RLC phosphorylation amplifies Ca2+ sensitivity of force in myocardium from cMyBP-C knockout mice

Figures

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  Figure 1.. cMyBP-C loss contributes to interstitial fibrosis. (A and B) Myocardial sections from WT (A) and cMyBP-C KO (B) mice were stained with hematoxylin and eosin to characterize cellular organization and fibrosis. (C) Fibrosis levels were further quantified utilizing a hydroxyproline assay. Averages represent mean ± SEM, with individual data points shown for each heart. Individual data points represent the average of three technical replicates from five biological replicates per genotype.

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  Figure 2.. RLC phosphorylation increases Ca2+ sensitivity of the stress–pCa relationships. (A and B) Ca2+-activated stress–pCa relationships for myocardial strips from WT (A) and cMyBP-C KO (B) mice with AP or MLCK treatment (mean ± SEM). Lines represent three-parameter Hill fits to the average stress–pCa data. (C) Relative RLC phosphorylation quantification by SYPRO Ruby and Pro-Q Diamond. Inset: Representative gel image for AP and MLCK treatment. The number of biological and technical replicates is listed in Table 1. Source data are available for this figure: SourceData F2.

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  Figure 3.. RLC phosphorylation influences maximal force production and Ca2+ sensitivity. (A) Normalized tension plotted against pCa shows the relative differences in Ca2+ sensitivity of contraction between all groups. (B and C) Fmax, or maximal stress (fixed effects: genotype: P = 0.015; treatment: P = 0.001; genotype*treatment: P = 0.003; B), and pCa50 values from the three-parameter Hill fits (fixed effects: genotype: P = 0.015; treatment: P < 0.001; genotype*treatment: P = 0.005; C; Fig. 2 and Table 1). Averages represent mean ± SEM, with individual data points shown where applicable. The number of biological and technical replicates is listed in Table 1.

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  Figure 4.. RLC phosphorylation does not alter viscoelastic stiffness under relaxed conditions (pCa 8.0). (A–D) Elastic moduli (A and B) and viscous moduli (C and D) values plotted against frequency for AP and MLCK treated strips from WT and cMyBP-C KO mice (mean ± SEM). The number of biological and technical replicates is listed in Table 1.

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  Figure 5.. RLC phosphorylation and cMyBP-C loss alter viscoelasticity and crossbridge kinetics under maximally activated conditions (pCa 4.8). (A–D) Elastic moduli (A and B) and viscous moduli (C and D) values plotted against frequency for AP- and MLCK-treated strips from WT and cMyBP-C KO mice. (E and F) Frequency of the minimum viscous modulus represents a relative measure of crossbridge recruitment rate (fixed effects: genotype: P = 0.015; treatment: P = 0.001; genotype*treatment: P = 0.012; E) and frequency of the maximal viscous modulus represents a relative measure of the crossbridge detachment rate (fixed effects: genotype: P = 0.011; treatment: P = 0.001; genotype*treatment: P < 0.001; F). Averages represent mean ± SEM, with individual data points shown for each fiber where applicable. The number of biological and technical replicates is listed in Table 1.