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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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Effect of the E258K cMyBP-C on twitch force production. (A and B) Absolute (A) and normalized (B) averaged force traces of KO adWT (blue line) and KO ad258 (red line). (C) FMax produced. (D) Contraction time from electrical stimulus to FMax (CT100). (E) Relaxation RT50. (F) Relaxation RT50–90. Blue bars, KO adWT mECT; red bars, KO ad258 mECT. *, P < 0.05 vs. WT (Student’s t test; KO adWT n = 11, KO ad258 n = 13). Error bars indicate SEM.
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fig4: Effect of the E258K cMyBP-C on twitch force production. (A and B) Absolute (A) and normalized (B) averaged force traces of KO adWT (blue line) and KO ad258 (red line). (C) FMax produced. (D) Contraction time from electrical stimulus to FMax (CT100). (E) Relaxation RT50. (F) Relaxation RT50–90. Blue bars, KO adWT mECT; red bars, KO ad258 mECT. *, P < 0.05 vs. WT (Student’s t test; KO adWT n = 11, KO ad258 n = 13). Error bars indicate SEM.

Mentions: Fig. S1 show DNA, RNA, and protein content in cMyBP-C−/− mECT and monolayer cardiomyocytes transduced with adWT and ad258. Figs. S2–S4 show immunofluorescent histology of KO adWT and KO ad258 ECT confirming appropriate incorporation of exogenous human cMyBP-C into the mouse sarcomere. Fig. S5 shows the phosphorylation status of Ser275, Ser284, and Ser304 in monolayer cMyBP-C−/− cardiomyocytes transduced with adWT and ad258. Fig. S6 show the phosphorylation status of Ser284 in KO adWT and KO ad258 mECT, as well as in WT neonatal and adult mouse hearts. Detailed data regarding mECT twitch force production and cMyBP-C phosphorylation depicted in Figs. 4, 5, and S5 are provided in Tables S1 and S2. Online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.201311018/DC1.


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)

Effect of the E258K cMyBP-C on twitch force production. (A and B) Absolute (A) and normalized (B) averaged force traces of KO adWT (blue line) and KO ad258 (red line). (C) FMax produced. (D) Contraction time from electrical stimulus to FMax (CT100). (E) Relaxation RT50. (F) Relaxation RT50–90. Blue bars, KO adWT mECT; red bars, KO ad258 mECT. *, P < 0.05 vs. WT (Student’s t test; KO adWT n = 11, KO ad258 n = 13). Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Effect of the E258K cMyBP-C on twitch force production. (A and B) Absolute (A) and normalized (B) averaged force traces of KO adWT (blue line) and KO ad258 (red line). (C) FMax produced. (D) Contraction time from electrical stimulus to FMax (CT100). (E) Relaxation RT50. (F) Relaxation RT50–90. Blue bars, KO adWT mECT; red bars, KO ad258 mECT. *, P < 0.05 vs. WT (Student’s t test; KO adWT n = 11, KO ad258 n = 13). Error bars indicate SEM.
Mentions: Fig. S1 show DNA, RNA, and protein content in cMyBP-C−/− mECT and monolayer cardiomyocytes transduced with adWT and ad258. Figs. S2–S4 show immunofluorescent histology of KO adWT and KO ad258 ECT confirming appropriate incorporation of exogenous human cMyBP-C into the mouse sarcomere. Fig. S5 shows the phosphorylation status of Ser275, Ser284, and Ser304 in monolayer cMyBP-C−/− cardiomyocytes transduced with adWT and ad258. Fig. S6 show the phosphorylation status of Ser284 in KO adWT and KO ad258 mECT, as well as in WT neonatal and adult mouse hearts. Detailed data regarding mECT twitch force production and cMyBP-C phosphorylation depicted in Figs. 4, 5, and S5 are provided in Tables S1 and S2. Online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.201311018/DC1.

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