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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

Cardiomyocyte Action Potential Measurement(A) Typical examples of action potential (AP) traces from single spontaneously active cardiomyocytes maintained in the three conditions indicated.(B–G) Average data of maximum diastolic potential (MDP), AP amplitude, AP upstroke velocity, and AP duration at 50% (APD50) and 90% (APD90) repolarization and AP frequency. SGKi, 5-day co-incubation with 6 μM GSK650394. Data are mean ± SEM. Actual AP values and n values are shown in Table 1. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test ∗p < 0.05.
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fig3: Cardiomyocyte Action Potential Measurement(A) Typical examples of action potential (AP) traces from single spontaneously active cardiomyocytes maintained in the three conditions indicated.(B–G) Average data of maximum diastolic potential (MDP), AP amplitude, AP upstroke velocity, and AP duration at 50% (APD50) and 90% (APD90) repolarization and AP frequency. SGKi, 5-day co-incubation with 6 μM GSK650394. Data are mean ± SEM. Actual AP values and n values are shown in Table 1. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test ∗p < 0.05.

Mentions: To test whether the bioenergetic changes and improved resting ΔΨp resulted in improvements in electrophysiological function, action potentials were measured in spontaneously active cells. To focus on a possible effect of glucocorticoid signaling, we measured cells maintained in three conditions: vehicle-only, T3+IGF-1, and TID. The results are shown in Figure 3 and summarized in Table 1. The MDP was increased by both T3+IGF-1 and TID (Figure 3B), aligning well with our estimates by TMRM uptake. The amplitude of the action potential (AP) was also increased progressively by both treatments (Figure 3C). Interestingly, while the upstroke velocity was not significantly increased by T3+IGF-1 it was markedly increased by TID, from 16 ± 5 to 59 ± 6 V/s (Figure 3D; p < 0.05). This specific effect was significantly blocked by 6 μM GSK650394, an inhibitor of the serum- and glucocorticoid inducible kinase SGK1 (Sherk et al., 2008), which was previously shown to block cardiac sodium channel degradation, and which is activated by IGF-1 (Boehmer et al., 2003). We confirmed SGK1 upregulation in response to Dex (Figure 3G). SCN5A expression was not increased by dexamethasone (Figure S2), and the inhibitor did not significantly affect any other aspect of the AP, together supporting this specific mechanistic explanation. Despite the increased MDP, AP frequency was increased by TID in this single-cell format (Figure 3F), differing from measurements in the monolayer format in Figure 2A. In summary, the AP data also support the conclusion that cardiomyocyte function was improved by the action of 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)

Cardiomyocyte Action Potential Measurement(A) Typical examples of action potential (AP) traces from single spontaneously active cardiomyocytes maintained in the three conditions indicated.(B–G) Average data of maximum diastolic potential (MDP), AP amplitude, AP upstroke velocity, and AP duration at 50% (APD50) and 90% (APD90) repolarization and AP frequency. SGKi, 5-day co-incubation with 6 μM GSK650394. Data are mean ± SEM. Actual AP values and n values are shown in Table 1. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test ∗p < 0.05.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644234&req=5

fig3: Cardiomyocyte Action Potential Measurement(A) Typical examples of action potential (AP) traces from single spontaneously active cardiomyocytes maintained in the three conditions indicated.(B–G) Average data of maximum diastolic potential (MDP), AP amplitude, AP upstroke velocity, and AP duration at 50% (APD50) and 90% (APD90) repolarization and AP frequency. SGKi, 5-day co-incubation with 6 μM GSK650394. Data are mean ± SEM. Actual AP values and n values are shown in Table 1. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test ∗p < 0.05.
Mentions: To test whether the bioenergetic changes and improved resting ΔΨp resulted in improvements in electrophysiological function, action potentials were measured in spontaneously active cells. To focus on a possible effect of glucocorticoid signaling, we measured cells maintained in three conditions: vehicle-only, T3+IGF-1, and TID. The results are shown in Figure 3 and summarized in Table 1. The MDP was increased by both T3+IGF-1 and TID (Figure 3B), aligning well with our estimates by TMRM uptake. The amplitude of the action potential (AP) was also increased progressively by both treatments (Figure 3C). Interestingly, while the upstroke velocity was not significantly increased by T3+IGF-1 it was markedly increased by TID, from 16 ± 5 to 59 ± 6 V/s (Figure 3D; p < 0.05). This specific effect was significantly blocked by 6 μM GSK650394, an inhibitor of the serum- and glucocorticoid inducible kinase SGK1 (Sherk et al., 2008), which was previously shown to block cardiac sodium channel degradation, and which is activated by IGF-1 (Boehmer et al., 2003). We confirmed SGK1 upregulation in response to Dex (Figure 3G). SCN5A expression was not increased by dexamethasone (Figure S2), and the inhibitor did not significantly affect any other aspect of the AP, together supporting this specific mechanistic explanation. Despite the increased MDP, AP frequency was increased by TID in this single-cell format (Figure 3F), differing from measurements in the monolayer format in Figure 2A. In summary, the AP data also support the conclusion that cardiomyocyte function was improved by the action of 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