Although well known as the location of the mechanism by which the cardiac sarcomere is activated by Ca2+ to generate force and shortening, the thin filament is now also recognized as a vital component determining the dynamics of contraction and relaxation. Molecular signaling in the thin filament involves steric, allosteric, and cooperative mechanisms that are modified by protein phosphorylation, sarcomere length and load, the chemical environment, and isoform composition. Approaches employing transgenesis and mutagenesis now permit investigation of these processes at the level of the systems biology of the heart. These studies reveal that the thin filaments are not merely slaves to the levels of Ca2+ determined by membrane channels, transporters and exchangers, but are actively involved in beat to beat control of cardiac function by neural and hormonal factors and by the Frank-Starling mechanism.

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.

Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca(2+) sensitivities and slower Ca(2+) dissociation rates from troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac troponin C to sense and respond to Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.

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-⑀ or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergentskinned 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.

Abstract-Striated muscle contraction is regulated by the binding of Ca 2ϩ to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, ␤-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca 2ϩ -dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca 2ϩ in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca 2ϩ sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca 2ϩ -sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca 2ϩ -binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca 2ϩ -binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through ␤-adrenergic signaling as well as the development of cardiomyopathy.