Abstract
There is evidence that multi-site phosphorylation of cardiac troponin I (cTnI) by protein kinase C is important in both long- and short-term regulation of cardiac function. To determine the specific functional effects of these phosphorylation sites (Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI was replaced with cTnI phosphorylated by protein kinase C-epsilon or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergent-skinned mouse cardiac fiber bundles to measure changes in Ca(2+)-dependence of force. Compared with controls, fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E were desensitized to Ca(2+), and maximum tension was as much as 27% lower, whereas fibers reconstituted with cTnI-T144E showed no change. In the in vitro motility assay actin filaments regulated by troponin complexes containing phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in Ca(2+) sensitivity and maximum sliding speed compared with controls, whereas filaments regulated by cTnI-S43E/S45E showed only decreased maximum sliding speed and filaments regulated by cTnI-T144E demonstrated only desensitization to Ca(2+). Our results demonstrate novel site specificity of effects of PKC phosphorylation on cTnI function and emphasize the complexity of modulation of the actin-myosin interaction by specific changes in the thin filament.
Highlights
Sliding speed of actin filaments regulated by Tn complexes containing cTnI-S43E/S45E decreased by as much as 30% at various pCa values; Vmax decreased by 15% compared with controls, whereas the change in pCa50 was not statistically different compared with controls (Fig. 4C and Table I)
Previous data indicated that phosphorylation of the Nterminal protein kinase A sites is able to decrease Ca2ϩ sensitivity with no effect on maximum tension and to increase shortening velocity and cross-bridge cycling rate [6, 11,12,13]
We report here that phosphorylation of Ser-43 and Ser-45 dominates regulation of the level of maximum tension
Summary
Protein Purification and Preparation—Expression and purification of recombinant human cTnC, mouse cTnI (wild-type (WT) and mutant), and mouse cTnT was previously described [21]. Phosphorylation of cTnI for in vitro motility assay studies was accomplished by first forming a 10 M cTnI-cTnC complex in 50 mM Tris-HCl (pH 8.0), 1 M KCl, 10 mM DTT, and 0.2 M phenylmethylsulfonyl fluoride. Initial maximum isometric force was measured in activating solution (pCa 4.5) containing 20 mM MOPS (pH 7.0), 33.8 mM KCl, 10 mM EGTA, 9.96 mM CaCl2, 5.39 mM ATP, 6.47 mM MgCl2, 12 mM creatine phosphate, 10 IU/ml creatine kinase, and 1 mM DTT. The cTnT-cTnI-treated fiber was treated for 90 min with a TnC reconstitution solution, prepared as previously described [25], followed by measurement of maximum Ca2ϩ-activated force in pCa 4.5 solution and subsequent force measurements ranging from pCa 8 to pCa 4.5. Fits to the Hill equation were made from a regression using all of the observed speeds so that a fit was generated from over 1200 filaments
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