Donald A. Martyn

Length-dependent Ca 2+ activation in cardiac muscle: some remaining questions

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank–Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca2+ sensitivity. How sarcomere length modulates Ca2+ sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca2+ sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca2+ sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin–myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca2+ sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca2+ sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca2+ sensitivity.

Calcium Regulation of Tension Redevelopment Kinetics with 2-Deoxy-ATP or Low [ATP] in Rabbit Skeletal Muscle

The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.5 mM, respectively. The activation dependence of k(tr) and unloaded shortening velocity (Vu) was measured with each substrate. With 5.0 mM ATP, Vu depended linearly on tension (P), whereas k(tr) exhibited a nonlinear dependence on P, being relatively independent of P at submaximum levels and rising steeply at P > 0.6-0.7 of maximum tension (Po). With dATP, Vu was 25% greater than control at Po and was elevated at all P > 0.15Po, whereas Po was unchanged. Furthermore, the Ca(+2) sensitivity of both k(tr) and P increased, such that the dependence of k(tr) on P was not significantly different from control, despite an elevation of Vu and maximal k(tr). In contrast, lowering [ATP] caused a slight (8%) elevation of Po, no change in the Ca(+2) sensitivity of P, and a decrease in Vu at all P. Moreover, k(tr) was decreased relative to control at P > 0.75Po, but was elevated at P < 0.75Po. These data demonstrate that the cross-bridge cycling rate dominates k(tr) at maximum but not submaximum levels of Ca(2+) activation.

Isometric force redevelopment of skinned muscle fibers from rabbit activated with and without Ca2

Fiber isometric tension redevelopment rate (kTR) was measured during submaximal and maximal activations in glycerinated fibers from rabbit psoas muscle. In fibers either containing endogenous skeletal troponin C (sTnC) or reconstituted with either purified cardiac troponin C (cTnC) or sTnC, graded activation was achieved by varying [Ca2+]. Some fibers were first partially, then fully, reconstituted with a modified form of cTnC (aTnC) that enables active force generation and shortening in the absence of Ca2+. kTR was derived from the half-time of tension redevelopment. In control fibers with endogenous sTnC, kTR increased nonlinearly with [Ca2+], and maximal kTR was 15.3 +/- 3.6 s-1 (mean +/- SD; n = 26 determinations on 25 fibers) at pCa 4.0. During submaximal activations by Ca2+, kTR in cTnC reconstituted fibers was approximately threefold faster than control, despite the lower (60%) maximum Ca(2+)-activated force after reconstitution. To obtain submaximal force with aTnC, eight fibers were treated to fully extract endogenous sTnC, then reconstituted with a mixture of a TnC and cTnC (aTnC:cTnC molar ratio 1:8.5). A second extraction selectively removed cTnC. In such fibers containing aTnC only, neither force nor kTR was affected by changes in [Ca2+]. Force was 22 +/- 7% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 24 +/- 8% (mean +/- SD; n = 8) at pCa 4.0, whereas kTR was 98 +/- 14% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 96 +/- 15% (mean +/- SD; n = 8) at pCa 4.0. Maximal reconstitution of fibers with aTnC alone increased force at pCa 9.2 to 69 +/- 5% of maximum control (mean + SD; n = 22 determinations on 13 fibers) and caused a small but significant reduction of kTR to 78 +/- 8% of maximum control (mean +/- SD; n = 22 determinations on 13 fibers); neither force nor krR was significantly affected by Ca>2(pCa 4.0). Taken together, we interpret our results to indicate that kTR reflects the dynamics of activation of individual thin filament regulatory units and that modulation of kTR by Ca> is effected primarily by Ca>+ binding to TnC.

Length and myofilament spacing-dependent changes in calcium sensitivity of skeletal fibres: effects of pH and ionic strength

