Ca2+ regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A7TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca2+binding sites on TnC, conformational changes resulting from Ca2+ binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca2+ binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1–2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca2+-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the “open” probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A7TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca2+ binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca2+ regulates the strong binding of M·ADP·Pi to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca2+ activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A7TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca2+]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca2+ binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. These results and conclusions suggest possible explanations for differences between skeletal and cardiac muscle regulation and delineate the paths future research may take toward a better understanding of striated muscle regulation.

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady‐state Ca2+‐activated force was studied. Native troponin C (TnC) was extracted from single, de‐membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca2+ coordination at N‐terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca2+, both sTnC and xxsTnC bind with similar apparent affinity to sTnC‐extracted thin filaments. Endogenous sTnC‐extracted fibres reconstituted with 100 % xxsTnC did not develop Ca2+‐activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca2+‐activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca2+ binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10–12 actins. As the number of FUs was decreased, Ca2+ sensitivity of force (pCa50) decreased proportionally. The slope of the force‐pCa relation (the Hill coefficient, nH) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, nH was reduced to 1.7 ± 0.2, compared with 3.8 ± 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near‐neighbour FUs along the thin filament (via head‐to‐tail tropomyosin interactions).

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.

Changes in thin filament structure induced by Ca(2+) binding to troponin and subsequent strong cross-bridge binding regulate additional strong cross-bridge attachment, force development, and dependence of force on sarcomere length in skeletal and cardiac muscle. Variations in activation properties account for functional differences between these muscle types.