Spatially resolving how cMyBP-C phosphorylation and haploinsufficiency in porcine and human myofibrils affect β-cardiac myosin activity

Figures

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  Figure S1.. Residual and cumulative frequency graphs for determining the optimal number of fitted exponentials. (Left) In this example, untreated all zones data are shown (squares) fitted to an increasing number of exponentials (dashed lines). (Right) For the corresponding fit, we show the residuals (fitted values minus data values). A larger amplitude on the y axis means a poorer fit, and it can be seen that the amplitudes decrease as the number of fitted exponentials increases. However, a clear deviation from the data is seen (arrows on left graphs) and the systematic noise (arrows on right graphs) remains until three exponentials are used. In the latter case, the maximum noise after fitting is <0.5%.

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  Figure 1.. Single-molecule tracking of fluorescently labeled ATP turnover in porcine cardiac myofibrils. (A) Schematic representation of a single sarcomere. P-, C-, and D-zones of the thick filament are labeled accordingly. Alexa 488–labeled α-actinin is denoted in magenta, while Cy3-ATP binding to the thick filament is represented as cyan circles. (B) Enlarged section of a representative porcine cardiac LV myofibril as observed following the overlay of 561-nm illuminated Cy3-ATP (cyan) and 488-nm illuminated α-actinin–containing Z-disks (magenta). (C) Distribution of sarcomere length for untreated porcine myofibrils with a mean of 1.82 µm ± 0.04 SD (N = 3 pigs, 647 sarcomeres) at 21°C. Distribution of sarcomere lengths for PKA-treated samples can be found in Fig. S2. The red dotted line indicates the threshold for the smallest sarcomere that was analyzed for all datasets. (D) Cumulative residence time histograms showing the distribution of ATP attachment event durations in untreated porcine cardiac myofibrils (circles; N = 3 pigs, n = 3,638 events) and PKA-treated porcine cardiac myofibrils (squares; N = 3 pigs, n = 1,463 events). Dashed lines show triple-exponential fits. Note the…

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  Figure 2.. Subsarcomeric resolution of myosin activity reveals cMyBP-C phosphorylation reduces SRX in the P- & C-zones, compensated for by an increase in the D-zone. Cy3-ATP turnover rates were calculated as an average across all zones of the sarcomere, as well as individually for the P-, C-, and D-zones. The Cy3-ATP turnover rates per zone were used to calculate the relative population amplitude of SRX myosin heads based on triple-exponential fits. Data were collected from untreated porcine myofibrils (N = 3 pigs, n = 3,638 events) or myofibrils treated with 62.5 U/μl of PKA (N = 3 pigs, n = 1,463 events). Data are shown as the mean and 95% CI errors. Significance was calculated using bootstrap analysis and indicated by *. Table 1 provides the values used in the graph. PKA phosphorylation affects the distribution of SRX and DRX in porcine myofibrils.

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  Figure 3.. Quantification of cMyBP-C phosphorylation after PKA treatment of porcine cardiac myofibrils. Protein was isolated from PKA-treated myofibrils and compared with untreated myofibrils. (A) Samples were run on a 4–20% gradient SDS-PAGE gel and stained with Pro-Q Diamond Phosphoprotein Gel Stain (right) followed by Coomassie blue stain (left) to quantify total protein. (B) Densitometry analysis was performed on the cMyBP-C Pro-Q bands normalized to the same band in the Coomassie gel, and cMyBP-C treatment with PKA results in a ∼2.5-fold increase in phosphorylation relative to the untreated group (N = 1, n = 2). Data shown are the mean ± SEM. Significance was calculated with a one-tailed t test, *P = 0.025. Source data are available for this figure: SourceData F3.

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  Figure 4.. MYBPC3-c.772G>A mutation alters regulation of the thick filament by accelerating ATP turnover in the C-zone of the sarcomere. (A) Representative image of a human cardiac myofibril illustrating 561-nm illuminated Cy3-ATP (cyan) and 488-nm illuminated α-actinin–containing Z-disks (magenta). (B) Normalized cumulative residence time histogram for all ATP binding events across the entire thick filament for mutation-negative control (squares; N = 2, n = 1,161 events) and MYBPC3-c.772G>A human ventricular myofibrils (circles; N = 1, n = 1,813 events). Dashed lines describe the cumulative exponential fits for both datasets. Cumulative frequency, y axis, is plotted on a logarithmic scale. (C) Percentage of myosin heads quantified to be in the SRX biochemical state in mutation-negative control human myectomy myofibrils and HCM MYBPC3-c.772G>A human myectomy myofibrils. The plot includes all zones and subsarcomeric zones of the thick filament. Data are shown as the mean and 95% CI errors, with significance calculated using bootstrap analysis and indicated by *. Table 2 provides the values used in the graph.

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  Figure S2.. Distribution of sarcomere lengths for PKA-treated porcine cardiac myofibrils. Histogram of the distribution of sarcomere lengths for PKA-treated myofibrils fitted to a Gaussian distribution (dotted line) provides a mean and SD 1,766 ± 38.5 nm.