SummaryThe calcium sensitivity of force was measured in glycerinated rabbit psoas fibres at sarcomere lengths (SL) from 2.3 to 3.4 Μm. Increasd SL caused calcium sensitivity to increase and the slope of force-calcium relations to decrease. We have hypothesized that length-dependent changes in myofilament lattice spacing and the presence of fixed charge on the myofilaments are important in determining calcium sensitivity. Lattice spacing changes were monitored by measuring fibre diameter (D). D was decreased by increasing SL, decreasing bathing solution pH and by osmotic compression with 3% PVP. 3% PVP caused D to decrease by about 15% at all SLs and pH values tested. Force-calcium relations were measured at different SLs and pH values, with and without 3% PVP in the bathing solutions. At all pH values D at SL 2.3 Μm with 3% PVP was comparable to the value at 3.4 Μm, without PVP. At pH 7.5 and 7.0 calcium sensitivity was about the same at both SL, although the slope of the force-calcium relation was less at longer SL. The similarity of the calcium sensitivity at the same D, but much different SL, indicates that lattice spacing is important in determining calcium sensitivity, while SL and the degree of myofilament overlap are important in determing the slope of force-calcium relations. In order to test for the role of myofilament charge in determining calcium sensitivity, pH and ionic strength were varied. Decreasing pH caused decreased maximum force and calcium sensitivity. In addition, the influence of SL on calcium sensitivity decreased as pH was lowered, with minimal SL dependence at pH 5.5; even though lattice spacing still decreased with increasing SL. When D was decreased with PVP, calcium sensitivity increased at all SLs in pH 7.5 and 7.0 while the same lattice spacing changes at pH 6.0 and 5.5 resulted in greatly reduced shifts in calcium sensitivity. These results indicate that the effect of lattice spacing on calcium sensitivity depends on myofilament charge. At pH 6.0, even though osmotic compression of the lattice has no effect, increasing SL causes about half the shift in calcium sensitivity seen at pH 7.0. Lowering ionic strength from 200 to 110 mM caused an increase in both the magnitude and length dependence of calcium sensitivity at pH 7.0, while at pH 5.5 both decreased. Although changes in pH and ionic strength can alter crossbridge binding and kinetics, and the binding of calcium to troponin, as well as the magnitude of myofilament charge, the results indicate the importance of both lattice spacing and fixed myofilament charge in determining contractile sensitivity to calcium.

Investigation of thin filament near‐neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle

Ca2+‐dependent activation of striated muscle involves cooperative interactions of cross‐bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild‐type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca2+‐activated force (Fmax) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where Fmax increased in a greater than linear manner with WT sTnC, and suggests that Ca2+ binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca2+ sensitivity of force and rate of force redevelopment (ktr) was leftward shifted by 0.1–0.2 −log [Ca2+] (pCa) units as WT cTn content was increased, but the slope of the force–pCa relation and maximal ktr were unaffected by loss of near‐neighbour RU interactions. Cross‐bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy‐ATP) had no greater effect in cardiac muscle with disruption of near‐neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca2+ dissociation was found to be > 2‐fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca2+ binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca2+ binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca2+ binding affinity.

Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit.

1. We examined the effects of aluminofluoride (AlFx) and orthovanadate (Vi), tightly binding analogues of orthophosphate (Pi), on the mechanical properties of glycerinated fibres from rabbit psoas muscle. Maximum Ca(2+)‐activated force, stiffness, and unloaded shortening velocity (Vus) were measured under conditions of steady‐state inhibition (up to 1 mM of inhibitor) and during the recovery from inhibition. 2. Stiffness was measured using either step or sinusoidal (1 kHz) changes in fibre length. Sarcomere length was monitored continuously by helium‐neon laser diffraction during maximum Ca2+ activation. Stiffness was determined from the changes in sarcomere length and the corresponding changes in force. Vus was measured using the slack test method. 3. AlF chi and Vi each reversibly inhibited force, stiffness and Vus. Actively cycling cross‐bridges were required for reversal of these inhibitory effects. Recovery from inhibition by AlF chi was 3‐ to 4‐fold slower than that following removal of V1. 4. At various degrees of inhibition, AlF chi and Vi both inhibited steady‐state isometric force more than either Vus or stiffness. For both AlF chi and Vi, the relatively greater inhibition of force over stiffness persisted during recovery from steady‐state inhibition. We interpret these results to indicate that the cross‐bridges with AlF chi or Vi bound are analogous to those which occur early in the cross‐bridge cycle.

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.

A simple model with myofilament compliance predicts activation-dependent crossbridge kinetics in skinned skeletal fibers.

