Biomedical subjects

D A Martyn

Publications and source records attributed to D A Martyn.

At least 19 recordsLinked to original sources

PubMed2004

Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle.

In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).

Adenosine Triphosphate↗

PubMed2002

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.

Actin Cytoskeleton↗

PubMed2001

Ca2+ - and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle.

Linear dichroism of 5'-tetramethylrhodamine (5'ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca2+-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the C-terminus with a cysteine inserted at site 98 (cTnC-C35S,C84S,S98C, cTnC(C98)). With 5'ATR-labeled cTnC(C84) and cTnC(C98) dichroism increased with increasing [Ca2+], while rigor cross-bridges caused dichroism to increase more with 5'ATR-labeled cTnC(C84) than cTnC(C98). The pCa50 values and n(H) from Hill analysis of the Ca2+-dependence of force and dichroism were 6.4 (+/-0.02) and 1.08 (+/-0.04) for force and 6.3 (+/-0.04) and 1.02 (+/-0.09) (n = 5) for dichroism in cTnC(C84) reconstituted trabeculae. Corresponding data from cTnC(C98) reconstituted trabeculae were 5.53 (+/-0.03) and 3.1 (+/-0.17) for force, and 5.39 (+/-0.03) and 1.87 (+/-0.17) (n = 5) for dichroism. The contribution of active cycling cross-bridges to changes in cTnC structure was determined by inhibition of force to 6% of pCa 4.0 controls with 1.0 mM sodium vanadate (Vi). With 5'ATR-labeled cTnC(C84) Vi caused both the pCa50)of dichroism and the maximum value at pCa 4.0 to decrease, while with 5'ATR-labeled cTnC(C98) the pCa50 of dichroism decreased with no change of dichroism at pCa 4.0. The dichroism of 5'ATR-labeled cTnC(C35) was insensitive to either Ca2+ or strong cross-bridges. These data suggest that both Ca2+ and cycling cross-bridges perturb the N-terminal structure of cTnC at Cys-84, while C-terminal structure is altered by site II Ca2+-binding, but not cross-bridges.

Animals↗

PubMed2001

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle.

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.

Animals↗

PubMed1999

Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition.

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].

Actomyosin↗

PubMed1999

Contractile properties of rabbit psoas muscle fibres inhibited by beryllium fluoride.

The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg.ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeFx vs. A1F4- or Vi). The BeFx bound state is thought to mimic the weak actin-binding M.ATP complex, while the states with A1F4- or Vi bound mimic the M.ADP.Pi state. While the effects of A1F4- and Vi on fibre mechanics have been previously described (Chase et al., 1994, 1993), the effects of BeFx have not been characterized in detail. At pCa 4.5 (12 degrees C), we measured (i) steady-state isometric tension, (ii) stiffness (KS; 1 kHz sinusoids), and (iii) unloaded shortening velocity (Vu; slack test) in single skinned muscle fibres from rabbit psoas. Results were compared when tension was inhibited with either BeFx or 2,3-butanedione-monoxime (BDM) or modulated by altering myoplasmic [Ca2+]. With 3 mM total fluoride, 1 mM BeFx inhibited both tension and KS by approximately 50% (compared to 7-10 mM BDM and 50-100 microM A1F4-). Increasing [BeFx] to 10 mM further reduced tension to approximately 15% P0, but had little further effect on KS; with BDM and altered [Ca2+], KS scaled more proportionately with tension. Inhibition of tension and KS by BeFx was more rapidly reversible, compared with slow recovery from tension inhibition with A1F4- or Vi. Vu exhibited a complex dependence on [BeFx], being relatively unaffected by concentrations < or = 1 mM, and becoming inhibited steeply for [BeFx] above this level. With BDM, Vu co-varied more directly with force. Our results suggest that BeFx may induce a different cross-bridge state in fibres than do A1F4- or Vi, but all three analogues of Pi form complexes that mimic crossbridge states that follow ATP hydrolysis.

Animals↗

PubMed1998

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.

Adenosine Triphosphate↗

PubMed1997

Characterization of troponin-C interactions in skinned barnacle muscle: comparison with troponin-C from rabbit striated muscle.

