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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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Diagrammatic representation of the domain structure of cMyBP-C. Ig domains are shown as hexagons, fibronectin-domains are shown as circles, and regions with a less organized domain structure are shown as light blue filled rectangles. Cardiac-specific regions are shown in red. Ig and fibronectin domains are numbered as C0–C10 from the N terminus to the C terminus. The positions of the three physiologically relevant phosphorylation sites (Ser275, Ser284, and Ser304) in the MyBPC motif (M-motif) are indicated by the yellow ovals. The position of the E258K mutation in the C1 domain, adjacent to the MyBPC motif is shown above the diagram. The binding sites for light meromyosin (LMM), myosin-S2 (S2), and titin, as well as the region essential for correct A-band incorporation and the region of N-terminal cMyBP-C implicated in interactions with components of the thin filament, are indicated by the double-headed arrows below the diagram.
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fig1: Diagrammatic representation of the domain structure of cMyBP-C. Ig domains are shown as hexagons, fibronectin-domains are shown as circles, and regions with a less organized domain structure are shown as light blue filled rectangles. Cardiac-specific regions are shown in red. Ig and fibronectin domains are numbered as C0–C10 from the N terminus to the C terminus. The positions of the three physiologically relevant phosphorylation sites (Ser275, Ser284, and Ser304) in the MyBPC motif (M-motif) are indicated by the yellow ovals. The position of the E258K mutation in the C1 domain, adjacent to the MyBPC motif is shown above the diagram. The binding sites for light meromyosin (LMM), myosin-S2 (S2), and titin, as well as the region essential for correct A-band incorporation and the region of N-terminal cMyBP-C implicated in interactions with components of the thin filament, are indicated by the double-headed arrows below the diagram.

Mentions: The E258K mutation results in a substitution of the amino acid lysine for glutamic acid at position 258 in cMyBP-C. It is one of the most prevalent disease-causing mutations in MYBPC3, with 39 probands identified in 11 independent studies (Niimura et al., 1998; Alders et al., 2003; Nanni et al., 2003; Richard et al., 2003; Van Driest et al., 2004; Song et al., 2005; Ehlermann et al., 2008; Olivotto et al., 2008; Page et al., 2012). The E258K mutation is highly penetrant, with six out of seven mutation carriers in one well-studied family showing clinical symptoms of HCM (Niimura et al., 1998). The phenotype is often severe with a high incidence of HCM-related death occurring in carriers of the mutation (Niimura et al., 1998; Page et al., 2012). Although HCM is often a disease that manifests during adulthood, this mutation has also resulted in childhood onset of HCM (Morita et al., 2008). The E258K mutation affects the last residue of the C1 IgC2 domain, at the junction between C1 and the MyBPC motif (Fig. 1; Carrier et al., 1997; Niimura et al., 1998; Ababou et al., 2008; Govada et al., 2008).


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)

Diagrammatic representation of the domain structure of cMyBP-C. Ig domains are shown as hexagons, fibronectin-domains are shown as circles, and regions with a less organized domain structure are shown as light blue filled rectangles. Cardiac-specific regions are shown in red. Ig and fibronectin domains are numbered as C0–C10 from the N terminus to the C terminus. The positions of the three physiologically relevant phosphorylation sites (Ser275, Ser284, and Ser304) in the MyBPC motif (M-motif) are indicated by the yellow ovals. The position of the E258K mutation in the C1 domain, adjacent to the MyBPC motif is shown above the diagram. The binding sites for light meromyosin (LMM), myosin-S2 (S2), and titin, as well as the region essential for correct A-band incorporation and the region of N-terminal cMyBP-C implicated in interactions with components of the thin filament, are indicated by the double-headed arrows below the diagram.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Diagrammatic representation of the domain structure of cMyBP-C. Ig domains are shown as hexagons, fibronectin-domains are shown as circles, and regions with a less organized domain structure are shown as light blue filled rectangles. Cardiac-specific regions are shown in red. Ig and fibronectin domains are numbered as C0–C10 from the N terminus to the C terminus. The positions of the three physiologically relevant phosphorylation sites (Ser275, Ser284, and Ser304) in the MyBPC motif (M-motif) are indicated by the yellow ovals. The position of the E258K mutation in the C1 domain, adjacent to the MyBPC motif is shown above the diagram. The binding sites for light meromyosin (LMM), myosin-S2 (S2), and titin, as well as the region essential for correct A-band incorporation and the region of N-terminal cMyBP-C implicated in interactions with components of the thin filament, are indicated by the double-headed arrows below the diagram.
Mentions: The E258K mutation results in a substitution of the amino acid lysine for glutamic acid at position 258 in cMyBP-C. It is one of the most prevalent disease-causing mutations in MYBPC3, with 39 probands identified in 11 independent studies (Niimura et al., 1998; Alders et al., 2003; Nanni et al., 2003; Richard et al., 2003; Van Driest et al., 2004; Song et al., 2005; Ehlermann et al., 2008; Olivotto et al., 2008; Page et al., 2012). The E258K mutation is highly penetrant, with six out of seven mutation carriers in one well-studied family showing clinical symptoms of HCM (Niimura et al., 1998). The phenotype is often severe with a high incidence of HCM-related death occurring in carriers of the mutation (Niimura et al., 1998; Page et al., 2012). Although HCM is often a disease that manifests during adulthood, this mutation has also resulted in childhood onset of HCM (Morita et al., 2008). The E258K mutation affects the last residue of the C1 IgC2 domain, at the junction between C1 and the MyBPC motif (Fig. 1; Carrier et al., 1997; Niimura et al., 1998; Ababou et al., 2008; Govada et al., 2008).

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