Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow

Abstract

Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34-59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 μm/s at 0.1 μM, while a recombinant peptide that excludes residues 21-59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.

Keywords: MYBPC1; actin; contractility; skeletal muscle.

Figure 1.

sMyBP-C is a complex subfamily of proteins. A) Schematic representation of the 5 variants of sMyBP-C expressed in mouse skeletal muscle: v1, v2, v3, v4, and v002. sMyBP-C consists of tandem Ig (open ovals) and Fn-III (gray ovals) domains with the Pro/Ala-rich region and M motif shown as dark gray and open horizontal rectangles, respectively. Alternatively spliced regions are indicated by colored vertical lines. Point mutations linked to DA-1 myopathy are highlighted in yellow. B) Variant distribution of sMyBP-C in mouse FDB, TA, and soleus muscles is shown as a percentage of total sMyBP-C as calculated by averaging the percentage of variant expression obtained from RT-PCR. C) Wild-type and mutant DA-1 recombinant proteins used in this study containing the NH₂ terminus, domain C7, and COOH terminus.

Figure 2.

The NH₂ terminus of sMyBP-C differentially modulates actomyosin interactions in a variant-specific manner. A) Equivalent amounts of NH₂-terminal sMyBP-C wild-type fusion constructs, including NH_2aa1–285, NH₂Δ34–59_aa1–285, and NH₂Δ21–59_aa1–285, were bound to glutathione matrices, incubated with protein homogenates prepared from adult mouse FDB muscle, and examined for their ability to retain endogenous myosin and actin. Total lysates were also included in the Western blot. Coomassie blue staining of the input proteins is shown in the bottom panel to indicate equal loading. B, C) Far-Western blot (B) and slot blot (C) assays were performed to assay direct interactions between the NH₂-terminal wild-type sMyBP-C constructs and myosin or actin. For the far-Western blot assays, actin (1 μM) and the HMM portion of myosin (1 μM) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with equivalent amounts of sMyBP-C NH₂-terminal proteins (NH_2aa1–285, NH₂Δ34–59_aa1–285, and NH₂Δ21–59_aa1–285). For the slot blot assays, actin and HMM (500 ng) were blotted onto nitrocellulose and incubated with equivalent amounts of sMyBP-C NH₂-terminal proteins. Positive interactions in both assays were determined by immunoprobing for the GST moiety of recombinant sMyBP-C proteins. D) Relative binding of recombinant NH₂-terminal proteins to HMM (solid bars) and actin (open bars) was quantified by densitometry of the immunoreactive bands detected in the far-Western blots; percentage (%) binding is reported relative to NH_2aa1–285, which was set to 100%. Percentages were calculated from 3 independent experiments. Error bars = sd (t test, P<0.01). E) In vitro motility assays were used to measure the sliding velocity of actin filaments over an HMM-coated surface in the presence and absence of NH₂-terminal sMyBP-C wild-type constructs (NH_2aa1–285, NH₂Δ34–59_aa1–285, NH₂Δ21–59_aa1–285). sMyBP-C constructs were added to the final motility buffer at various concentrations, including 0, 0.1, 0.5, 1, 2.5, and 5 μM. F) Bar graphs depicting the percentage of actin filaments available at the gliding surface 75 s following addition of the indicated proteins. Insets: representative snapshots of the gliding surface 75 s following the addition of the wild-type proteins at 1 μM. Error bars = sem. *P < 0.01 vs. sham treatment; t test.

Figure 3.

The NH₂ terminus of sMyBP-C fails to modulate actomyosin interaction and sliding in the presence of the W236R DA-1 mutation. A) GST pulldown assays were used to assess the ability of the sMyBP-C NH₂-terminal variants (NH₂W236R_aa1–285, NH₂Δ34–59W236R_aa1–285, and NH₂Δ21–59W236R_aa1–285) to interact with endogenous myosin and actin in the presence of the W236R DA-1 mutation. B) In vitro motility assays were used to measure the sliding velocity of actin filaments over a surface coated with HMM in the presence or absence of the NH₂-terminal variants containing the W236R mutation (NH₂ W236R_aa1–285, NH₂Δ34–59W236R_aa1–285, and NH₂Δ21–59W236R_aa1–285), as described in Fig. 2. Bar graphs indicate the percentage change in the sliding velocity of actin filaments normalized to sham conditions. C) Percentage ± se of available actin at the gliding surface 75 s following addition of the mutant NH₂-terminal proteins. Insets: representative snapshots of the gliding surface 75 s following the addition of the mutant proteins at 1 μM. *P < 0.01 vs. sham treatment, ^#P < 0.01 vs wild-type proteins; t test.

Figure 4.

Alternative splicing within the C7 domain has no effect on actomyosin interactions; however, alternative splicing within the C10 domain affects its ability to interact with thick filaments. A, D) C7 (A) and C10 (D) domain constructs of sMyBP-C (C7_aa720–823 and C7Δ763–780_aa720–823, A; and C10_{aa1016–1127} and C10Δ1107–1127_{aa1016–1127}, D) were assessed for their ability to interact with endogenous myosin and actin in GST pulldown assays. E, F) Far-Western blot (E) and slot blot (F) assays were also performed with the C10 domain constructs (C10_{aa1016–1127} and C10Δ1107–1127_{aa1016–1127}) to assay for direct interactions with actin and HMM. Although the COOH terminus of sMyBP-C is complexed with myosin and actin filaments in a variant-specific manner (D), it is not able to directly interact with HMM or actin (B). B, C, G, H) In vitro motility assays were used to measure the sliding velocity of actin filaments over a surface coated with HMM in the presence or absence of the C7 (C7_aa720–823 and C7Δ763–780_aa720–823; B, C) and C10 (C10_{aa1016–1127} and C10Δ1107–1127_{aa1016–1127}; G, H) domain proteins. B, G) Bar graphs indicate the percentage change in the sliding velocity of actin filaments normalized to sham conditions. C, H) Bar graphs depict the percentage ± se of available actin at the gliding surface 75 s following addition of the sMyBP-C proteins. Insets: representative snapshots of the gliding surface 75 s following the addition of the proteins at 1 μM. *P < 0.01 vs. sham treatment; t test.

Figure 5.

COOH terminus of sMyBP-C mediates binding to thick filaments but fails to support binding in the presence of the Y856H DA-1 mutation. A) GST pulldown assays were used to assess the ability of the extended COOH-terminal constructs of sMyBP-C (C8–C10_aa854–1127 and C8–C10Δ1107–1127_aa854–1127) to interact with endogenous myosin and actin. B) COOH-terminal constructs of sMyBP-C carrying the Y856H DA-1 mutation (C8–C10Y856H_aa854–1127 and C8–C10Δ1107–1127 Y856H_aa854–1127) were assessed for their ability to interact with endogenous myosin and actin. The presence of the Y856H DA-1 mutation abolishes all binding activity.

Figure 6.

sMyBP-C modulates the formation of actomyosin cross-bridges via its NH₂ terminus in a variant-specific manner, and binds to thick filaments through its COOH terminus. Both regulatory properties are abolished by single-point mutations linked to DA-1 myopathy. A) Proposed model depicting the different roles of the sMyBP-C variants within the sarcomeric A and M bands. B) Introduction of the single DA-1 point mutations within the NH₂ and COOH termini of MYBPC1 (W236R and Y856H, respectively) abolishes the ability of sMyBP-C to regulate actomyosin binding and sliding.