FIGURE 4.

Cardiac TnI Ser-150 phosphorylation increases myofilament Ca²⁺-sensitive force development and blunts cTnI PKA dependent desensitization. Skinned mouse cardiac fiber bundles were exchanged with human cTn containing either wild-type (Tn WT), Ser-150 (Tn S150D), Ser-23/24 (Tn S23D/S24D), or combined Ser-150 and PKA (Tn S23D/S24D/S150D) pseudo-phosphorylated cTnI. Fiber bundles from individual force experiments were analyzed for cTn exchange by Western blot with a TnT antibody. Representative fibers demonstrate an average of 67% incorporation of the larger molecular mass, exogenous human TnT band that was not different in any of the exchanged groups. A, average tension-Ca²⁺ measurements of fiber bundles at 2.2 μm exchanged with Tn S150D (gray triangle, dashed line) exhibit increased Ca²⁺ sensitivity compared with Tn WT (black circles, dashed line) exchange in the absence of an effect on Hill coefficient or maximal force development (inset). B, fiber bundles exchanged with Tn S23D/S24D (black square, solid line) demonstrate the expected decrease in Ca²⁺ sensitivity compared with Tn WT. Exchange of the cTnI Tn S23D/S24D/S150D (gray diamond, solid line) did not exhibit altered Ca²⁺ sensitivity compared with Tn WT; however, Tn S23D/S24D/S150D Ca²⁺ sensitivity was increased compared with that of Tn S23D/S24D exchange. Tn S23D/S24D/S150D exchange did not alter the Hill coefficient or maximal force development (inset). C, the change in EC₅₀ at 2.2 μm of the various exchanged cTn from that of WT demonstrates Tn S150D (gray bar) increased EC₅₀ by 0.51 μm from Tn WT, whereas combination of S150D with S23D/S24D phosphorylation (hatched bar) was not different from Tn WT and blunted the −1.05 μm decrease in EC₅₀ of cTnI S23D/S24D (white bar) exchange alone. Gray, Tn S150D; white, Tn S23D/S24D; hatched, Tn S23D/S24D/S150D. *, ANOVA Bonferroni EC₅₀ p < 0.05 versus WT.