Regulatory roles of MgADP and calcium in tension development of skinned cardiac muscle

We investigated the regulatory roles of MgADP and free Ca2+ in isometric tension development in skinned bovine cardiac muscle. We found that, in the relaxed state without free Ca2+, MgADP elicited a sigmoidal increase in active tension, as is the case in skeletal muscle (ADP-contraction). The critical MgADP concentration, at which the tension increment became half-maximal, increased in proportion to MgATP concentration, with a slope of approximately 1 for cardiac and 4 for skeletal muscle. Raising the free Ca2+ concentration decreased the critical MgADP concentration in proportion to the free Ca2+ concentration. In addition, the apparent Ca2+ sensitivity of tension development increased with MgADP, while decreasing with inorganic phosphate (Pi); MgADP suppressed the Ca2+- desensitizing effect of Pi in a concentration-dependent manner. These activating effects of MgADP were quantitatively assessed by means of a model based upon the kinetic scheme of actomyosin ATPase. These experimental results and model simulation suggest that the state of thin filaments is synergistically regulated by both the binding of Ca2+ to troponin and the formation of the actomyosin–ADP complex.     

Regulation of contraction in striated muscle

Ca(2+) 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 (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) 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 Ca(2+)-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 A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) 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 Ca(2+) 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 A(7)TmTn 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 ([Ca(2+)]) 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 Ca(2+) 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. (ABSTRACT TRUNCATED)     

Filament overlap affects TnC extraction from skinned muscle fibres

Summary Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca²⁺/Mg²⁺ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 m were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 m with 20 mm Tris, pH 7.8, 5 mm EDTA. The decrease in the post-extraction maximum Ca²⁺ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40–120 m, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca²⁺ binding and that under the conditions used here, they retard its extraction.     

The troponin I: inhibitory peptide uncouples force generation and the cooperativity of contractile activation in mammalian skeletal muscle

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca²⁺-activated force and reduces the effectiveness Ca²⁺ as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca²⁺-activated force and increase [Ca²⁺] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca²⁺ and RXB activated muscle contraction. Because maximum force can be reduced with no change—or even an increase—in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.     

Inorganic Phosphate Affects the pCa–Force Relationship More than the pCa–ATPase by Increasing the Rate of Dissociation of Force Generating Cross-Bridges in Skinned Fibers from Both EDL and Soleus Muscles of the Rat

The effect of inorganic phosphate (Pi) on Ca2+ -activation of actomyosin ATPase activity and force in permeabilized (skinned) single extensor digitorum longus (EDL) and soleus muscle fibers of the rat were investigated. Increasing concentrations of Pi decreased force more than ATPase rate at all Ca2+ concentrations and this effect was more pronounced at submaximal Ca2+ -activation. Increasing Pi caused both the normalized pCa-ATPase and pCa-force relationship to be shifted to a higher Ca2+ concentration. At all Ca2+ concentrations ATPase was activated at a lower concentration of Ca2+ than force and this difference in Ca2+ concentration required for the activation of ATPase and force was greater in fast-twitch (EDL) than in slow twitch (soleus) muscle. Soleus muscle pCa-ATPase and pCa-force curves were more sensitive to Ca2+ (pCa50 = 5.97 and 5.89, respectively) than EDL (pCa50 = 5.68 and 5.54, respectively). Finally the shape of the pCa-ATPase and pCa-force curves was similar and not affected by Pi. Analysis shows that Pi increases the rate of dissociation of force generating myosin cross-bridges (ratio of ATPase/force (g(app at all Ca2+ concentration, especially at submaximal Ca2+ -activation levels. Pi effects on g(app) are discussed in terms Pi interacting with the isomerization high force AM*ADP states to form high force transitional AM*ADP*Pi* states which facilitate the dissociation of ADP from AM*ADP. Increasing Ca2+ during Ca2+ -activation of the fibers is associated with a progressive decrease in rate of dissociation of force generating myosin cross-bridges g(app).     

Calcium- and myosin-dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca²⁺-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca²⁺ dependence as active force production, with a Hill coefficient ( n _H) close to 3, consistent with a single co-operative transition controlled by Ca²⁺ binding. Complete inhibition of active force by 25 μ m blebbistatin had very little effect on the Ca²⁺-dependent structural changes and in particular did not significantly reduce the value of n _H. Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca²⁺ also changed the orientation of the C and E helices, and addition of Ca²⁺ in rigor produced further changes characterized by increased Ca²⁺ affinity but with n _H close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca²⁺ regulation of cardiac contractility must therefore be intrinsic to the thin filaments.     

Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

Role of myofibrillar creatine kinase in the relaxation of rigor tension in skinned cardiac muscle

In the absence of creatine phosphate, MgATP produced relaxation of rigor tension in chemically-skinned right papillary muscles of the rat, the half maximal effect being obtained at 1.8 mM MgATP. In the presence of 12 mM creatine phosphate and 250 M ADP, a decrease in MgATP concentration even to 10^–9 M never induced rigor tension. At a very low MgATP concentration (10^–6 M), the half maximal relaxing effect was obtained with 2 mM creatine phosphate, a value close to theK _m of isolated MM-creatine kinase for this substrate, or with 14 M MgADP, a value 5 times lower than the reportedK _m. An exogenous MgATP regenerating system (phosphoenol pyruvate + pyruvate kinase) was not able to fully relax the fibres. When MM-creatine kinase was inhibited by fluorodinitrobenzene, the dependency of rigor tension on MgATP became the same as it was without creatine phosphate. After washing out the fluorodinitrobenzene the addition of exogenous MM-creatine kinase for half an hour fully relaxed rigor tension; moreover, this effect persisted even after prolonged washout. These results show that endogenous MM-creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a localized high MgATP/MgADP ratio; they also suggest the existence of rapidly exchangeable binding sites for MM-creatine kinase in cardiac myofibrils.     

