Molecular dynamics provides new insights into the mechanism of calcium signal transduction and interdomain interactions in cardiac troponin.

Understanding the regulation of cardiac muscle contraction at a molecular level is crucial for the development of therapeutics for heart conditions. Despite the availability of atomic structures of the protein components of cardiac muscle thin filaments, detailed insights into their dynamics and response to calcium are yet to be fully depicted. In this study, we used molecular dynamics simulations of the core domains of the cardiac muscle protein troponin to characterize the equilibrium dynamics of its calcium-bound and calcium-free forms, with a focus on elements of cardiac muscle contraction activation and deactivation, that is, calcium binding to the cardiac troponin Ca -binding subunit (TnC) and the release of the switch region of the troponin inhibitory subunit (TnI) from TnC.

The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the Intermediate "C" State of Regulated Actin under High- and Low-Free Ca(2+) Conditions.

The R146G mutation of troponin I (TnI) is associated with hypertrophic cardiomyopathy in humans. Earlier data pointed to stabilization of the intermediate, C state, of actin-tropomyosin-troponin by this mutant. Because cardiac disorders appear to be linked to changes in regulated actin distributions, we determined the extent to which the R146G TnI mutant alters the distribution of states at low and high Ca(2+) concentrations.

Green Tea Catechin Normalizes the Enhanced Ca2+ Sensitivity of Myofilaments Regulated by a Hypertrophic Cardiomyopathy-Associated Mutation in Human Cardiac Troponin I (K206I).

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease characterized by thickening of ventricular walls and decreased left ventricular chamber volume. The majority of HCM-associated mutations are found in genes encoding sarcomere proteins. Herein, we set out to functionally characterize a novel HCM-associated mutation (K206I-TNNI3) and elucidate the mechanism of dysfunction at the level of myofilament proteins.

Cardiac muscle activation blunted by a mutation to the regulatory component, troponin T.

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca(2+) sensor (TnC) and the actin filament.

Conserved Asp-137 is important for both structure and regulatory functions of cardiac α-tropomyosin (α-TM) in a novel transgenic mouse model expressing α-TM-D137L.

α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions.

Calcium induced regulation of skeletal troponin--computational insights from molecular dynamics simulations.

The interaction between calcium and the regulatory site(s) of striated muscle regulatory protein troponin switches on and off muscle contraction. In skeletal troponin binding of calcium to sites I and II of the TnC subunit results in a set of structural changes in the troponin complex, displaces tropomyosin along the actin filament and allows myosin-actin interaction to produce mechanical force. In this study, we used molecular dynamics simulations to characterize the calcium dependent dynamics of the fast skeletal troponin molecule and its TnC subunit in the calcium saturated and depleted states.

Integration of troponin I phosphorylation with cardiac regulatory networks.

We focus here on the modulation of thin filament activity by cardiac troponin I phosphorylation as an integral and adaptive mechanism in cardiac homeostasis and as a mechanism vulnerable to maladaptive response to stress. We discuss a current concept of cardiac troponin I function in the A-band region of the sarcomere and potential signaling to cardiac troponin I in a network involving the ends of the thin filaments at the Z-disk and the M-band regions. The cardiac sarcomere represents a remarkable set of interacting proteins that functions not only as a molecular machine generating the heartbeat but also as a hub of signaling.

New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I.

Previous structural studies indicated a special functional role for an acidic region composed 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).

The C-terminus of troponin T is essential for maintaining the inactive state of regulated actin.

Striated muscle contraction is regulated by the actin binding proteins tropomyosin and troponin. Defects in these proteins lead to myopathies and cardiomyopathies. Deletion of the 14 C-terminal residues of cardiac troponin T leads to hypertrophic cardiomyopathy.

Phosphorylation of cardiac troponin I at protein kinase C site threonine 144 depresses cooperative activation of thin filaments.

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.

Ala scanning of the inhibitory region of cardiac troponin I.

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.

Sites of intra- and intermolecular cross-linking of the N-terminal extension of troponin I in human cardiac whole troponin complex.

Our previous studies (Howarth, J. W., Meller, J.

Some cardiomyopathy-causing troponin I mutations stabilize a functional intermediate actin state.

We examined four cardiomyopathy-causing mutations of troponin I that appear to disturb function by altering the distribution of thin filament states. The R193H (mouse) troponin I mutant had greater than normal actin-activated myosin-S1 ATPase activity in both the presence and absence of calcium. The rate of ATPase activity was the same as that of the wild-type at near-saturating concentrations of the activator, N-ethylmaleimide-S1.

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins.

Reductive methylation of lysine residues in proteins offers a way to introduce 13C methyl groups into otherwise unlabeled molecules. The 13C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of 13C methyl groups in NMR spectra.

Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I.

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions.

Cardiac thin filament regulation.

Myocardial contraction is initiated upon the release of calcium into the cytosol from the sarcoplasmic reticulum following membrane depolarization. The fundamental physiological role of the heart is to pump an amount blood that is determined by the prevailing requirements of the body. The physiological control systems employed to accomplish this task include regulation of heart rate, the amount of calcium release, and the response of the cardiac myofilaments to activator calcium ions.

Increased cross-bridge cycling kinetics after exchange of C-terminal truncated troponin I in skinned rat cardiac muscle.

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn.

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation.

We review development of evidence and current perceptions of the multiple and significant functions of cardiac troponin I in regulation and modulation of cardiac function. Our emphasis is on the unique structure function relations of the cardiac isoform of troponin I, especially regions containing sites of phosphorylation. The data indicate that modifications of specific regions cardiac troponin I by phosphorylations either promote or reduce cardiac contractility.

Negative charges at protein kinase C sites of troponin I stabilize the inactive state of actin.

Alterations in the troponin complex can lead to increases or decreases in contractile activity. Most mutations of troponin that cause hypertrophic cardiomyopathy increase the activity of cardiac muscle fibers. In at least some cases these mutants stabilize the active state of regulated actin.

The troponin C G159D mutation blunts myofilament desensitization induced by troponin I Ser23/24 phosphorylation.

Striated muscle contraction is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, beta-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(2+)-dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae.

Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C.

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.

Identification of a region of troponin I important in signaling cross-bridge-dependent activation of cardiac myofilaments.

Force generating strong cross-bridges are required to fully activate cardiac thin filaments, but the molecular signaling mechanism remains unclear. Evidence demonstrating differential extents of cross-bridge-dependent activation of force, especially at acidic pH, in myofilaments in which slow skeletal troponin I (ssTnI) replaced cardiac TnI (cTnI) indicates the significance of a His in ssTnI that is an homologous Ala in cTnI. We compared cross-bridge-dependent activation in myofilaments regulated by cTnI, ssTnI, cTnI(A66H), or ssTnI(H34A).

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure.

It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor.

Increased Ca2+ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I.

To understand the molecular mechanisms whereby cardiomyopathy-related cardiac troponin I (cTnI) mutations affect myofilament activity, we have investigated the Ca2+ binding properties of various assemblies of the regulatory components that contain one of the cardiomyopahty-related mutant cTnI. Acto-S1 ATPase activities in reconstituted systems were also determined. We investigated R145G and R145W mutations from the inhibitory region and D190H and R192H mutations from the second actin-tropomyosin-binding site.