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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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Model of the interaction between cMyBP-C C1-C2 and the distal 126 aa of S2. (A) Detailed view of the interactions of C1 and S2. C1 is shown in blue and S2 is shown in red. Important side chains in the interaction are colored by atom type (carbon, green; oxygen, red; nitrogen, blue), and amino acid labels are colored by protein. Amino acids at the C1–S2 interface that, when mutated, are implicated in the pathogenesis of HCM are underlined, and the position of the cMyBP-C E258K mutation is highlighted by the arrow. (B) Diagrammatic representation of the proposed orientation of the C1C2 region of cMyBP-C on S2. Domains C1 and C2 of MyBP-C are shown in orange and the MyBPC-motif in gray. The three phosphorylation sites in the linker are shown in purple, residues in the MyBPC-motif mutated in HCM are shown in yellow with labels, and charged residues are colored according to their charge. S2 is shown with solvent-accessible surface colored by a simple electrostatic potential with the N terminus on the left (hidden by C1) and the C terminus on the right. Reproduced from Ababou et al. (2008) with permission from Elsevier.
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fig7: Model of the interaction between cMyBP-C C1-C2 and the distal 126 aa of S2. (A) Detailed view of the interactions of C1 and S2. C1 is shown in blue and S2 is shown in red. Important side chains in the interaction are colored by atom type (carbon, green; oxygen, red; nitrogen, blue), and amino acid labels are colored by protein. Amino acids at the C1–S2 interface that, when mutated, are implicated in the pathogenesis of HCM are underlined, and the position of the cMyBP-C E258K mutation is highlighted by the arrow. (B) Diagrammatic representation of the proposed orientation of the C1C2 region of cMyBP-C on S2. Domains C1 and C2 of MyBP-C are shown in orange and the MyBPC-motif in gray. The three phosphorylation sites in the linker are shown in purple, residues in the MyBPC-motif mutated in HCM are shown in yellow with labels, and charged residues are colored according to their charge. S2 is shown with solvent-accessible surface colored by a simple electrostatic potential with the N terminus on the left (hidden by C1) and the C terminus on the right. Reproduced from Ababou et al. (2008) with permission from Elsevier.

Mentions: Collectively our phosphorylation data and Y2H data suggest a model in which the E258K mutation abolishes the C1C2–S2 interaction by altering the intrinsic binding properties of C1C2 (Ababou et al., 2008). The importance of the glutamic acid residue at position 258 cMyBP-C is supported by its 100% conservation among species and among the MyBP-C isoforms (Govada et al., 2008). The glutamic acid residue at position 258 of WT human cMyBP-C forms part of an acidic patch on the surface of the C-terminal end of the C1 domain that constitutes the MyBPC-C1–S2 interface (Fig. 7; Ababou et al., 2008; Govada et al., 2008). E258 is thought to form a hydrogen bond with T857 of myosin (Fig. 7 A), and the importance of this bond is indicated by the fact that the chemical shift perturbation at this residue as a result of S2 binding is among the highest of any residues in the C1 domain (Ababou et al., 2008). It is therefore plausible that the charge reversal brought about by the E258K may, in addition to abolishing this one hydrogen bond, also cause a repulsion between the C1 binding surface and myosin S2. Furthermore, as the binding site for the C2 domain has been mapped to the region directly adjacent to E258 (E258 in C1 interacting with T857 of S2 and D431 in C2 interacting with R858; Ababou et al., 2007, 2008), a charge reversal in this region may destabilize the whole C1C2–S2 interaction. Although the present study is the first to show that the E258K mutation abolishes the C1C2–S2 interaction, abolition or weakening of this interaction as a result of HCM-causing mutations in cMyBP-C and myosin S2 is not unprecedented (Gruen and Gautel, 1999; Ababou et al., 2007, 2008). Another example can be found with the charge reversal caused by the E924K mutation in S2 abolishing its interaction with N-terminal cMyBP-C (Gruen and Gautel, 1999). Additionally, the myosin S2 E846K, R870H, and cMyBP-C (C1) D228N mutations severely weakened the interaction (Gruen and Gautel, 1999; Ababou et al., 2007, 2008). Finally, introduction of negative charges through phosphorylation of serine residues 275, 284, and 304 in the MyBPC motif cause a repulsion of the C1C2–S2 interaction (Gruen et al., 1999).


