Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner

Figures

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  Figure 1. Schematic diagram demonstrates the association between myosin and cMyBP-C.A, schematic diagram of myosin and cMyBP-C with their interaction domains. Myosin has been divided into N-terminal heavy meromyosin (HMM), which is further divided into S1 and S2 and the C-terminal light meromyosin (LMM). The long helical S2 is marked with proximal region of S2 (126 amino acids), three HCM-causing mutations, R870H, E924K, and E930Δ, and interacting regions with C0-C2 of cMyBP-C. The S1 head of myosin with essential light chain (ELC) and regulator light chain (RLC) proteins. B, in cMyBP-C, the diagram further displays cardiac-specific C0 domain, phosphorylation M domain, and a small inset in C5 domain (green-coded), proline-alanine (P/A)-rich region, interaction region with RLC, immunoglobulin domains from C1-C5, C8, and C10, and fibronectin domains, C6, C7, and C9. The phosphorylated M domain includes three major phosphorylation motifs (Ser-273, Ser-282, and Ser-302) that dynamically regulate the interaction with S2 region of myosin. C, schematic diagram of C0-C2 domains with WT, phospho-ablation (AAA), and phospho-mimetic (DDD), in which the phosphorylation sites Ser-273, Ser-282, and…

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  Figure 2. Mutant hS2 proteins display increased affinity by decreasing Kdto phosphorylated hC0-C2 protein by SPBA. The binding curve between hS2Wt and hC0-C2 (solid green), hS2Wt and hC0-C2p (dashed green), (A) hS2R870H and hC0-C2 (solid blue), hS2R870H and hC0-C2p (dashed blue), (B) hS2E924K and hC0-C2 (solid orange), hS2E924K and hC0-C2p (dashed orange), (C) hS2E930Δand hC0-C2 (solid red), and hS2E930Δand hC0-C2p (dashed red). D, relative maximal binding capacity (Bmax) was determined for each listed combination and compared against hS2Wt and C0-C2 proteins. E, binding affinity or dissociation constant, Kd, compared against hS2Wt and hC0-C2 proteins. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison test with single pooled variance. n = 3 with triplicates for each concentration. Data are expressed as the mean ± SEM. See Table 1 for analysis of main factors and interactions. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus hS2Wt/hC0-C2 controls. hS2E924K, human recombinant proximal S2 protein with E924K mutation; hS2E930Δ, human recombinant proximal S2 protein with E930Δ mutation; hS2R870H, human recombinant proximal S2 protein with R870H mutation; hS2Wt,…

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  Figure 3. Increased affinity of mutant hS2 proteins to mC0-C2DDDprotein by SPBA.A, relative Bmax and B, relative Kd for mC0-C2Wt (green), mC0-C2AAA (blue), and mC0-C2DDD (red) treated with hS2Wt (solid line) and S2R870H (dashed line). C, relative Bmax and D, relative Kd for mC0-C2Wt (green), mC0-C2AAA (blue), and mC0-C2DDD (red) treated with hS2Wt (solid line) and hS2E924K (dashed line). E, relative Bmax and F, relative Kd for mC0-C2Wt (green), mC0-C2AAA (blue), and mC0-C2DDD (red) treated with hS2Wt (solid line) and hS2E930Δ (dashed line). Data were fit to Michaelis–Menten binding fit, and one-way ANOVA with Tukey’s pooled multiple variance test were used to calculate the significance between the Kd values. n = 3 with replicates of three for each n value. Data are expressed as the mean ± SEM. See Table 2 for analysis of main factors and interactions. ∗p < 0.05, ∗∗p < 0.01 versus hS2Wt to mC0-C2Wt control. AAA, phospho-ablated cMyBP-C or phospho-ablation; DDD, phospho-mimetic cMyBP-C; hS2E930Δ, human recombinant proximal S2 protein with E930Δ mutation; hS2Wt, human recombinant proximal S2 WT protein; Kd, dissociation constant; mC0-C2, mouse recombinant C0-C2 protein; SPBA, solid-phase…

