Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin

Figures

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  Figure 1.. The SRX state of myosin assessed in skinned PMs from 7–10-mo-old mouse models of hypertrophic (HCM-A57G) and restrictive (RCM-E143K) cardiomyopathy and in a near-physiological Δ43 ELC model compared with WT-ELC mice. (A) Fluorescence decay curves and simulated single-exponential curves of mant-ATP release. (B) Comparison of fluorescence decay curves for WT-ELC (11 mice, 5 M and 6 F), HCM-A57G (8 mice, 4 M and 4 F), Δ43 (10 mice, 5 M and 5 F), and RCM-E143K (7 mice, 4 M and 3 F) animals (left), redistribution of cross-bridges (in %) between the DRX (P1) state (middle) and the SRX (P2) state (right). Open symbols depict female mice, and closed symbols depict male mice. Data are mean ± SD; *, P < 0.05 for mutant versus WT-ELC; $, P < 0.05 for E143K versus A57G; and &, P < 0.05 for Δ43 versus A57G calculated with one-way ANOVA with Tukey’s multiple comparison test. Note that the percentage of DRX is increased in HCM-A57G compared with WT-ELC, Δ43, and RCM-E143K mice, and this increase is coupled with a decrease in percentage of SRX for HCM-A57G mice. (C) Myosin head redistribution between the DRX (P1) versus SRX (P2) states plotted as a function of genotype and sex. Data are mean ±…

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  Figure 2.. Force transients in intact PMs from 8–10-mo-old HCM-A57G (5 M and 3 F), Δ43 (3 M and 3 F), and RCM-E143K (3 M and 5 F) compared with WT-ELC (7 M and 4 F). The data for A57G and Δ43 animals were taken, with permission, from Sitbon et al. (2020). (A) Left: Normalized force transients for WT-ELC (blue), HCM-A57G (green), Δ43 (red), and RCM-E143K (black) mice. Right: Force (in millinewtons per square millimeter) was calculated as a ratio of peak force to physiological cross-sectional area (see Materials and methods) and plotted as a function of genotype and sex. (B) Time points t25, t50, and t75 depicting the time (in milliseconds) from peak force to 25%, 50%, and 75% baseline for male and female mice of all genotypes. Open symbols depict female mice, and closed symbols depict male mice. Data are mean ± SD of male or female mice analyzed by two-way ANOVA followed by Tukey’s or Sidak’s multiple comparison test. *, P < 0.05 for mutant versus WT; $, P < 0.05 for A57G versus E143K; and &, P < 0.05 for A57G versus Δ43.

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  Figure S1.. Calcium transients in intact PMs from HCM-A57G, RCM-E143K, and Δ43 versus WT-ELC hearts. Data for HCM-A57G and Δ43 were taken, with permission, from Sitbon et al. (2020). (A) Normalized calcium transients for HCM-A57G (green), RCM-E143K (black), Δ43 (red), and WT (blue) mice. (B) Time points t25, t50, and t75, depicting the time (in milliseconds) from peak [Ca2+] to 25%, 50%, and 75% baseline for all four genotypes. Open symbols depict female mice and closed symbols depict male mice. The hearts of n = 8 (5 M, 3 F) HCM-A57G, n = 6 (3 M, 3 F) Δ43, n = 8 (3 M, 5 F) RCM-E143K, and n = 15 (10 M, 5 F) WT-ELC mice were tested. Values are mean ± SD and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Significance was depicted as *, P < 0.05 for mutant versus WT-ELC; and &, P < 0.05 for RCM-E143K versus Δ43. Note delayed relaxation times for HCM-A57G (t50) and RCM-E143K (t25, t50, and t75) compared with WT-ELC mice.

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  Figure 3.. Myosin RLC, cMyBP-C, and TnI phosphorylation in ventricular CMFs purified from the hearts of WT, HCM-A57G, RCM-E143K, and Δ43 mice. The hearts of 4 M and 3 F (7.5–10-mo-old) WT-ELC, 3 M and 5 F (8–10-mo-old) A57G, 3 M and 3 F (6–9-mo-old) E143K, and 4 M and 2 F (7.5–10-mo-old) Δ43 mice were used. (A) SDS-PAGE and sarcomeric protein phosphorylation monitored by ProQ/Coomassie. (B) Quantification of myosin RLC, cMyBP-C, and TnI phosphorylation in CMFs from all genotypes. Open symbols depict female mice and closed symbols depict male mice. Data are mean ± SD and were analyzed using one-way ANOVA with Tukey’s comparison test with significance depicted as *, P < 0.05 between mutant versus WT; $$$, P < 0.001 for A57G versus E143K; and &, P < 0.05 for E143K versus Δ43 mice. Abbreviations: +P-RLC, phosphorylated form of mouse ventricular RLC; +P-MyBP-C, phosphorylated form of mouse cMyBP-C; +P-TnI, phosphorylated form of TnI; F/A, F-actin; ELCmse, mouse ventricular essential light chain; ELChu, human transgenic ELC; Δ43 ELC, N-terminally truncated ELC protein standard.

