Myosin-binding protein C stabilizes, but is not the sole determinant of SRX myosin in cardiac muscle

Figures

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  Figure 1.. Structure and regulation of myosin, cMyBP-C, and the sarcomere. (A) Myosin’s biochemical states. (B) Cartoon of the sarcomere, showing thick and thin filaments, delineation of C-, D-, P-, and bare zones, as well as myosin and cMyBP-C (yellow). (C) Domain structure of cMyBP-C, truncated variant (ΔC0C1f), and null (t/t).

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  Figure 2.. ATP binding events and overall lifetime distributions. (A) Kymograph showing multiple fluorescent nucleotide binding events (white streaks). (B) Overall lifetime distributions for WT, t/t, and ΔC0C1f transgenic. Inset shows the proportions of SRX and DRX events. Error bars denote the standard error of the fit. Both t/t and ΔC0C1f lifetime distributions are significantly different than WT (P = 0.00000002 and P = 0.0002 by KS test, respectively). * denotes P < 0.05. WT events are combined from 19 technical replicates taken from 6 biological replicates. t/t events are combined from 16 technical replicates taken from 5 biological replicates. ΔC0C1f events are combined from 10 technical replicates taken from 3 biological replicates.

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  Figure 3.. Event localizations and distributions. (A) Top: Combined fluorescence image showing anti-myomesin (red) and BODIPY-ATP (green), combined from 100 s of acquisition. Bottom: X-Y plot of resulting localization data for the same interval. (B) Scatterplot showing distance from M-line and lifetime for each localized event. WT (black), ΔC0C1f (green), and t/t (red). (C) Histograms showing event frequency as a function of distance from the M-line. (D) Fitting the double-exponential model within a sliding window shows a gradient in SRX abundance for all three mouse models. Linear regressions were fitted through data in the bare and P-zones and separately through the C- and D-zones for all three models. Fit parameters are reported in Table S1. (E) Fractions of SRX/DRX events for events when binned by zone with lifetime distribution plots for these results in Fig. S1. * denotes P < 0.05 by KS test. Error bars denote the standard error of the fit. The C-zones of both t/t and ΔC0C1f were significantly different than WT (P = 0.02 and P = 0.01, respectively), as were the D-zones (P = 0.0007 and P = 0.04). D-zone event distributions were significantly different from the C-zone for WT (P = 0.04),…

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  Figure S1.. Zone-specific lifetime distributions and fits. Fluorescent nucleotide binding events were parsed into P-, C-, and D-zones according to distance from the M-line (see text and Fig. 3 E). Lifetime distributions were then fit with a double exponential model, as described in Materials and methods.

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  Figure S2.. RLC phosphorylation. Top: SDS-PAGE gel was run on myofibril preparations from three mouse models (WT, t/t, and ΔC0C1f) under three loading conditions. The gel was then stained with Pro-Q Diamond (phosphoprotein stain), followed by SYPRO Ruby (total protein stain). Both stains were performed according to the manufacturer’s instructions (Molecular Probes/Invitrogen) and imaged in a Bio-Rad Chemidoc (Bio-Rad Products). Bottom: Fluorescence intensity ratios were determined using Gel Analysis Tools in ImageJ and normalized according to the mean intensity ratio for WT according to (Pro-Q Intensity:SYPRO Intensity)/(WT Pro-Q Intensity:WT SYPRO Intensity). Error bars denote SD. Values shown in the bar chart represent six technical replicates from two biological replicates for each condition. Source data are available for this figure: SourceData FS2.