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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

Using TMRM to Identify Modifiers of ΔΨp in hESC-Derived NKX2-5+ Cardiomyocytes(A) Time-course measurement of TMRM accumulation after cardiac differentiation of NKX2-5eGFP/w hESCs. fluorescence-activated cell sorting (FACS) plots show eGFP and TMRM fluorescence after 0, 4, 8, 13, and 22 min of loading with TMRM. A minor cross-bleed correction has been applied to all.(B) Median TMRM fluorescence intensity in eGFP+ cells plotted against loading time.(C) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a vehicle-only control after 5 days of incubation with the factors shown (n = 6–34).(D) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3-treated control after 5 days of incubation with the factors shown (n = 10–22).(E) Upper panel: example FACS plots showing measurement of a vehicle-only control alongside a T3+IGF-1+Dex-treated sample. Lower panel: histograms of eGFP and TMRM intensity in the eGFP+ populations as marked on the dot plots above.(F) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3+IGF-1+Dex-treated sample after 5 days of incubation with the factors shown (n = 11).Data are mean ± SEM. The n signifies biological replicates. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction, #p < 0.05 for eGFP fluorescence; ∗p < 0.05 for TMRM fluorescence. See also Figures S1 and S2.
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fig1: Using TMRM to Identify Modifiers of ΔΨp in hESC-Derived NKX2-5+ Cardiomyocytes(A) Time-course measurement of TMRM accumulation after cardiac differentiation of NKX2-5eGFP/w hESCs. fluorescence-activated cell sorting (FACS) plots show eGFP and TMRM fluorescence after 0, 4, 8, 13, and 22 min of loading with TMRM. A minor cross-bleed correction has been applied to all.(B) Median TMRM fluorescence intensity in eGFP+ cells plotted against loading time.(C) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a vehicle-only control after 5 days of incubation with the factors shown (n = 6–34).(D) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3-treated control after 5 days of incubation with the factors shown (n = 10–22).(E) Upper panel: example FACS plots showing measurement of a vehicle-only control alongside a T3+IGF-1+Dex-treated sample. Lower panel: histograms of eGFP and TMRM intensity in the eGFP+ populations as marked on the dot plots above.(F) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3+IGF-1+Dex-treated sample after 5 days of incubation with the factors shown (n = 11).Data are mean ± SEM. The n signifies biological replicates. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction, #p < 0.05 for eGFP fluorescence; ∗p < 0.05 for TMRM fluorescence. See also Figures S1 and S2.

Mentions: Populations of cardiomyocytes were generated by monolayer differentiation of NKX2-5eGFP/w hESCs, and these were maintained until day 16. To screen for modulators of resting plasma membrane potential (ΔΨp), we assessed short-term accumulation of the cationic fluorescent probe tetramethylrhodamine methyl ester (TMRM) in cell suspensions. TMRM follows Nernstian behavior across cell membranes, the early phase of TMRM accumulation into cells is dominated by the ΔΨp, while with longer loading times the mitochondrial membrane potential and matrix volume contribute increasingly to total accumulation (Gerencser et al., 2012). Time-course measurement of TMRM accumulation was performed from control conditions (Figures 1A and 1B). Based on this, 8-min loading time was chosen for relative assessments of ΔΨp, since this was in the linear phase of accumulation but gave signal substantially above background. Additionally, total cellular eGFP fluorescence was used as an estimate of cell size/volume, on the assumption that eGFP levels should correlate with total cell protein. There are several caveats to this reasoning, but modulators can be subsequently validated with more direct assays. In support of a general correlation, fetal calf serum exposure, known to increase the size of these cells (Birket et al., 2013), increased eGFP levels after 5 days of exposure (Figure S1).


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)

Using TMRM to Identify Modifiers of ΔΨp in hESC-Derived NKX2-5+ Cardiomyocytes(A) Time-course measurement of TMRM accumulation after cardiac differentiation of NKX2-5eGFP/w hESCs. fluorescence-activated cell sorting (FACS) plots show eGFP and TMRM fluorescence after 0, 4, 8, 13, and 22 min of loading with TMRM. A minor cross-bleed correction has been applied to all.(B) Median TMRM fluorescence intensity in eGFP+ cells plotted against loading time.(C) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a vehicle-only control after 5 days of incubation with the factors shown (n = 6–34).(D) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3-treated control after 5 days of incubation with the factors shown (n = 10–22).(E) Upper panel: example FACS plots showing measurement of a vehicle-only control alongside a T3+IGF-1+Dex-treated sample. Lower panel: histograms of eGFP and TMRM intensity in the eGFP+ populations as marked on the dot plots above.(F) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3+IGF-1+Dex-treated sample after 5 days of incubation with the factors shown (n = 11).Data are mean ± SEM. The n signifies biological replicates. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction, #p < 0.05 for eGFP fluorescence; ∗p < 0.05 for TMRM fluorescence. See also Figures S1 and S2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4644234&req=5

fig1: Using TMRM to Identify Modifiers of ΔΨp in hESC-Derived NKX2-5+ Cardiomyocytes(A) Time-course measurement of TMRM accumulation after cardiac differentiation of NKX2-5eGFP/w hESCs. fluorescence-activated cell sorting (FACS) plots show eGFP and TMRM fluorescence after 0, 4, 8, 13, and 22 min of loading with TMRM. A minor cross-bleed correction has been applied to all.(B) Median TMRM fluorescence intensity in eGFP+ cells plotted against loading time.(C) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a vehicle-only control after 5 days of incubation with the factors shown (n = 6–34).(D) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3-treated control after 5 days of incubation with the factors shown (n = 10–22).(E) Upper panel: example FACS plots showing measurement of a vehicle-only control alongside a T3+IGF-1+Dex-treated sample. Lower panel: histograms of eGFP and TMRM intensity in the eGFP+ populations as marked on the dot plots above.(F) eGFP and TMRM fluorescence values in eGFP+ cardiomyocytes relative to a T3+IGF-1+Dex-treated sample after 5 days of incubation with the factors shown (n = 11).Data are mean ± SEM. The n signifies biological replicates. Statistical significance compared to the vehicle-only control was calculated using a one-way ANOVA with Dunnett’s correction, #p < 0.05 for eGFP fluorescence; ∗p < 0.05 for TMRM fluorescence. See also Figures S1 and S2.
Mentions: Populations of cardiomyocytes were generated by monolayer differentiation of NKX2-5eGFP/w hESCs, and these were maintained until day 16. To screen for modulators of resting plasma membrane potential (ΔΨp), we assessed short-term accumulation of the cationic fluorescent probe tetramethylrhodamine methyl ester (TMRM) in cell suspensions. TMRM follows Nernstian behavior across cell membranes, the early phase of TMRM accumulation into cells is dominated by the ΔΨp, while with longer loading times the mitochondrial membrane potential and matrix volume contribute increasingly to total accumulation (Gerencser et al., 2012). Time-course measurement of TMRM accumulation was performed from control conditions (Figures 1A and 1B). Based on this, 8-min loading time was chosen for relative assessments of ΔΨp, since this was in the linear phase of accumulation but gave signal substantially above background. Additionally, total cellular eGFP fluorescence was used as an estimate of cell size/volume, on the assumption that eGFP levels should correlate with total cell protein. There are several caveats to this reasoning, but modulators can be subsequently validated with more direct assays. In support of a general correlation, fetal calf serum exposure, known to increase the size of these cells (Birket et al., 2013), increased eGFP levels after 5 days of exposure (Figure S1).

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