Unloaded shortening of skinned muscle fibers from rabbit activated with and without Ca2+ (original) (raw)
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Isometric force redevelopment of skinned muscle fibers from rabbit activated with and without Ca2+
Biophysical Journal, 1994
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 [Call]. 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 Ca2+-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 aTnC 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 krR 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. .
Thin-filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres
The Journal of Physiology, 2007
Thin-filament regulation of isometric force redevelopment (k tr) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I-and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca 2+-activated force (rF max) decreased as the fraction of sTnC in sTnC : xxsTnC mixtures was reduced, but maximal k tr was unaffected until rF max was <0.2 of pre-extracted F max. In contrast, reconstitution with cTnC or xsTnC reduced maximal k tr to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF max of 0.68 ± 0.03 and 0.25 ± 0.02 F max. The k tr-pCa relation of fibres containing sTnC : xxsTnC mixtures (rF max > 0.2 F max) was little effected, though k tr was slightly elevated at low Ca 2+ activation. The magnitude of the Ca 2+-dependent increase in k tr was greatly reduced following cTnC or xsTnC reconstitution because k tr at low levels of Ca 2+ was elevated and maximal k tr was reduced. Solution Ca 2+ dissociation rates (k off) from whole Tn complexes containing sTnC (26 ± 0.1 s −1), cTnC (38 ± 0.9 s −1) and xsTnC (50 ± 1.2 s −1) correlated with k tr at low Ca 2+ levels and were inversely related to rF max. At low Ca 2+ activation, k tr was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k tr at any [Ca 2+ ] and suggest Ca 2+ activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.
The Journal of Physiology, 2005
We studied the relative contributions of Ca 2+ binding to troponin C (TnC) and myosin binding to actin in activating thin filaments of rabbit psoas fibres. The ability of Ca 2+ to activate thin filaments was reduced by replacing native TnC with cardiac TnC (cTnC) or a site I-inactive skeletal TnC mutant (xsTnC). Acto-myosin (crossbridge) interaction was either inhibited using N -benzyl-p-toluene sulphonamide (BTS) or enhanced by lowering [ATP] from 5.0 to 0.5 mM. Reconstitution with cTnC reduced maximal force (F max ) by ∼1/3 and the Ca 2+ sensitivity of force (pCa 50 ) by 0.17 unit (P < 0.001), while reconstitution with xsTnC reduced F max by ∼2/3 and pCa 50 by 0.19 unit (P < 0.001). In both cases the apparent cooperativity of activation (n H ) was greatly decreased. In control fibres 3 µM BTS inhibited force to 57% of F max while in fibres reconstituted with cTnC or xsTnC, reconstituted maximal force (rF max ) was inhibited to 8.8% and 14.3%, respectively. Under control conditions 3 µM BTS significantly decreased the pCa 50 , but this effect was considerably reduced in cTnC reconstituted fibres, and eliminated in xsTnC reconstituted fibres. In contrast, when crossbridge cycle kinetics were slowed by lowering [ATP] from 5 to 0.5 mM in xsTnC reconstituted fibres, pCa 50 and n H were increased towards control values. Combined, our results demonstrate that when the ability of Ca 2+ binding to activate thin filaments is compromised, the relative contribution of strong crossbridges to maintain thin filament activation is increased. Furthermore, the data suggest that at low levels of Ca 2+ , the level of thin filament activation is determined primarily by the direct effects of Ca 2+ on tropomyosin mobility, while at higher levels of Ca 2+ the final level of thin filament activation is primarily determined by strong cycling crossbridges.
The Journal of Physiology, 2008
The influence of Ca 2+ binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca 2+ dissociation rates (k off ) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca 2+ at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca 2+ , the rate of tension redevelopment (k TR ) was not altered for fibres containing sTnC mutants with decreased k off or mixtures of sTnC:xxsTnC. We examined the influence of k off on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca 2+ ]. In myofibrils with M80Q sTnC F27W (decreased k off ), maximal tension, activation rate (k ACT ), k TR and rates of relaxation were not altered. With I60Q sTnC F27W (increased k off ), maximal tension, k ACT and k TR decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased k off , respectively. For all sTnC reconstitution conditions, Ca 2+ dependence of k TR in fibres showed Ca 2+ sensitivity of k TR (pCa 50 ) shifted parallel to tension and low-Ca 2+ k TR was elevated. Together the data suggest the Ca 2+ -dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by k off of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca 2+ .
