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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 mutation on the interactions of N-terminal cMyBP-C with myosin S2 and cardiac actin. (A–H) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and the 126 distal amino acids of human myosin S2. (A) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (B) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (C) pGBKT7 × pGADT7 (negative control). (D) pGBKT7 × pGADT7-S2 (negative control). (E) pGBKT7-C1C2WT × pGADT7 (negative control). (F) pGBKT7-C1C2E258K × pGADT7 (negative control). (G) pGBKT7-C1C2WT × pGADT7-S2 (interaction of interest). (H) pGBKT7-C1C2E258K × pGADT7-S2 (interaction of interest). (I–N) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and α cardiac actin (ACTC1), as well as their appropriate control interaction tests. (I) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (J) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (K) pGBKT7 × pGADT7-ACTC1 (negative control). (L) pGBKT7-53 × pGADT7-ACTC1 (negative control). (M) pGBKT7-C1C2WT × pGADT7-ACTC1 (interaction of interest). (N) pGBKT7-C1C2E258K × pGADT7-ACTC1 (interaction of interest). Yeast were grown on minimal selection media lacking leucine, tryptophan, histidine, and adenine. (O) Quantitative β-galactosidase assay performed in diploid yeast containing pGBKT7-C1C2WT × pGADT7-S2 (blue bar) and pGBKT7-C1C2E258K × pGADT7-S2 (red bar). n = 3. Error bars indicate SEM.
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fig6: Effect of the E258K mutation on the interactions of N-terminal cMyBP-C with myosin S2 and cardiac actin. (A–H) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and the 126 distal amino acids of human myosin S2. (A) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (B) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (C) pGBKT7 × pGADT7 (negative control). (D) pGBKT7 × pGADT7-S2 (negative control). (E) pGBKT7-C1C2WT × pGADT7 (negative control). (F) pGBKT7-C1C2E258K × pGADT7 (negative control). (G) pGBKT7-C1C2WT × pGADT7-S2 (interaction of interest). (H) pGBKT7-C1C2E258K × pGADT7-S2 (interaction of interest). (I–N) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and α cardiac actin (ACTC1), as well as their appropriate control interaction tests. (I) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (J) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (K) pGBKT7 × pGADT7-ACTC1 (negative control). (L) pGBKT7-53 × pGADT7-ACTC1 (negative control). (M) pGBKT7-C1C2WT × pGADT7-ACTC1 (interaction of interest). (N) pGBKT7-C1C2E258K × pGADT7-ACTC1 (interaction of interest). Yeast were grown on minimal selection media lacking leucine, tryptophan, histidine, and adenine. (O) Quantitative β-galactosidase assay performed in diploid yeast containing pGBKT7-C1C2WT × pGADT7-S2 (blue bar) and pGBKT7-C1C2E258K × pGADT7-S2 (red bar). n = 3. Error bars indicate SEM.

Mentions: The effect of the E258K missense mutation on the interaction between N-terminal cMyBP-C and myosin S2 was tested using a Y2H interaction assay. In these tests, either WT human C1C2 (C1C2WT) or human C1C2 into which the E258K missense mutation was engineered (C1C2E258K) were fused to the GAL4-DNA binding domain (C1C2WT/C1C2E258K-BD). The 126 distal amino acids of myosin S2 were also expressed as a fusion peptide with the GAL4 activation domain (S2-AD). Additionally, as myosin S2 is known to form coiled-coil dimers (Blankenfeldt et al., 2006), we also expressed an S2-BD fusion peptide. Coexpression of BD and AD fusion peptides was achieved through yeast mating, and selection for interaction between fusion peptides was assessed by the ability of yeast to grow on minimal selection media lacking leucine, tryptophan, histidine, and adenine (QDO). Robust growth, resulting from activation of the HIS3 and ADE2 nutritional reporter genes, was noted when positive control 53-BD and T-AD fusion peptides were expressed (Fig. 6 A). Additionally, consistent with the known dimerization of myosin S2 (Blankenfeldt et al., 2006), coexpression of S2-BD and S2-AD also resulted in growth (Fig. 6 B). Coexpression of unfused BD and AD peptides did not result in reporter gene activation, as indicated by lack of growth on QDO selection media (Fig. 6 C). Coexpression of unfused BD and S2-AD, or C1C2WT/C1C2E258K-BD and AD, did not result in growth on QDO selection media (Fig. 6, D–F), which suggests that S2-AD, C1C2WT-BD, and C1C2E258K-BD do not auto-activate expression of nutritional reporter genes. Coexpression of C1C2WT-BD and S2-AD fusion peptides resulted in robust growth on QDO selection media, which indicates a strong interaction between C1C2WT and S2, resulting in robust activation of the HIS3 and ADE2 nutritional reporter genes (Fig. 6 G). In contrast, no growth was observed when C1C2E258K-BD and S2-AD were coexpressed (Fig. 6 H), which suggests that the C1C2E258K is unable to interact with myosin-S2.


