We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.

There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K T (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca 2؉ -bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the ؉Ca 2؉ state of the two systems is similar whereas the ؊Ca 2؉ state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.

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