The Journal of General Physiology, 1985
The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction ...
The Journal of general …, 1990
Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC 2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca 2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC 2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KC1, and either imidazole or PO42-as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC~ resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca 2+] by as much as 0.2 pCa units after LC~ extraction. At low concentrations of Ca 2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC~ to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC~. LCz extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca 2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-$2 hinge region of myosin.
Ca 2+ dependence of loaded shortening in rat skinned cardiac myocytes and skeletal muscle fibres
The Journal of Physiology, 2000
This study examined the effects of activator Ca¥ on loaded shortening and power output in skinned rat cardiac myocyte preparations, and fast-and slow-twitch skeletal muscle fibres at 12°C. 2. Shortening velocities were slowed at nearly all relative loads when Ca¥ activation levels were reduced to •70% maximal isometric force (P4•5) in cardiac myocyte preparations, as well as in fast-twitch and slow-twitch skeletal muscle fibres. 3. Peak absolute power outputs declined significantly as Ca¥ activation levels were progressively reduced from maximal to 30% P4•5 in all three striated muscle types, with the greatest change in fast-twitch fibres. In cardiac myocyte preparations, even peak relative power output progressively fell when Ca¥ activation levels were lowered to •70, 50 and 30% P4•5. Peak relative power output also progressively fell in fast-twitch fibres as Ca¥ activation levels were lowered from maximal down to 50% P4•5. However, in slow-twitch fibres, peak relative power output decreased only at 70% P4•5 and then remained unchanged with further reductions in Ca¥ activation levels. The greater Ca¥ dependence of peak relative power output in cardiac myocytes and fast-twitch fibres may arise from a shared mechanism such as cooperative inactivation of the thin filament, which is likely to be slowest in less cooperative slow-twitch fibres. 4. During submaximal Ca¥ activations, the time course of shortening became markedly curvilinear during isotonic shortening in all three muscle types. The progressive slowdown in shortening velocity during isotonic contractions was greatest in fast-twitch fibres, consistent with the higher degree of cooperativity of Ca¥ activation in fast-twitch fibres. Additionally, fast-twitch and slow-twitch fibre stiffness decreased in concert with the curvature of length traces during loaded shortening. These results are consistent with the idea that cooperative inactivation of the thin filament occurs during loaded shortening and such a mechanism may contribute to the progressive slowing and overall Ca¥ dependence of loaded shortening velocity.
The Journal of physiology, 2007
We studied how enhanced skeletal troponin C (sTnC) Ca2+-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca2+ dissociation, resulting in the following order of rates: wild-type (WT) sTnC-sTn>sTnC(F27W)-sTn>M80Q sTnC-sTn>M80Q sTnCF27W-sTn. Reconstitution of sTnC-extracted fibres increased Ca2+ sensitivity of steady-state force (pCa(50)) by 0.08 for M80Q sTnC, 0.15 for sTnCF27W and 0.32 for M80Q sTnCF27W with minimal loss of slope (nH, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca2+ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC...
Biophysical Journal, 1999
In maximally activated skinned fibers, the rate of tension redevelopment (k tr ) following a rapid release and restretch is determined by the maximal rate of cross-bridge cycling. During submaximal Ca 2ϩ activations, however, k tr regulation varies with thin filament dynamics. Thus, decreasing the rate of Ca 2ϩ dissociation from TnC produces a higher k tr value at a given tension level (P), especially in the [Ca 2ϩ ] range that yields less than 50% of maximal tension (P o ). In this study, native rabbit TnC was replaced with chicken recombinant TnC, either wild-type (rTnC) or mutant (NHdel), with decreased Ca 2ϩ affinity and an increased Ca 2ϩ dissociation rate (k off ). Despite marked differences in Ca 2ϩ sensitivity (Ͼ0.5 ⌬pCa 50 ), fibers reconstituted with either of the recombinant proteins exhibited similar k tr versus tension profiles, with k tr low (1-2 s Ϫ1 ) and constant up to ϳ50% P o , then rising sharply to a maximum (16 Ϯ 0.8 s Ϫ1 ) in fully activated fibers. This behavior is predicted by a four-state model based on coupling between cross-bridge cycling and thin filament regulation, where Ca 2ϩ directly affects only individual thin filament regulatory units. These data and model simulations confirm that the range of k tr values obtained with varying Ca 2ϩ can be regulated by a rate-limiting thin filament process.