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)

Model of the interaction between cMyBP-C C1-C2 and the distal 126 aa of S2. (A) Detailed view of the interactions of C1 and S2. C1 is shown in blue and S2 is shown in red. Important side chains in the interaction are colored by atom type (carbon, green; oxygen, red; nitrogen, blue), and amino acid labels are colored by protein. Amino acids at the C1–S2 interface that, when mutated, are implicated in the pathogenesis of HCM are underlined, and the position of the cMyBP-C E258K mutation is highlighted by the arrow. (B) Diagrammatic representation of the proposed orientation of the C1C2 region of cMyBP-C on S2. Domains C1 and C2 of MyBP-C are shown in orange and the MyBPC-motif in gray. The three phosphorylation sites in the linker are shown in purple, residues in the MyBPC-motif mutated in HCM are shown in yellow with labels, and charged residues are colored according to their charge. S2 is shown with solvent-accessible surface colored by a simple electrostatic potential with the N terminus on the left (hidden by C1) and the C terminus on the right. Reproduced from Ababou et al. (2008) with permission from Elsevier.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Model of the interaction between cMyBP-C C1-C2 and the distal 126 aa of S2. (A) Detailed view of the interactions of C1 and S2. C1 is shown in blue and S2 is shown in red. Important side chains in the interaction are colored by atom type (carbon, green; oxygen, red; nitrogen, blue), and amino acid labels are colored by protein. Amino acids at the C1–S2 interface that, when mutated, are implicated in the pathogenesis of HCM are underlined, and the position of the cMyBP-C E258K mutation is highlighted by the arrow. (B) Diagrammatic representation of the proposed orientation of the C1C2 region of cMyBP-C on S2. Domains C1 and C2 of MyBP-C are shown in orange and the MyBPC-motif in gray. The three phosphorylation sites in the linker are shown in purple, residues in the MyBPC-motif mutated in HCM are shown in yellow with labels, and charged residues are colored according to their charge. S2 is shown with solvent-accessible surface colored by a simple electrostatic potential with the N terminus on the left (hidden by C1) and the C terminus on the right. Reproduced from Ababou et al. (2008) with permission from Elsevier.
Mentions: Collectively our phosphorylation data and Y2H data suggest a model in which the E258K mutation abolishes the C1C2–S2 interaction by altering the intrinsic binding properties of C1C2 (Ababou et al., 2008). The importance of the glutamic acid residue at position 258 cMyBP-C is supported by its 100% conservation among species and among the MyBP-C isoforms (Govada et al., 2008). The glutamic acid residue at position 258 of WT human cMyBP-C forms part of an acidic patch on the surface of the C-terminal end of the C1 domain that constitutes the MyBPC-C1–S2 interface (Fig. 7; Ababou et al., 2008; Govada et al., 2008). E258 is thought to form a hydrogen bond with T857 of myosin (Fig. 7 A), and the importance of this bond is indicated by the fact that the chemical shift perturbation at this residue as a result of S2 binding is among the highest of any residues in the C1 domain (Ababou et al., 2008). It is therefore plausible that the charge reversal brought about by the E258K may, in addition to abolishing this one hydrogen bond, also cause a repulsion between the C1 binding surface and myosin S2. Furthermore, as the binding site for the C2 domain has been mapped to the region directly adjacent to E258 (E258 in C1 interacting with T857 of S2 and D431 in C2 interacting with R858; Ababou et al., 2007, 2008), a charge reversal in this region may destabilize the whole C1C2–S2 interaction. Although the present study is the first to show that the E258K mutation abolishes the C1C2–S2 interaction, abolition or weakening of this interaction as a result of HCM-causing mutations in cMyBP-C and myosin S2 is not unprecedented (Gruen and Gautel, 1999; Ababou et al., 2007, 2008). Another example can be found with the charge reversal caused by the E924K mutation in S2 abolishing its interaction with N-terminal cMyBP-C (Gruen and Gautel, 1999). Additionally, the myosin S2 E846K, R870H, and cMyBP-C (C1) D228N mutations severely weakened the interaction (Gruen and Gautel, 1999; Ababou et al., 2007, 2008). Finally, introduction of negative charges through phosphorylation of serine residues 275, 284, and 304 in the MyBPC motif cause a repulsion of the C1C2–S2 interaction (Gruen et al., 1999).

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