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E258K HCM-causing mutation in cardiac MyBP-C reduces contractile force and accelerates twitch kinetics by disrupting the cMyBP-C and myosin S2 interaction.

De Lange WJ, Grimes AC, Hegge LF, Spring AM, Brost TM, Ralphe JC - J. Gen. Physiol. (2013)

Bottom Line: Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT).Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere.Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA.

ABSTRACT
Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C-S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

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Expression of WT and E258K cMyBP-C in cardiomyocytes and mECT. (A) Effect of adenoviral transduction on cardiac cell viability in neonatal mouse cMyBP-C−/− cardiac cells in monolayer culture, transduced with adWT (blue bars) and ad258 (red bars) at MOIs of 0, 5, 10, 20, 50, and 100 as assessed by an MTT assay. (B) Western blot showing total cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. (C) Densitometric quantification of cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. The arrow in A indicates an MOI = 20 used to transduce mECT. *, P < 0.05 (one-way ANOVA with a Tukey’s post-hoc test; n = 3). Error bars indicate SEM.
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fig2: Expression of WT and E258K cMyBP-C in cardiomyocytes and mECT. (A) Effect of adenoviral transduction on cardiac cell viability in neonatal mouse cMyBP-C−/− cardiac cells in monolayer culture, transduced with adWT (blue bars) and ad258 (red bars) at MOIs of 0, 5, 10, 20, 50, and 100 as assessed by an MTT assay. (B) Western blot showing total cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. (C) Densitometric quantification of cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. The arrow in A indicates an MOI = 20 used to transduce mECT. *, P < 0.05 (one-way ANOVA with a Tukey’s post-hoc test; n = 3). Error bars indicate SEM.

Mentions: We previously demonstrated that transduction of cardiac cells with adWT or a scrambled control adenovirus at an MOI up to fivefold higher than used in these experiments did not adversely affect cell survival (de Lange et al., 2011). To confirm this finding and to assess the effect of E258K cMyBP-C on cell survival, 2D monolayer cultures of mouse neonatal cMyBP-C−/− cardiac cells were transduced with adWT and ad258 at MOIs of 0, 5, 10, 20, 50, and 100. Cardiac cell number and viability were assessed by both total RNA yield/105 cells and the MTT viability assay, and showed no significant differences between cardiac cells transduced with either virus at an MOI of up to 100 (Fig. 2 A and Fig. S1 A), which suggests that neither the virus nor expression of E258K cMyBP-C adversely affect cardiac cell survival. Because these data agreed with observations in similar experiments with the adWT virus (de Lange et al., 2011), KO adWT and KO ad258 mECT were produced from KO cardiac cells transduced at an MOI of 20 during the gyration culture. We subsequently assessed expression levels of human cMyBP-C in KO adWT and KO ad258 mECT and compared it to the levels of endogenous mouse cMyBP-C in untransduced WT mECT. Our data show equivalent expression levels of cMyBP-C between these three groups, confirming appropriate cMyBP-C levels in KO adWT and KO ad258 mECT (Fig. 2, B and C).


E258K HCM-causing mutation in cardiac MyBP-C reduces contractile force and accelerates twitch kinetics by disrupting the cMyBP-C and myosin S2 interaction.

De Lange WJ, Grimes AC, Hegge LF, Spring AM, Brost TM, Ralphe JC - J. Gen. Physiol. (2013)

Expression of WT and E258K cMyBP-C in cardiomyocytes and mECT. (A) Effect of adenoviral transduction on cardiac cell viability in neonatal mouse cMyBP-C−/− cardiac cells in monolayer culture, transduced with adWT (blue bars) and ad258 (red bars) at MOIs of 0, 5, 10, 20, 50, and 100 as assessed by an MTT assay. (B) Western blot showing total cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. (C) Densitometric quantification of cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. The arrow in A indicates an MOI = 20 used to transduce mECT. *, P < 0.05 (one-way ANOVA with a Tukey’s post-hoc test; n = 3). Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753599&req=5

fig2: Expression of WT and E258K cMyBP-C in cardiomyocytes and mECT. (A) Effect of adenoviral transduction on cardiac cell viability in neonatal mouse cMyBP-C−/− cardiac cells in monolayer culture, transduced with adWT (blue bars) and ad258 (red bars) at MOIs of 0, 5, 10, 20, 50, and 100 as assessed by an MTT assay. (B) Western blot showing total cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. (C) Densitometric quantification of cMyBP-C protein levels in WT, KO adWT, and KO ad258 mECT. The arrow in A indicates an MOI = 20 used to transduce mECT. *, P < 0.05 (one-way ANOVA with a Tukey’s post-hoc test; n = 3). Error bars indicate SEM.
Mentions: We previously demonstrated that transduction of cardiac cells with adWT or a scrambled control adenovirus at an MOI up to fivefold higher than used in these experiments did not adversely affect cell survival (de Lange et al., 2011). To confirm this finding and to assess the effect of E258K cMyBP-C on cell survival, 2D monolayer cultures of mouse neonatal cMyBP-C−/− cardiac cells were transduced with adWT and ad258 at MOIs of 0, 5, 10, 20, 50, and 100. Cardiac cell number and viability were assessed by both total RNA yield/105 cells and the MTT viability assay, and showed no significant differences between cardiac cells transduced with either virus at an MOI of up to 100 (Fig. 2 A and Fig. S1 A), which suggests that neither the virus nor expression of E258K cMyBP-C adversely affect cardiac cell survival. Because these data agreed with observations in similar experiments with the adWT virus (de Lange et al., 2011), KO adWT and KO ad258 mECT were produced from KO cardiac cells transduced at an MOI of 20 during the gyration culture. We subsequently assessed expression levels of human cMyBP-C in KO adWT and KO ad258 mECT and compared it to the levels of endogenous mouse cMyBP-C in untransduced WT mECT. Our data show equivalent expression levels of cMyBP-C between these three groups, confirming appropriate cMyBP-C levels in KO adWT and KO ad258 mECT (Fig. 2, B and C).

Bottom Line: Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT).Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere.Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA.

ABSTRACT
Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C-S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

Show MeSH
Related in: MedlinePlus