2A). Under standard conditions (15 mM glucose, 0.5 mM sodium pyruvate) normalized to total cell protein, T3 mildly increased the oligomycin-sensitive respiration rate as previously reported (Yang et al., 2014), indicating increased mitochondrial ATP turnover (Figures 2B and 2D). Neither IGF-1 nor Dex stimulated this further, but the combination of TID did have a further stimulatory effect (TID: 213 ± 4 versus vehicle: 124 ± 6 pmol O2/min/10 μg cell protein; p < 0.05). The anaerobic glycolytic rate (calculated as previously described by Mookerjee et al., 2015) was also significantly increased by TID (TID: 68 ± 4 versus vehicle: 34 ± 4 pmol H+/min/10 μg cell protein; p < 0.05) (Figure 2C). A conversion of these values to ATP production rates (see Experimental Procedures) showed a large increase (1.7-fold) in combined ATP turnover with TID (Figure 2D). The oligomycin-inhibited rates, the maximum uncoupled respiration rates induced by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and the non-mitochondrial rates were not significantly different between conditions (Figure 2B). These results support a synergistic effect for the actions of IGF-1 and Dex on basal cell activity. Inhibition of both excitation and contraction by co-injection of nifedipine and blebbistatin decreased the mitochondrial respiration rate by 39.3% ± 4.2% in the vehicle-only condition and 43.9% ± 9.0% in TID (Figure 1E) after subtraction of a minor buffer-only injection effect. As the starting respiration rate was higher with TID, this showed that the activity of these processes had increased at least proportionally by TID.

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