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 mutation on the interactions of N-terminal cMyBP-C with myosin S2 and cardiac actin. (A–H) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and the 126 distal amino acids of human myosin S2. (A) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (B) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (C) pGBKT7 × pGADT7 (negative control). (D) pGBKT7 × pGADT7-S2 (negative control). (E) pGBKT7-C1C2WT × pGADT7 (negative control). (F) pGBKT7-C1C2E258K × pGADT7 (negative control). (G) pGBKT7-C1C2WT × pGADT7-S2 (interaction of interest). (H) pGBKT7-C1C2E258K × pGADT7-S2 (interaction of interest). (I–N) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and α cardiac actin (ACTC1), as well as their appropriate control interaction tests. (I) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (J) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (K) pGBKT7 × pGADT7-ACTC1 (negative control). (L) pGBKT7-53 × pGADT7-ACTC1 (negative control). (M) pGBKT7-C1C2WT × pGADT7-ACTC1 (interaction of interest). (N) pGBKT7-C1C2E258K × pGADT7-ACTC1 (interaction of interest). Yeast were grown on minimal selection media lacking leucine, tryptophan, histidine, and adenine. (O) Quantitative β-galactosidase assay performed in diploid yeast containing pGBKT7-C1C2WT × pGADT7-S2 (blue bar) and pGBKT7-C1C2E258K × pGADT7-S2 (red bar). n = 3. Error bars indicate SEM.
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fig6: Effect of the E258K mutation on the interactions of N-terminal cMyBP-C with myosin S2 and cardiac actin. (A–H) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and the 126 distal amino acids of human myosin S2. (A) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (B) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (C) pGBKT7 × pGADT7 (negative control). (D) pGBKT7 × pGADT7-S2 (negative control). (E) pGBKT7-C1C2WT × pGADT7 (negative control). (F) pGBKT7-C1C2E258K × pGADT7 (negative control). (G) pGBKT7-C1C2WT × pGADT7-S2 (interaction of interest). (H) pGBKT7-C1C2E258K × pGADT7-S2 (interaction of interest). (I–N) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and α cardiac actin (ACTC1), as well as their appropriate control interaction tests. (I) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (J) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (K) pGBKT7 × pGADT7-ACTC1 (negative control). (L) pGBKT7-53 × pGADT7-ACTC1 (negative control). (M) pGBKT7-C1C2WT × pGADT7-ACTC1 (interaction of interest). (N) pGBKT7-C1C2E258K × pGADT7-ACTC1 (interaction of interest). Yeast were grown on minimal selection media lacking leucine, tryptophan, histidine, and adenine. (O) Quantitative β-galactosidase assay performed in diploid yeast containing pGBKT7-C1C2WT × pGADT7-S2 (blue bar) and pGBKT7-C1C2E258K × pGADT7-S2 (red bar). n = 3. Error bars indicate SEM.
Mentions: The effect of the E258K missense mutation on the interaction between N-terminal cMyBP-C and myosin S2 was tested using a Y2H interaction assay. In these tests, either WT human C1C2 (C1C2WT) or human C1C2 into which the E258K missense mutation was engineered (C1C2E258K) were fused to the GAL4-DNA binding domain (C1C2WT/C1C2E258K-BD). The 126 distal amino acids of myosin S2 were also expressed as a fusion peptide with the GAL4 activation domain (S2-AD). Additionally, as myosin S2 is known to form coiled-coil dimers (Blankenfeldt et al., 2006), we also expressed an S2-BD fusion peptide. Coexpression of BD and AD fusion peptides was achieved through yeast mating, and selection for interaction between fusion peptides was assessed by the ability of yeast to grow on minimal selection media lacking leucine, tryptophan, histidine, and adenine (QDO). Robust growth, resulting from activation of the HIS3 and ADE2 nutritional reporter genes, was noted when positive control 53-BD and T-AD fusion peptides were expressed (Fig. 6 A). Additionally, consistent with the known dimerization of myosin S2 (Blankenfeldt et al., 2006), coexpression of S2-BD and S2-AD also resulted in growth (Fig. 6 B). Coexpression of unfused BD and AD peptides did not result in reporter gene activation, as indicated by lack of growth on QDO selection media (Fig. 6 C). Coexpression of unfused BD and S2-AD, or C1C2WT/C1C2E258K-BD and AD, did not result in growth on QDO selection media (Fig. 6, D–F), which suggests that S2-AD, C1C2WT-BD, and C1C2E258K-BD do not auto-activate expression of nutritional reporter genes. Coexpression of C1C2WT-BD and S2-AD fusion peptides resulted in robust growth on QDO selection media, which indicates a strong interaction between C1C2WT and S2, resulting in robust activation of the HIS3 and ADE2 nutritional reporter genes (Fig. 6 G). In contrast, no growth was observed when C1C2E258K-BD and S2-AD were coexpressed (Fig. 6 H), which suggests that the C1C2E258K is unable to interact with myosin-S2.

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