Research ArticleBIOPHYSICS

Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling

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Science Advances  20 Feb 2015:
Vol. 1, no. 1, e1400205
DOI: 10.1126/sciadv.1400205
  • Fig. 1 Sarcomeric organization and MyBP-C.

    (A) Cardiac muscle sarcomere with interdigitating thick and thin filaments. MyBP-C localized to the C-zone, whereas the ryanodine receptors are localized in puncta (CRUs) along the Z-lines, forming the boundaries of each sarcomere. (B) Schematic diagram of cardiac MyBP-C’s Ig-like (oval) and fibronectin-like (rectangular) domains with four phosphorylation sites (P) in the M-domain and C0C3 fragment (dashed box) used in the 3D EM and in vitro motility experiments. (C) Negatively stained EM image of a sarcomere within a mouse ventricular myocyte labeled with antibodies to MyBP-C. (D) Two-color dSTORM super-resolution image of sarcomeres as in (C), labeled with Alexa Fluor–conjugated antibodies to MyBP-C and ryanodine receptors.

  • Fig. 2 Sarcomeric calcium gradients in ventricular myocytes.

    (A) Single cardiac myocyte labeled with a lipophilic wheat germ agglutinin conjugated to Alexa Fluor 594 to identify t-tubule CRUs. Yellow box indicates area expanded in (B). (B) Line of yellow arrow represents confocal line scan across several CRUs. Only confirmed CRUs and their adjacent M-lines were scanned. Yellow boxes represent 5-pixel regions of interest (ROIs) (500 nm) over the Z- and M-line regions. (C) Rising phase of the calcium transient ([Ca2+]i) from scanned region depicted in (B) immediately after stimulation of ventricular myocyte loaded with fluorescent calcium indicator. Certain M-lines are flanked by two adjacent CRUs that release calcium immediately upon stimulation (for example, M2 and M3), whereas others are adjacent to only one CRU that is triggered (for example, M1 and M4). The inset depicts the spatial profile of [Ca2+]i 13 ms after stimulation (vertical yellow line). (D) Average [Ca2+]i transient ± error bars (SEM) rising phase time course from 59 Z-line CRUs (green, n = 12 cells) and their adjacent (adj.) M-lines when flanked by two triggered CRUs (blue, n = 59 sites, 12 cells) and a single triggered CRU (red, n = 53 sites, 12 cells). (E) Z- to M-line [Ca2+]i gradient calculated by subtracting M-line from Z-line traces in (D). (F) Z- to M-line [Ca2+]i gradient ± SEM 5, 10, 15, and 20 ms after stimulation in sarcomeres. *P < 0.01, **P < 0.001, one-sample t test.

  • Fig. 3 The effects of MyBP-C on native thin filament activation.

    (A) Illustration of a thin-filament shard traveling over one-half of a native thick filament, as in TIRFM experiments. (B) Displacement-time plots for five thin filaments on wild-type thick filaments at pCa 5 demonstrating two phases of velocity (black, fast; blue, slow). (C) Trajectory length ± SEM–pCa and percent trajectories with biphasic velocities (as in B)–pCa histograms for thin filaments on wild-type thick filaments. (D) As in (B) for five filaments at pCa 7 demonstrating movement only within the thick filament C-zone. (E) Fraction of thin filaments moving ± SEM–pCa plots for thin filament motion in the absence of (black) and presence of wild-type (blue) and λ-phosphatase–treated MyBP-C (red). Data fitted with sigmoidal dose-response curves with differing pCa50 (P < 0.001, extra sum-squares F test). Dephos, dephosphorylated with λ-phosphatase. (F and G) 3D structure of native thin filaments with tropomyosin (Tm) in the (F) “blocked” position in the absence of and (G) “closed” position in the presence of calcium. Atomic structures of actin with Tm in the “blocked” position (red) and “closed” position (green) were fitted to each 3D structure. (H and I) 3D structure of native thin filaments decorated with MyBP-C N-terminal fragments in the absence of calcium. Position of tropomyosin with addition of (H) C0C3 and (I) phosphomimetic C0C3-4D fragments. The 3D structure fitted with atomic structures showed Tm’s movement in thin filament decorated with C0C3 from “blocked” to “closed” position. In contrast, the fitted structures for the thin filament decorated with C0C3-4D showed only partial movement of Tm toward the “closed” position.

