Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments.

The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca levels and for limiting activation at saturating Ca. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT.

Dual thick and thin filament linked regulation of stretch- and L-NAME-induced tone in young and senescent murine basilar artery.

Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC)-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone.

Hypertrophic Cardiomyopathy Mutations of Troponin Reveal Details of Striated Muscle Regulation.

Striated muscle contraction is inhibited by the actin associated proteins tropomyosin, troponin T, troponin I and troponin C. Binding of Ca to troponin C relieves this inhibition by changing contacts among the regulatory components and ultimately repositioning tropomyosin on the actin filament creating a state that is permissive for contraction. Several lines of evidence suggest that there are three possible positions of tropomyosin on actin commonly called Blocked, Closed/Calcium and Open or Myosin states.

C-Terminal Basic Region of Troponin T Alters the Ca-Dependent Changes in Troponin I Interactions.

The C-terminal 14-16 residues of human troponin T are required for full inactivation, and they prevent full activation at saturating Ca. Basic residues within that C-terminal region of TnT are essential for its function, but the mechanism of action is unknown. That region of TnT is natively disordered and does not appear in reconstructions of the troponin structure.

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms.

Structure of the shutdown state of myosin-2.

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads, which prevents filament formation and inactivates the motors. The mechanism by which this happens is unclear.

The Positively Charged C-Terminal Region of Human Skeletal Troponin T Retards Activation and Decreases Calcium Sensitivity.

Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca activation.

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca and to eliminate one of the inactive states at low Ca.

Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers.

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart.

Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium.

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca and for limiting its activation at saturating Ca Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe.

Stepwise C-Terminal Truncation of Cardiac Troponin T Alters Function at Low and Saturating Ca.

Activation of striated muscle contraction occurs in response to Ca binding to troponin C. The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin-catalyzed ATP hydrolysis. It now appears that the C-terminal 14 amino acids of cardiac troponin T (TnT) control the level of activity at both low and high Ca.

Troponin C Mutations Partially Stabilize the Active State of Regulated Actin and Fully Stabilize the Active State When Paired with Δ14 TnT.

Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca and activating cross-bridges (i.e.

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.

Calpain inhibition rescues troponin T3 fragmentation, increases Cav1.1, and enhances skeletal muscle force in aging sedentary mice.

Loss of strength in human and animal models of aging can be partially attributed to a well-recognized decrease in muscle mass; however, starting at middle-age, the normalized force (force/muscle cross-sectional area) in the knee extensors and single muscle fibers declines in a curvilinear manner. Strength is lost faster than muscle mass and is a more consistent risk factor for disability and death. Reduced expression of the voltage sensor Ca(2+) channel α1 subunit (Cav1.

Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein.

Organization of F-actin by Fesselin (avian smooth muscle synaptopodin 2).

Fesselin or avian synaptopodin 2 is a member of the synaptopodin family of actin binding proteins. Fesselin promotes G-actin polymerization and the formation of large actin complexes that can be collected by low-speed centrifugation. Because of the potential role of fesselin in some cancers and its effects on actin, we further investigated the effect of fesselin on actin.

Structural dynamics of troponin I during Ca2+-activation of cardiac thin filaments: a multi-site Förster resonance energy transfer study.

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca(2+)-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm.

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites.

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca(2+) binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin-tropomyosin-actin complex. Herein, we report a calorimetric study of Ca(2+) binding to HcTnC.

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.

Disease causing mutations of troponin alter regulated actin state distributions.

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies.

Michael Bárány: a recollection.

In this special edition of the Journal of Muscle Research and Cell Motility, we recall the lives and scientific contributions of Michael and Kate Bárány, who died in 2011. Michael and Kate were Holocaust survivors who went on to become leading researchers in muscle contraction. Their research topics included myosin isoforms, phosphorylation as a regulator of muscle contraction and the application of NMR to study muscle metabolism.

Acrylodan-labeled smooth muscle tropomyosin reports differences in the effects of troponin and caldesmon in the transition from the active state to the inactive state.

Changes in the orientation of tropomyosin on actin are important for the regulation of striated muscle contraction and could also be important for smooth muscle regulation. We showed earlier that acrylodan-labeled skeletal muscle tropomyosin reports the kinetics of the reversible transitions among the active, intermediate, and inactive states when S1 is rapidly detached from actin-tropomyosin. We now show that acrylodan-labeled smooth muscle tropomyosin reports similar transitions among states of actin-tropomyosin.