Previously it was shown that when troponin-C (TnC) is extracted from barnacle myofibrillar bundles they lose their Ca2+ sensitivity, which can be restored by adding back barnacle TnC (either isoform, BTnC1 or BTnC2). Thus barnacle muscle shows thin filament regulation, as does rabbit psoas skeletal muscle. In this paper we compare the interactions of barnacle and rabbit fast muscle TnC in their respective muscles. We demonstrate that muscle fibres from the giant barnacle, Balanus nubilus, contain about 186 microM kg-1 muscle tissue of BTnC1 plus BTnC2 compared to about 91 microM kg-1 of TnC in rabbit psoas muscle fibres. Extraction of BTnC is achieved using similar low ionic strength, low divalent ion Ca(2+)-low Mg2+ conditions which are required for TnC extraction in rabbit psoas skinned muscle fibres; extraction was prevented by 1 mM Mg2+. Full reconstitution of Ca(2+)-sensitivity was achieved by adding back BTnC (1 + 2, or 2). Reconstitution of barnacle muscle with rabbit fast skeletal TnC (RTnC) was more complex, with partial recovery of Ca(2+)-sensitivity with reconstitution in the presence of 3 mM Mg2+ and more fully with reconstitution in the presence of activating Ca2+ (pCa 4.0). This suggests that the barnacle TnC-TnI (troponin I) recognition sites may be more complex than in rabbit because the barnacle sites appear to have at least two different conformations or types, in which one recognizes RTnC in the presence of Mg2+ and the other only in the presence of Ca2+ and Mg2+. This is consistent with the presence of several TnI isoforms in barnacle striated myofibrils. RTnC has two C-terminal Ca(2+)-Mg2+ binding sites that are thought to be involved in the Mg(2+)-sensitive binding of RTnC in rabbit muscle, yet it has been suggested that this site in barnacle muscle does not bind Mg2+, even though Mg2+ stabilizes BTnC binding in barnacle muscle. Consistent with this stabilizing action of Mg2+, using fluorescent probes IAANS or IAE on isolated BTnC2 we demonstrate that BTnC2 binds both Ca2+ and Mg2+, but the data do not suggest direct competition. Consistent with the C-terminal sites on BTnC being Ca(2+)-specific, BTnC1 + 2 could only reconstitute low levels of force (about 1/3) in TnC-extracted rabbit skinned muscle fibers in the presence of pCa 4.0 (not just Mg2+) and only at low ionic strengths (0.09 M). Ca(2+)-activation of contraction was further examined using fluorescently labelled BTnC2 (labelled with IANBD) incorporated into skinned barnacle myofibrillar bundles. Maximal Ca2+ binding produced structural changes in BTnC which resulted in a 45% decrease in the fluorescence compared to the value at pCa 9.2. The magnitude of the fluorescence decrease paralleled the increase in force with increasing Ca2+. The Hill fits to the data gave pCa1/2 and n of 5.61 +/- 0.02 and 2.06 +/- 0.12 for force, and 5.52 +/- 0.02 and 1.88 +/- 0.10 for fluorescence. Removing MgATP to induce rigor in the fibre decreased BTnC2-NBD fluorescence only about 11%, but the addition of Ca2+ in rigor further decreased the fluorescence to a slightly larger extent than under maximal Ca2+ activating conditions. These fluorescence changes are qualitatively similar to the fluorescence enhancement seen with Ca(2+)-activation and rigor with RTnCDanz exchanged into rabbit psoas skinned muscle fibres. The data support a similar model for Ca(2+)-activation of force in barnacle muscle and in rabbit psoas skeletal muscle fibres.

Animals↗

PubMed1996

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.

Actomyosin↗

PubMed1996

Influence of Ca2+ on force redevelopment kinetics in skinned rat myocardium.

The influence of Ca2+ on isometric force kinetics was studied in skinned rat ventricular trabeculae by measuring the kinetics of force redevelopment after a transient decrease in force. Two protocols were employed to rapidly detach cycling myosin cross-bridges: a large-amplitude muscle length ramp followed by a restretch back to the original length or a 4% segment length step. During the recovery of force, the length of the central region of the muscle was controlled by using a segment marker technique and software feedback control. Tension redevelopment was fit by a rising exponential governed by the rate constant ktr for the ramp/restretch protocol and kstep for the step protocol. ktr and kstep averaged 7.06 s-1 and 15.7 s-1, respectively, at 15 degrees C; neither ktr nor kstep increased with the level of Ca2+ activation. Similar results were found at submaximum Ca2+ levels when sarcomere length control by laser diffraction was used. The lack of activation dependence of ktr contrasts with results from fast skeletal fibers, in which ktr varies 10-fold from low to high activation levels, and suggests that Ca2+ does not modulate the kinetics of cross-bridge attachment or detachment in mammalian cardiac muscle.

Adenosine Triphosphate↗

PubMed1995

Faster force transient kinetics at submaximal Ca2+ activation of skinned psoas fibers from rabbit.