  • Fig. 4 Effect of [Ca2+]i on thin filament activation in the absence of MyBP-C near the Z-line and presence of MyBP-C within the C-zone.

    (A) Fraction of thin filaments moving ± SEM–pCa plots within the Z- and M-line regions of the sarcomere in the (black) absence and (blue) presence of wild-type (WT) MyBP-C. [Ca2+]i in the Z- and M-line regions 10 ms after cardiac excitation indicated (green dashed lines). (B to D) Model generated estimates of thin filament activation after calcium release. Thin filament activation–time plot generated for regions of the thin filaments near the Z-lines (black) and within the C-zone, near the center of the sarcomere. (B) Model assumes that the C-zone lacks MyBP-C and thus predicts that thin filament activation in the C-zone (gray) would lag behind the Z-line. (C) Model assumes normally phosphorylated wild-type MyBP-C in C-zone (blue) and predicts that C-zone activation level would no longer lag behind the Z-line. (D) Model assumes dephosphorylated MyBP-C within C-zone (red), resulting in a sensitized C-zone that would contribute to significant activation even during relaxation.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/1/1/e1400205/DC1

    Materials and Methods

    Fig. S1. Two-color dSTORM super-resolution imaging of MyBP-C and ryanodine receptors (RyR2).

    Fig. S2. Treatment with the contractile inhibitor blebbistatin does not obscure sarcomeric calcium gradients.

    Fig. S3. Calcium-dependent regulation of native thin filament motion.

    Fig. S4. Histograms for thin filaments on λ-phosphatase–treated thick filaments containing dephosphorylated MyBP-C (trajectory length ± SEM and percent trajectories with biphasic velocities versus pCa).

    Fig. S5. 3D reconstructions of low-Ca2+ native thin filaments decorated with C0C3 (actin/C0C3, 1:3) in which the four phosphorylatable serines have been replaced with alanines (fully dephosphorylated, C0C3-4A) or with aspartic acid residues (phosphomimetic, C0C3-4D).

    Fig. S6. Transverse sections of the respective low-Ca2+ reconstructions in fig. S5, in which native thin filaments have been decorated with C0C3-4A and C0C3-4D.

    Fig. S7. Phosphorylation of MyBP-C N-terminal domains modulates their ability to activate native thin filament motion in the in vitro motility assay on a bed of monomeric myosin.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Two-color dSTORM super-resolution imaging of MyBP-C and ryanodine receptors (RyR2).
    • Fig. S2. Treatment with the contractile inhibitor blebbistatin does not obscure sarcomeric calcium gradients.
    • Fig. S3. Calcium-dependent regulation of native thin filament motion.
    • Fig. S4. Histograms for thin filaments on λ-phosphatase–treated thick filaments containing dephosphorylated MyBP-C (trajectory length ± SEM and percent trajectories with biphasic velocities versus pCa).
    • Fig. S5. 3D reconstructions of low-Ca2+ native thin filaments decorated with C0C3 (actin/C0C3,1:3) in which the four phosphorylatable serines have been replaced with alanines (fully dephosphorylated, C0C3-4A) or with aspartic acid residues (phosphomimetic, C0C3-4D).
    • Fig. S6. Transverse sections of the respective low-Ca2+ reconstructions in fig. S5, in which native thin filaments have been decorated with C0C3-4A and C0C3-4D.
    • Fig. S7. Phosphorylation of MyBP-C N-terminal domains modulates their ability to activate native thin filament motion in the in vitro motility assay on a bed of monomeric myosin.

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