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  Figure 4.. Myosin RLC and cMyBP-CSer282 phosphorylation in myofibrils from heart ventricles of WT-ELC, HCM-A57G, RCM-E143K, and Δ43 mice detected by Western blotting. (A) SDS-PAGE and sarcomeric protein phosphorylation monitored by Western blots and RLC-specific (left) or cMyBP-C–specific (right) antibodies (Aby). Phosphorylated forms of the mouse RLC and cMyBP-C (upper panels) were detected with +P-RLCmse or +P-MyBP-CSer282 antibodies, while total proteins were assessed with RLCtot (CT-1) or MyBP-Ctot antibodies (lower panels). (B) Quantification of myosin RLC and cMyBP-CSer282 phosphorylation in CMFs purified from WT-ELC (4 M, 3 F), HCM-A57G (3 M, 5 F), RCM-E143K (3 M, 3 F), and Δ43 (4 M, 2 F) mice. Open symbols depict female mice and closed symbols depict male mice. Data are mean ± SD and were analyzed using one-way ANOVA with Tukey’s comparison test. Significance was depicted as *, P < 0.05 and **, P < 0.01 between mutant versus WT-ELC; $$$$, P < 0.0001 for HCM-A57G versus RCM-E143K; and &&&&, P < 0.0001 for RCM-E143K versus Δ43 mice.

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  Figure S2.. Assessment of mitochondrial protein expression in heart homogenates from 7–11-mo-old HCM-A57G, Δ43, and RCM-E143K relative to WT-ELC mice. (A) Representative Western blots of mitochondrial and metabolism proteins in heart homogenates of mice. (B and C) Expression of CI, CIV, glycolysis, fatty acid subunits (HK1, HK2, and CPT1A), and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α; PPAR-γ coactivator and master regulator of mitochondrial biogenesis) for all four genotypes (B), and separately for female versus male mice (C). Values are mean ± SD of n = 8 (5 M, 3 F) WT-ELC; n = 8 (4 M, 4 F) HCM-A57G; n = 8 (4 M, 4 F) Δ43; and n = 8 (4 M, 4 F) RCM-E143K animals with significance depicted as *, P < 0.05 for mutant versus WT or between genders. $, P < 0.05 for HCM-A57G versus RCM-E143K; and &, P < 0.05 between HCM-A57G versus Δ43 analyzed by one-way ANOVA with Tukey’s multiple comparison test (B) and two-way ANOVA with two nominal variables (genotype, sex) followed by Tukey’s or Sidak’s multiple comparison test (C). Optical density was assessed with ImageJ. Each protein band was normalized to total protein content assessed for each blot before the primary antibody…

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  Figure S3.. Enzymatic activities in the hearts of ELC mice. (A and B) Spectrophotometric assessment of enzymatic activities in heart homogenates from HCM-A57G (3 M, 3 F), Δ43 (3 M, 3 F), and RCM-E143K (4 M, 2 F) relative to WT (3 M, 3 F) mice (A), and separated by sex (B). Values are mean ± SD and analyzed using two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test with significance depicted as &, P < 0.05 between HCM-A57G versus Δ43. Open symbols depict female mice and closed symbols depict male mice.

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  Figure S4.. CI-driven respiration profiles in the hearts of 8–10-mo-old WT, HCM-A57G, Δ43, and RCM-E143K mice. (A) Averaged traces of CI OCR in isolated mitochondria from WT-ELC (n = 7 mice), HCM-A57G (n = 7 mice), Δ43 (n = 5 mice), and RCM-E143K (n = 5 mice) using Seahorse XFp analyzer. (B) Characterization of mitochondrial OCR for state II, state IIIADP, state IVo, state IIIu, state IV, and the RCR, calculated as OCR-state IIIADP/OCR-state IVo. (C) Data from B separated by sex: WT (3 M, 4 F), HCM-A57G (3 M, 4 F), Δ43 (3 M, 2 F), and RCM-E143K (2 M, 3 F). Values are mean ± SD analyzed by two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test with significance *, P < 0.05 for female versus male Δ43 mice. Open symbols depict female mice and closed symbols depict male mice.

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  Figure 5.. Schematic representation of the antagonistic mechanism by which the HCM-linked A57G and RCM-associated E143K mutations in cardiac myosin ELC regulate the SRX state of myosin and myosin RLC phosphorylation. The HCM-A57G mutation promotes the SRX-to-DRX transition, and this process is coupled with a ∼40% increase in myosin RLC phosphorylation compared with the level of RLC phosphorylation in WT-ELC myocardium. The RCM-E143K model favors the SRX conformation, and its RLC phosphorylation is approximately twofold less than in WT-ELC hearts, as judged by Western blotting and RLC-specific antibodies. Mechanistically, it is proposed that N-ELC, which is missing in the Δ43 model, regulates the SRX-to-DRX transition and contributes to sterically blocking (RCM-E143K) or facilitating (HCM-A57G) myosin RLC phosphorylation. Fig. 5 was created with https://BioRender.com.