The early, rapid phase of tension recovery (phase 2) after a step change in sarcomere length is thought to reflect the force-generating transition of myosin bound to actin. We have measured the relation between the rate of tension redevelopment during phase 2 (r), estimated from the half-time of tension recovery during phase 2 (r = t0.5(-1)), and steady-state force at varying [Ca2+] in single fibers from rabbit psoas. Sarcomere length was monitored continuously by laser diffraction of fiber segments (length approximately 1.6 mm), and sarcomere homogeneity was maintained using periodic length release/restretch cycles at 13-15 degrees C. At lower [Ca2+] and forces, r was elevated relative to that at pCa 4.0 for both releases and stretches (between +/- 8 nm). For releases of -3.4 +/- 0.7 nm.hs-1 at pCa 6.6 (where force was 10-20% of maximum force at pCa 4.0), r was 3.3 +/- 1.0 ms-1 (mean +/- SD; N = 5), whereas the corresponding value of r at pCa 4.0 was 1.0 +/- 0.2 ms-1 for releases of -3.5 +/- 0.5 nm.hs-1 (mean +/- SD; N = 5). For stretches of 1.9 +/- 0.7 nm.hs-1, r was 1.0 +/- 0.3 ms-1 (mean +/- SD; N = 9) at pCa 6.6, whereas r was 0.4 +/- 0.1 ms-1 at pCa 4.0 for stretches of 1.9 +/- 0.5 (mean +/- SD; N = 14). Faster phase 2 transients at submaximal Ca(2+)-activation were not caused by changes in myofilament lattice spacing because 4% Dextran T-500, which minimizes lattice spacing changes, was present in all solutions. The inverse relationship between phase 2 kinetics and force obtained during steady-state activation of skinned fibers appears to be qualitatively similar to observations on intact frog skeletal fibers during the development of tetanic force. The data are consistent with models that incorporate a direct effect of [Ca2+] on phase 2 kinetics of individual cross-bridges or, alternatively, in which phase 2 kinetics depend on cooperative interactions between cross-bridges.

Animals↗

PubMed1994

Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride, a slowly dissociating analogue of inorganic phosphate, in chemically skinned fibres from rabbit psoas muscle.

To examine the mechanism by which aluminiofluoride, a tightly binding analogue of inorganic phosphate, inhibits force in single, chemically skinned fibres from rabbit psoas muscle, we measured the Ca(2+)-dependence of the kinetics of inhibitor dissociation and the kinetics of actomyosin interactions when aluminiofluoride was bound to the crossbridges. The relation between stiffness and the speed of stretch during small amplitude ramp stretches (< 5 nm per h.s.) was used to characterize the kinetic properties of crossbridges attached to actin; sarcomere length was assessed with HeNe laser diffraction. During maximum Ca(2+)-activation at physiological ionic strength (pCa 4.0, 0.2 M gamma/2), stiffness exhibited a steep dependence on the rate of stretch; aluminiofluoride inhibition at pCa 4.0 (0.2 M gamma/2) resulted in an overall decrease in stiffness, with stiffness at high rates of stretch (10(3)-10(4) nm per h.s. per s) being disproportionately reduced. Thus the slope of the stiffness-speed relation was reduced during aluminiofluoride inhibition of activated fibres. Relaxation of inhibited fibres (pCa 9.2, 0.2 M gamma/2) resulted in aluminiofluoride being 'trapped' and was accompanied by a further decrease in stiffness at all rates of stretch which was comparable to that found in control relaxed fibres. In relaxed, low ionic strength conditions (pCa 9.2, 0.02 M gamma/2) which promote weak crossbridge binding, stiffness at all rates of stretch was significantly inhibited by aluminiofluoride 'trapped' in the fibre. To determine the Ca(2+)-dependence of inhibitor dissociation, force was regulated independent of Ca2+ using an activating troponin C (aTnC). Results obtained with a TnC-activated fibres confirmed that there is no absolute requirement for Ca2+ for recovery from force inhibition by inorganic phosphate analogues in skinned fibres; the only requirement is thin filament activation which enables active crossbridge cycling. These results indicate that aluminiofluoride preferentially inhibits rapid equilibrium or weak crossbridge attachment to actin, that aluminiofluoride-bound crossbridges attach tightly to the activated thin filament, and that, at maximal (or near-maximal) activation, crossbridge attachment to actin prior to inorganic phosphate analogue dissociation is the primary event regulated by Ca2+.

Actomyosin↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1993

Force-calcium relations in skinned twitch and slow-tonic frog muscle fibres have similar sarcomere length dependencies.

The sarcomere length (SL) dependence of the calcium sensitivity of force was measured in skinned single twitch and slow-tonic muscle fibres from frog and toad. Twitch and slow-tonic fibres were characterized by location, appearance, physiological response to calcium and by protein band patterns from sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Force-calcium relations were determined for each fibre type at two sarcomere lengths, 2.4 and 3.1 microns. Bathing solution ionic strength (IS) was 200 mM and solution pH was 7.0, 6.0 or 5.5; experiments were also done at IS = 120 mM and pH 7.0. At all pHs and ionic strengths tested, slow-tonic fibres exhibited a slower time course of force development and were more sensitive to calcium than were twitch fibres. Lowering IS increased calcium sensitivity and lowering pH decreased calcium sensitivity in both fibre types. Increasing SL increased the calcium sensitivity of force in both twitch and slow-tonic fibres at pH 7.0 and at both 200 and 120 mM IS. Lowering pH caused a decrease in the length dependence of calcium sensitivity of both fibre types; at pH 5.5 the calcium sensitivity of force in slow-tonic fibres exhibited a slight decrease with increasing SL.

Animals↗

PubMed1993

Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C.

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.

Animals↗

PubMed1993

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.

Aluminum↗

PubMed1993

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.

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