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  Figure 4. Mutant hS2 displays a higher rate of heat change upon binding to phosphorylated hC0-C2 protein by ITC assay.A, representative traces for change in heat observed upon titrating hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and E930Δ (red) proteins to hC0-C2 proteins. B, representative traces for change in heat observed upon titrating hS2Wt (green dashed), hS2R870H (blue dashed), hS2E924K (orange dashed), and hS2E930Δ (red dashed) proteins to hC0-C2p proteins. C, sigmoidal curve for titration of hS2Wt to hC0-C2 yields the dissociation constant (1/slope) and stoichiometry of the reaction. Titration of hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins against dephosphorylated hC0-C2 proteins. D, sigmoidal curve for titration of hS2Wt (green dashed), hS2R870H (blue dashed), hS2E924K (orange dashed), and hS2E930Δ (red dashed) proteins to phosphorylated hC0-C2 protein. E, change in affinity for hS2Wt to hC0-C2 (solid) and hC0-C2p (half solid) proteins. F, change in slope for hS2Wt proteins to hC0-C2 (solid) and hC0-C2p (half solids) protein calculated by four-parameter logistic curve. Statistical analyses were performed by one-way ANOVA with Tukey’s…

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  Figure 5. Rate of force redevelopment (ktr) was enhanced by mutant S2 proteins that were permeabilized in fibersPKA.A, raw ktr traces for nontransgenic (NTg) fibers treated with all four S2 proteins with a concentration range from 9 to 45 μM, which determined that the 9 μM concentration of S2 proteins was sufficient to differentiate the effect of hS2Wt to all mutant hS2 proteins. B, raw ktr traces and C, values for fiberscontrol that were untreated (black) and treated with 9 μM hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins. D, raw ktr traces and E, values for fibersPKA that were untreated (black) and treated with 9 μM hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins. F, ktr traces and G, values for fibersλ-phosphatase that were untreated (black) and treated with 9 μM hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison test with single pooled variance. n = 5 fibers/3 mice (12 weeks old, FVB/N, mixed sex) were used at submaximal pCa 5.7 and sarcomere length 2.0 μM in the study. Data are expressed as the mean ± SEM.…

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  Figure 6. Rate of force redevelopment (ktr) was elevated by mutant hS2 proteins in fibers expressing cMyBP-C DDD in transgenic (Tg) protein.A, raw ktr traces and B, values for fibers that expressed cMyBP-C AAA without treatment (black) and fibers treated with 9 μM hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins. C, raw ktr traces and D, values for fibers that expressed cMyBP-C DDD without treatment (black) and treated with 9 μM hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins. Statistical analyses were performed by one-way ANOVA, and Tukey’s pooled multiple variance test was used to calculate the significance between the ktr values. n = 5 fibers/3 mice (12 weeks old, FVB/N, mixed sex), where n is each fiber treated with a single concentration of myosin S2 protein and measured ktr at submaximal pCa 5.7 and sarcomere length 2.0 μM. Data are expressed as the mean ± SEM. See Table 5 for analysis of main factors and interactions. ∗∗p < 0.01, and ∗∗∗p < 0.001 versus untreated controls. AAA, phospho-ablated cMyBP-C or phospho-ablation; cMyBP-C, cardiac myosin-binding protein-C; DDD, phospho-mimetic cMyBP-C; hS2E924K, human recombinant…

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  Figure 7. Mutant S2 proteins were unable to keep coiled coil structure intact under high-salt buffer conditions.A, the α-helical coiled coil spectra for hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins in the absence of urea. B, the ratio of ellipticity (222/208) for S2 in a low-salt buffer with no change in coiled coil structure. C, the effect of 25% or 2 M urea on α-helical coiled coil spectra for hS2Wt (green), hS2R870H (blue), hS2E924K (orange), and hS2E930Δ (red) proteins. D, the ratio of ellipticity (222/208) for hS2 in 2 M urea buffer with a significant change in coiled coil structure to independent α-helices. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparison test with single pooled variance (n = 3). Data are expressed as the mean ± SEM. See Table 6 for analysis of main factors and interactions. ∗∗p < 0.01, and ∗∗∗p < 0.001 versus hS2Wt controls. hS2, human cardiac myosin S2; hS2E924K, human recombinant proximal S2 protein with E924K mutation; hS2E930Δ, human recombinant proximal S2 protein with E930Δ mutation; hS2R870H, human recombinant proximal S2 protein with R870H mutation; hS2Wt, human recombinant proximal S2 WT…