The contribution of thick and thin filaments to skeletal muscle fiber compliance has been shown to be significant. If similar to the compliance of cycling cross-bridges, myofilament compliance could explain the difference in time course of stiffness and force during the rise of tension in a tetanus as well as the difference in Ca(2+) sensitivity of force and stiffness and more rapid phase 2 tension recovery (r) at low Ca(2+) activation. To characterize the contribution of myofilament compliance to sarcomere compliance and isometric force kinetics, the Ca(2+)-activation dependence of sarcomere compliance in single glycerinated rabbit psoas fibers, in the presence of ATP (5.0 mM), was measured using rapid length steps. At steady sarcomere length, the dependence of sarcomere compliance on the level of Ca(2+)-activated force was similar in form to that observed for fibers in rigor where force was varied by changing length. Additionally, the ratio of stiffness/force was elevated at lower force (low [Ca(2+)]) and r was faster, compared with maximum activation. A simple series mechanical model of myofilament and cross-bridge compliance in which only strong cross-bridge binding was activation dependent was used to describe the data. The model fit the data and predicted that the observed activation dependence of r can be explained if myofilament compliance contributes 60-70% of the total fiber compliance, with no requirement that actomyosin kinetics be [Ca(2+)] dependent or that cooperative interactions contribute to strong cross-bridge binding.

Ca2+ and segment length dependence of isometric force kinetics in intact ferret cardiac muscle.

The influence of Ca2+ and sarcomere length on myocardial crossbridge kinetics was studied in ferret papillary muscle by measuring the rate of force redevelopment following a rapid length step that dropped the force to zero. Tetanic stimulation with 5 mumol/L ryanodine was used to obtain a steady-state contraction, and segment length was measured and controlled using a sense-coil technique that measures changes in the cross-sectional area of the central region of the muscle. The rate constant for the recovery of force (ktr) following a rapid length release was obtained by fitting the data with a single exponential function. Contrary to results from skinned skeletal fibers in which ktr increases almost 10-fold from low to maximal activation levels, ktr was found not to increase at higher activation levels in this study. Similarly, although force increased with segment length under all conditions, ktr never increased with length. Data presented here are consistent with a model of myocardial Ca2+ activation in which Ca2+ modulates the number of crossbridges interacting with the thin filament and are inconsistent with a model in which Ca2+ modulates the kinetics of transitions to force producing states within the actomyosin cycle. Differences in the activation dependence of the force redevelopment rate between cardiac and skeletal muscle suggest that there are fundamental differences in the mechanism of Ca2+ activation between these two muscle types.

UNLOADED SHORTENING OF SKINNED MUSCLE FIBERS FROM RABBIT ACTIVATED WITH AND WITHOUT CA2

Unloaded shortening velocity (VUS) was determined by the slack method and measured at both maximal and submaximal levels of activation in glycerinated fibers from rabbit psoas muscle. Graded activation was achieved by two methods. First, [Ca2+] was varied in fibers with endogenous skeletal troponin C (sTnC) and after replacement of endogenous TnC with either purified cardiac troponin C (cTnC) or sTnC. Alternatively, fibers were either partially or fully reconstituted with a modified form of cTnC (aTnC) that enables force generation and shortening in the absence of Ca2+. Uniformity of the distribution of reconstituted TnC across the fiber radius was evaluated using fluorescently labeled sTnC and laser scanning fluorescence confocal microscopy. Fiber shortening was nonlinear under all conditions tested and was characterized by an early rapid phase (VE) followed by a slower late phase (VL). In fibers with endogenous sTnC, both VE and VL varied with [Ca2+], but VE was less affected than VL. Similar results were obtained after extraction of TnC and reconstitution with either sTnC or cTnC, except for a small increase in the apparent activation dependence of VE. Partial activation with aTnC was obtained by fully extracting endogenous sTnC followed by reconstitution with a mixture of aTnC and cTnC (aTnC:cTnC molar ratio 1:8.5). At pCa 9.2, VE and VL were similar to those obtained in fibers reconstituted with sTnC or cTnC at equivalent force levels. In these fibers, which contained aTnC and cTnC, VE and VL increased with isometric force when [Ca2+] was increased from pCa 9.2 to 4.0. Fibers that contained a mixture of a TnC and cTnC were then extracted a second time to selectively remove cTnC. In fibers containing aTnC only, VE and VL were proportional to the resulting submaximal isometric force compared with maximum Ca(2+)-activated control. With aTnC alone, force, VE, and VL were not affected by changes in [Ca2+]. The similarity of activation dependence of VUS whether fibers were activated in a Ca(2+)-sensitive or -insensitive manners implies that VUS is determined by the average level of thin filament activation and that, with sTnC or cTnC, VUS is affected by Ca2+ binding to TnC only.