The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca 2+ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosincrossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca 2+ -binding to TnC presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI with tropomyosin and actin that cause tropomyosin movement are unknown and thus the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI and then 3D-EM was used to resolve where troponin is anchored on actin-tropomyosin. EM-reconstruction showed how TnI-binding to both actin and tropomyosin at low-Ca 2+ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated stericinterference of actin-myosin interaction that regulates muscle contraction. Keywordsactin; troponin; tropomyosin; calcium; electron microscopy Author Information Correspondence should be addressed to W.L. (wlehman@bu.edu). * These authors contributed equally to this work. Supplementary DataSupplementary data associated with this article can be found in the online version, at … Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Contraction of skeletal and cardiac muscles is switched on and off by the thin filament proteins troponin and tropomyosin reacting in concert to changes in intracellular Ca 2+ levels. By shifting position along thin filaments in response to Ca 2+ binding to troponin, tropomyosin is known to either block or expose myosin-binding sites on actin, hence regulating myosin-crossbridge cycling and consequently contraction 1-5 ; however, the structural basis of troponin's effect on tropomyosin remains obscure. Troponin, itself a three-component complex, consists of TnI, the "inhibitory" subunit, TnC, the Ca 2+ -sensor that relieves...

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca 2؉ sensitivity of force development compared with cTnT3-and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca 2؉ was released from site II of cTnC in the cTnI⅐cTnC complex (122/s) was 12.5-fold faster than for the ssTnI⅐cTnC complex (9.8/s). Addition of cTnT3 to the cTnI⅐cTnC complex resulted in a 3.6-fold decrease in the Ca 2؉ dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI⅐cTnC complex resulted in a 1.9-fold increase in the Ca 2؉ dissociation rate from site II of cTnC. The rate at which Ca 2؉ dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca 2؉ dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.

Background: Changes in phosphorylation status of sarcomeric proteins allows rapid alteration of cardiac function. Results: Tropomyosin dephosphorylation results in myocyte hypertrophy with increases in SERCA2a (sarcoplasmic reticulum Ca 2ϩ ATPase 2a) expression and phospholamban phosphorylation but without functional changes. Conclusion: Tropomyosin phosphorylation can influence calcium regulatory proteins and cardiac remodeling in response to stress. Significance: This is the first report detailing that altering tropomyosin phosphorylation affects calcium handling proteins.

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). A drop from pH 7.0 to 6.5 induced enhanced cross-bridge-dependent activation in cTnI myofilaments, but depressed activation in cTnI(A66H) myofilaments. This same drop in pH depressed crossbridge-dependent activation in both ssTnI myofilaments and ssTnI(H34A) myofilaments. Compared with controls, cTnI(A66H) myofilaments were desensitized to Ca 2؉ , whereas there was no difference in the Ca 2؉ -force relationship between ssTnI and ssTnI(H34A) myofilaments. The mutations in cTnI and ssTnI did not affect Ca 2؉ dissociation rates from cTnC at pH 7.0 or 6.5. However, at pH 6.5, cTnI(A66H) had lower affinity for cTnT than cTnI. We also probed cross-bridge-dependent activation in myofilaments regulated by cTnI(Q56A). Myofilaments containing cTnI(Q56A) demonstrated cross-bridge-dependent activation that was similar to controls containing cTnI at pH 7.0 and an enhanced cross-bridge-dependent activation at pH 6.5. We conclude that a localized N-terminal region of TnI comprised of amino acids 33-80, which interacts with C-terminal regions of cTnC and cTnT, is of particular significance in transducing signaling of thin filament activation by strong cross-bridges.