Phosphorylation Mimetic of Myosin Regulatory Light Chain Mitigates Cardiomyopathy-Induced Myofilament Impairment in Mouse Models of RCM and DCM

Figures

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  Figure 1. Force measurements in skinned LVPM from Tg-WT ELC (A) and Tg-E143K ELC (B) mice reconstituted with recombinant RLC proteins. Maximal force (top panels), Ca2+-sensitivity of force (middle panels), and force–pCa relationship (bottom panels) were measured in LVPM fibers from Tg-WT ELC and Tg-E143K mice. The fibers were depleted of endogenous RLC and reconstituted with recombinant WT-RLC (black-filled symbols) and S15D-RLC (green-filled symbols) proteins. Data are the average ± SD of n = 7–8 reconstituted LVPM fibers from Tg-WT ELC (2 animals-1F, 1M) and n = 9–10 reconstituted LVPM fibers from Tg-E143K ELC (3 animals-2F, 1M). Significance (p values) was calculated by Student’s t-test.

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  Figure 2. Summary of the SRX study for LVPM fibers from Tg-WT ELC (A) and Tg-E143K ELC (B) reconstituted with WT-RLC versus phosphomimetic S15D-RLC. The proportion of myosin heads in SRX state (top panels) and DRX states (bottom panels) is shown for native (clear points), WT- (black-filled points), and S15D- (green-filled points) RLC reconstituted Tg fibers. Note that % SRX is increased in native Tg-E143K fibers compared with Tg-WT ELC native controls (~71% versus ~61%). For Tg-E143K fibers reconstituted with WT-RLC or S15D-RLC, reconstitution with the latter significantly decreased % of myosin heads in SRX (~56%) compared with WT-RLC-reconstituted (68%) or native Tg-E143K fibers (71%). Data are expressed as mean ± SD for n = 14 Tg-WT ELC native fibers (4 animals-2F, 2M) and n = 6–7 (4 animals-2F, 2M) for Tg-WT ELC fibers reconstituted with WT-RLC or S15D-RLC. For Tg-E143K set, n = 13 fibers for native Tg-E143K (4 animals-2F, 2M) and n = 10–11 fibers for Tg-E143K (5 animals-3F, 2M) reconstituted with WT-RLC or S15D-RLC were used. The significance was calculated using one-way ANOVA with Tukey’s multiple comparison test.

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  Figure 3. Summary of the SRX study performed on reconstituted LVPM fibers from the model of DCM, Tg-D94A mice. (A) SRX comparison between Tg-WT RLC and Tg-D94A RLC mice. (B) The proportion of myosin heads in the SRX (top) and DRX (bottom) states. LVPM fibers reconstituted with WT-RLC are shown in black-filled symbols, while those reconstituted with S15D-RLC are shown in green-filled symbols. D94A-RLC-reconstituted fibers are depicted with red-filled symbols. Data are expressed as mean ± SD of n = N° fibers: n = 13, 4 animals-3F, 1M for native Tg-D94A, n = 17, 5 animals-2F, 3M for Tg-WT native, and n = 8–13, 4 animals-1F, 3M for LVPM fibers from Tg-D94A reconstituted with RLC proteins. The significance was calculated by Student’s t-test (A) and one-way ANOVA with Tukey’s multiple-comparison test (B).

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  Figure 4. Contractile function in skinned LVPM fibers from Tg-WT ELC (A), Tg-E143K ELC (B), Tg-WT RLC (C), and Tg-D94A RLC (D) mice treated with 3 µM OM versus placebo (pCa 8 buffer). Maximal force (top panels), Ca2+-sensitivity of force (middle panels), and force–pCa relationships (bottom panels) were measured in fibers treated with 3 µM OM (cyan-filled points) compared with placebo (pCa 8 solution) (clear points). Data are the average ± SD of n = 4–8 fibers (1–2 mice) per group. p values were calculated by Student’s t-test.

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  Figure 5. Rescue of SRX↔DRX equilibrium in LVPM from RCM-ELC and DCM-RLC mouse models treated with 3 µM OM. Fibers treated with 3 µM OM (cyan-filled points) were compared with placebo-treated controls (clear points). (A) Tg-WT ELC, (B) Tg-E143K ELC, (C) Tg-WT RLC, and (D). Tg-D94A RLC mice. Data are the average ± SD of n = 9–14 fibers of 1–2 mice per group. p values were calculated by Student’s t-test.