There is evidence for PKC-dependent multisite phosphorylation of cardiac troponin I (cTnI) at Ser-23 and Ser-24 (also PKA sites) in the cardiac-specific N-terminal extension and at Thr-144, a unique residue in the inhibitory region. The functional effect of these phosphorylations in combination is of interest in view of data indicating intramolecular interaction between the N-terminal extension and the inhibitory region of cTnI. To determine the role of PKC-dependent phosphorylation of cTnI on sarcomeric function, we measured contractile regulation at multiple levels of complexity. Ca 2؉ binding to thin filaments reconstituted with either cTnI(wild-type) or pseudo-phosphorylated cTnI(S23D/S24D), cTnI(T144E), and cTnI(S23D/S24D/ T144E) was determined. Compared with controls regulated by cTnI(wild-type), thin filaments with cTnI(S23D/S24D) and cTnI(S23D/S24D/T144E) exhibited decreased Ca 2؉ sensitivity. In contrast, there was no significant difference between Ca 2؉ binding to thin filaments with cTnI(wild-type) and with cTnI(T144E). Studies of the pCa-force relations in skinned papillary fibers regulated by these forms of cTnI yielded similar results. However, in both the Ca 2؉ binding measurements and the skinned fiber tension measurements, the presence of cTnI(S23D/S24D/T144E) induced a much lower Hill coefficient than either wild type, S23D/S24D, or T144E. These data highlight the importance of thin filament-based cooperative mechanisms in cardiac regulation, with implications for mechanisms of control of function in normal and pathological hearts.In experiments reported here, we have investigated functional effects of multisite phosphorylation in cardiac troponin I (cTnI).3 cTnI is an inhibitory subunit of the cardiac troponin (cTn) complex, which consists also of troponin C (TnC), a Ca 2ϩ binding component and troponin T (TnT), a tropomyosin (Tm) binding component. cTnI functions as a key protein in the cardiac muscle contraction-relaxation cycle by relieving or restoring its inhibitory influence that is regulated by Ca 2ϩ association to or dissociation from the regulatory Ca 2ϩ binding site of TnC (1-4). It is now well recognized that cTnI has multiple sites, which are substrates for a variety of kinases, especially PKA and PKD (Ser-23 and Ser-24), and PKC . These phosphorylations have been demonstrated to occur in various signaling pathways that control cardiac dynamics and growth (for review, see Ref. 5). There is reasonable agreement that phosphorylation of Ser-23/Ser-24 located in the unique N terminus of cTnI enhances the "off" rate for Ca 2ϩ dissociation from cTnC and is important in the abbreviated cycle time associated with ␤-adrenergic stimulation of the heart. However, the functional significance of the PKC sites remains unclear and controversial.Previous work has focused on PKC-dependent phosphorylation of cTnI at Ser-43/Ser-45, which decreases maximum Ca 2ϩ -activated force, ATPase activity, and sliding velocity, and desensitizes myofilaments to [Ca 2ϩ ] by stabilizing the off-state of the thin...

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca 2؉ concentration. Tn is composed of TnC, TnI, and TnT. Ca 2؉ binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca 2؉ activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca 2؉ , which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca 2؉ NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca 2؉ , there was little difference in the activation of ATPase activity between these preparations.Striated muscle thin filaments exist in equilibrium among multiple states. Ca 2ϩ binding to the regulatory domain of troponin C (TnC) 2 along the thin filaments and strong crossbridge interactions with thick filaments are thought to shift the equilibrium. Ca 2ϩ binds to the regulatory domain of TnC, which regulates the interaction of troponin I (TnI) with actintropomyosin (Tm) and TnC (1-3). In the thin filaments, the inhibitory region of TnI (residues 104 -115 of rabbit fast skeletal TnI (fsTnI) or 138 -149 of mouse cardiac TnI (cTnI)) undergoes a structural transition depending on the Ca 2ϩ state of TnC (4, 5). In the absence of Ca 2ϩ at the regulatory site(s) of TnC, the inhibitory region interacts with actin to prevent activation of myosin ATPase activity. When Ca 2ϩ binds to the regulatory site(s) of TnC, the switch region of TnI, which is located at the C terminus of the inhibitory region, interacts with the newly exposed hydrophobic patch of the N-terminal regulatory domain of . This interaction causes the removal of the inhibitory region and the second actin-Tm binding region of TnI from the actin surface and allows actin to interact with myosin. In the presence of Ca 2ϩ at the regulatory sites of TnC, the inhibitory region and the central helical region of TnC are mutually stabilized, according to the recent x-ray crystal structure of the core domain of the fsTn complex (9). The sequence variations in the N-terminal and the C-terminal regions of TnT, another component of the Tn complex, are known to alter the Ca 2ϩ sensitivity of myofilament activity (10, 11). In addition, TnT is involved in the Ca 2ϩ -dependent interaction of the Tn complex with actin-...

Previous structural studies indicated a special functional role for an acidic region comprised of residues 1–10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-ε (PKCε) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca2+. In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca2+-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24).