Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice

Figures

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  Figure 1. In-silico prediction of phosphorylation sites of the human cardiac RLC. (A) Phosphorylation of Ser/Tyr/Thr sites in the RLC was predicted by PhosphoSVM search http://sysbio.unl.edu/PhosphoSVM/prediction.php/. (B) Amino acid sequence of the human cardiac RLC (NCBI_P10916) with the location of predicted pSer, pTyr, and pThr residues. Bolded and enlarged, the S15 (in red font) and T160 (in green font) sites are studied in this report.

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  Figure 2. Representative SDS-PAGE images of RLC-depleted and mutant-reconstituted cardiac muscle preparations from Tg-WT and Tg-R58Q mice. (A) Depletion and reconstitution experiments in mouse-purified cardiac myosin. (B) Myosin gels' quantification. (C) Depletion and reconstitution experiments in LVPM fibers. (D) LVPM gels' quantification. Cardiac preparations were CDTA/Triton-depleted of endogenous RLC and reconstituted with recombinant WT-RLC, R58Q, T160D, and S15D RLC proteins. Three to Four different myosin preparations from Tg-WT and 5–7 from Tg-R58Q mice were used to deplete 60 ± 6% endogenous RLC, followed by myosin reconstitution with recombinant RLC proteins at 140 ± 30% in Tg-WT and 155 ± 40% Tg-R58Q myosin (A,B). Eleven to Twenty LVPM fibers from Tg-WT and 9–20 from Tg-R58Q mice were employed to deplete 60 ± 12% RLC from LVPM, followed by 112 ± 20% reconstitution with recombinant RLC proteins in Tg-WT and 106 ± 21% in Tg-R58Q LVPM fibers (C,D). Data in (B,D) are presented as means ± SD.

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  Figure 3. Summary of the actin-activated myosin ATPase activity study. Titration isotherms (left panels), and Vmax (middle panels) and Km (right panels) parameters of Tg-WT (A) and Tg-R58Q (B) myosin reconstituted with recombinant WT-RLC (black), T160D (green), S15D (blue), and R58Q (red) proteins. Myosin (0.5 μM) was complexed with actin of increasing concentrations (0–15 μM), and the actin-activated ATPase activity was determined for all reconstituted myosin preparations. Data are expressed as mean ± SD of n = N° experiments. For Tg-WT reconstituted w/WT-RLC, n = 6, w/T160D, n = 8, and w/S15D, n = 6. For Tg-R58Q reconstituted w/WT-RLC, n = 11, w/T160D, n = 6, w/S15D, n = 6, and w/R58Q, n = 6. Statistical significance between titration isotherms was calculated by two-way ANOVA with Tukey's multiple comparisons, with the p-values presented in Table 2. One-way ANOVA with Tukey's multiple comparisons was used to calculate significant differences in Vmax and Km between the groups, and the p-values are presented in the graphs.

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  Figure 4. Contractile function in skinned LVPM from Tg-WT (A) and Tg-R58Q (B) mice reconstituted with recombinant RLC proteins. Maximal force (left panels), Calcium sensitivity of force (middle panels), and force-pCa relationships (right panels) were measured in LVPM from Tg-WT and Tg-R58Q preparations depleted of endogenous RLC and reconstituted with recombinant WT-RLC (black), T160D (green), S15D (blue), and R58Q (red) proteins. For Tg-WT fibers reconstituted w/WT, n = 7 fibers, w/T160D, n = 8, and w/S15D, n = 8, and Tg-R58Q reconstituted w/WT, n = 5, w/T160D, n = 6, w/S15D, n = 5, and w/R58Q, n = 5. Data are the average ± SD of n = N° fibers per group with the significance calculated by one-way ANOVA with Tukey's multiple comparison test (for Fmax and pCa50 values, p-values presented in the graph), or two-way ANOVA with Tukey's multiple comparison test (for the force-pCa relationships).

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  Figure 5. Summary of the super-relaxed (SRX) state study. (A) Fluorescence decay curves in RLC-depleted and mutant-reconstituted LVPM fibers from Tg-WT (upper panel) and Tg-R58Q (lower panel) hearts. (B) Distribution of myosin heads between the SRX and DRX states in Tg-WT fibers reconstituted w/WT-RLC (black), w/T160D (green), and w/S15D (blue). (C) Distribution of myosin heads between the SRX and DRX states in Tg-R58Q fibers reconstituted w/WT (black), w/T160D (green), w/S15D (blue), and w/R58Q (red). Data are the average ± SD of n fibers per group. For Tg-WT fibers reconstituted w/WT, n = 10, w/T160D, n = 9, and w/S15D, n = 9. For Tg-R58Q reconstituted w/WT, n = 7, w/T160D, n = 6, w/S15D, n = 7, and w/R58Q, n = 7. The significance was calculated by one-way ANOVA with Tukey's multiple comparison test with p values depicted in the graph. Note that the % SRX is increased in R58Q reconstituted Tg-R58Q fibers.

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  Figure 6. The S15 and T160 phosphorylation sites are visualized as phosphomimetics S15D and T160D in the human cardiac RLC-WT (A) and RLC-R58Q (B) proteins. I-TASSER derived secondary structures of the human cardiac RLC were built using protein templates selected from the Protein Data Bank (PDB): 5tbyE, 3dtpE, 3jvtB, 3pn7E, 3j04B, 3i5iB, 2w4aB, 2bl0C, 6iihA, and 6k7yI. Note that mild intramolecular rearrangements in the vicinity of phosphorylation sites at amino acids (aa) 15 and 160 are visible in the superimposed structures of WT and R58Q proteins.