Maria V. Razumova

Myosin Crossbridge Activation of Cardiac Thin Filaments: Implications for Myocardial Function in Health and Disease

At the level of the myofibrillar proteins, activation of myocardial contraction is thought to involve switch-like regulation of crossbridge binding to the thin filaments. A central feature of this view of regulation is that Ca2+ binding to the low-affinity (≈3 μmol/L) site on troponin C alters the interactions of proteins in the thin filament regulatory strand, which leads to movement of tropomyosin from its blocking position on the thin filament and binding of crossbridges to actin. Although Ca2+ binding is a critical step in initiating contraction, this event alone does not account for the activation dependence of contractile properties of myocardium. Instead, activation is a highly cooperative process in which initial crossbridge binding to the thin filaments recruits additional crossbridge binding to actin as well as increased Ca2+ binding to troponin C. This review addresses possible roles of thin filament cooperativity in myocardium as a process that modulates the activation dependence of force and the rate of force development and also possible mechanisms by which cooperative signals are transmitted along the thick filament. Emerging evidence suggests that such mechanisms could contribute to the regulation of fundamental mechanical properties of myocardium and alterations in regulation that underlie contractile disorders in diseases such as cardiomyopathies.

Nonlinear Myofilament Regulatory Processes Affect Frequency-Dependent Muscle Fiber Stiffness

To investigate the role of nonlinear myofilament regulatory processes in sarcomeric mechanodynamics, a model of myofilament kinetic processes, including thin filament on-off kinetics and crossbridge cycling kinetics with interactions within and between kinetic processes, was built to predict sarcomeric stiffness dynamics. Linear decomposition of this highly nonlinear model resulted in the identification of distinct contributions by kinetics of recruitment and by kinetics of distortion to the complex stiffness of the sarcomere. Further, it was established that nonlinear kinetic processes, such as those associated with cooperative neighbor interactions or length-dependent crossbridge attachment, contributed unique features to the stiffness spectrum through their effect on recruitment. Myofilament model-derived sarcomeric stiffness reproduces experimentally measured sarcomeric stiffness with remarkable fidelity. Consequently, characteristic features of the experimentally determined stiffness spectrum become interpretable in terms of the underlying contractile mechanisms that are responsible for specific dynamic behaviors.

Myofilament kinetics in isometric twitch dynamics.

AbstractTo better understand the relationship between kinetic processes of contraction and the dynamic features of an isometric twitch, studies were conducted using a mathematical model that included: (1) kinetics of cross bridge (XB) cycling; (2) kinetics of thin filament regulatory processes; (3) serial and feedback interactions between these two kinetic processes; and (4) time course of calcium activation. Isometric twitch wave forms were predicted, morphometric features of the predicted twitch wave form were evaluated, and sensitivities of wave form morphometric features to model kinetic parameters were assessed. Initially, the impulse response of the XB cycle alone was analyzed with the findings that dynamic constants of the twitch transient were much faster than turnover number of steady-state XB cycling, and, although speed and duration of the twitch wave form were sensitive to XB cycle kinetic constants, parameters of wave shape were not. When thin filament regulatory unit (RU) kinetics were added to XB cycle kinetics, the system impulse response was slowed with only little effect on wave shape. When cooperative neighbor interactions between RU and XB were added, twitch wave shape (as well as amplitude, speed and duration) proved to be sensitive to variation in cooperativity. Importantly, persistence and shape of the falling phase could be strongly modified. When kinetic coefficients of XB attachment were made to depend on sarcomere length, changes in wave shape occurred that did not occur when only sliding filament mechanisms were operative. Indeed, the force–length relationship proved to be highly sensitive to length-dependent XB attachment in combination with cooperative interactions. These model findings are the basis of hypotheses for the role of specific kinetic events of contraction in generating twitch wave form features.

Regulation of the Rate of Force Development in Heart and Skeletal Muscles

Contractions of heart and skeletal muscle cells in vivo are usually well matched to the loads the muscle must move or bear, but despite considerable similarity in motor and regulatory proteins, the detailed mechanisms of regulation differ in significant ways in the two muscle types. Other chapters in this volume address the molecular components of the thick and thin filaments in striated muscles and address the specific processes by which Ca2+ and cross-bridges regulate the activation state of the thin filament. This chapter deals with the physiological manifestations of these regulatory processes during force development in heart and skeletal muscles, with particular focus on the regulation of the kinetics of force development.

Both PKA Treatment and Cardiac Troponin-I N-Terminal Phosphorylation Alone Decrease Ca-Sensitivity and Eliminate Length-Dependent Activation in Skinned Cardiac Muscle

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility. PKA targets the cardiac troponin I (cTnI) N-terminus, cardiac myosin-binding protein C (cMyBP-C) and titin. To isolate cTnI and cMyBP-C /titin phosphorylation effects on force-[Ca2+] relations, endogenous cardiac troponin (Tn) was exchanged in rat, skinned trabeculae with either WT Tn or Tn containing a non-phosphorylatable mutant cTnI(S23/24A) or phosphomimetic cTnI(S23/24D). PKA cannot phosphorylate either cTnI mutant, leaving cMyBP-C and titin as sole PKA targets. Force-[Ca2+] relations and Ca2+-sensitivity (pCa50) were measured at 2.3 and 2.0 µm SL. Decreasing steady SL reduced maximal force (Fmax) and pCa50 similarly with WT Tn and Tn containing cTnI(S23/24A). PKA treatment of native, WT and cTnI(S23/24A) exchanged trabeculae reduced pCa50 at 2.3, but not 2.0 um SL, eliminating SL-dependence of pCa50. Reconstitution with Tn containing cTnI(S23/24D) reduced pCa50 at both SL (compared to WT and cTnI(S23,24A) and eliminated pCa50 SL-dependence; PKA did not significantly alter pCa50 at either SL. At each SL Fmax was similar with WT and mutant troponins, and was unaffected by PKA. Low angle x-ray diffraction experiments were performed to determine whether shifts in pCa50 were associated with changes in myofilament spacing (D1,0) or interaction. D1,0 at 2.3 um SL was similar in native trabeculae, with WT Tn and Tn containing either cTnI(S23,24A) or cTnI(S23,24D); PKA increased D1,0 in all cases. The results suggest that PKA phosphorylation of either cTnI or cMyBP-C /titin reduced the Ca2+-sensitivity of force and length-dependent activation. Supported by NIH HL067071-06.