Cross-bridge cycling in smooth muscle: a short review

This review is focused on the cross-bridge interaction of the organized contractile system of smooth muscle fibres. By using chemically skinned preparations the different enzymatic reactions of actin-myosin interaction have been associated with mechanical events. A rigor state has been identified in smooth muscle and the binding of ATP causes dissociation of rigor cross-bridges at rates slightly slower than those in skeletal muscle, but fast enough not to be rate-limiting for cross-bridge turn over in the muscle fibre. The release of inorganic phosphate (P_i) is associated with force generation, and this process is not rate-limiting for maximal shortening velocity (V_max) in the fully activated muscle. The binding of ADP to myosin is strong in the smooth muscle contractile system, a property that might be associated with the generally slow cross-bridge turn over. Both force and V_max are modulated by the extent of myosin light chain phosphorylation. Low levels of activation are considered to be associated with the recruitment of slowly cycling dephosphorylated cross-bridges which reduces shortening velocity. The attachment of these cross-bridge states in skinned smooth muscle can be regulated by cooperative mechanisms and thin filament associated systems. Smooth muscles exhibit a large diversity in their V_max and the individual smooth muscle tissue can alter its V_max under physiological conditions. The diversity and the long-term modulation of phenotype are associated with changes in myosin heavy and light chain isoform expression.     

The green tea polyphenol (−)-epigallocatechin-3-gallate inhibits magnesium binding to the C-domain of cardiac troponin C

Cardiac muscle contraction is activated via the single Ca²⁺-binding site (site II) in the N-domain of troponin C (cTnC). The two Ca²⁺/Mg²⁺ binding sites in the C-domain of cTnC (sites III and IV) have been considered to play a purely structural role in anchoring cTnC to the thin filament. However, several recent discoveries suggest a possible role of this domain in contractile regulation. The green tea polyphenol (?)-epigallocatechin 3-gallate (EGCg), which binds specifically to the C-domain of cTnC, reduces cardiac myofilament Ca²⁺ sensitivity along with maximum force and acto-myosin ATPase activity. We have determined the effect of EGCg on Ca²⁺ and Mg²⁺ binding to the C-domain of cTnC. In the absence of Mg²⁺ there was no significant effect of EGCg on the Ca²⁺–cTnC affinity. Surprisingly, in the presence of Mg²⁺ EGCg caused an increase in Ca²⁺ affinity for sites III and IV of cTnC. However, in the absence of Ca²⁺ the addition of EGCg caused a significant reduction in Mg²⁺–cTnC affinity. This reduction is presumably responsible for the increase in Ca²⁺–cTnC affinity produced by EGCg in the presence of Mg²⁺. We propose that the inhibitory effect of EGCg on myofilament Ca²⁺ activation may be related to an enhanced Ca²⁺–Mg²⁺exchange at sites III and IV of cTnC, which might reduce the myosin crossbridge dependent component of thin filament activation.     

Regulation of the cross-bridge cycle: the effects of MgADP,LC17 isoforms and telokin

This review summarizes the role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle and a potential physiological role for telokin. In tonic, compared with phasic, smooth muscles the affinity of cross-bridges is ～5 times higher for MgADP and the apparent second-order rate constant for MgATP is ～3 times lower. This gives rise to a large population of dephosphorylated cross-bridges in tonic smooth muscle. Such cross-bridges are thought to be major determinants of the different relaxation kinetics of the two types of smooth muscle and contribute to force maintenance at low levels of MLC₂₀ phosphorylation, termed ‘catch-like state’ (Somlyo & Somlyo 1967) or ‘latch’ (Dillon et al. 1981). The molecular basis of the different affinities for MgADP and MgATP between tonic and phasic smooth muscle myosin was explored by exchange of essential myosin light chain (LC₁₇) isoforms. In phasic bladder smooth muscle the exchange of LC_17b for LC_17a caused a significant decrease in the unloaded shortening velocity of non-phosphorylated, slowly cycling cross-bridges, suggesting that the LC₁₇ isoforms contribute to the nucleotide affinity of latch bridges. The role of telokin in Ca²⁺-desensitization in phasic smooth muscle is reviewed. Telokin, the independently expressed C-terminus of myosin light chain kinase, is extensively phosphorylated during forskolin- and 8-br-cGMP-induced relaxation in situ. Telokin accelerated dephosphorylation of the regulatory myosin light chain and relaxed rabbit ileum smooth muscle. The results suggest that telokin contributes to cAMP and/or cGMP kinase-mediated Ca²⁺-desensitization of phasic smooth muscles.     

Molecular mechanism of troponin-C function

Conclusions There is now a large body of evidence in support of the view that Ca²⁺ binding to the low affinity sites of TnC induces a movement of helices B and C away from helices A and D, thus opening a hydrophobic cavity, the site of interaction with TnI. Another site of similar structure is formed by the helical segments in the C-terminal domain. Both sites appear to interact with the inhibitory segment of TnI. Whereas the interactions at both sites are necessary for the full regulatory activity of TnC, the interaction at the C-terminal domain stabilizes the complex and that involving the N-terminal domain is directly linked to the release of inhibition. In the absence of Ca²⁺ the inhibitory region of TnI would preferentially bind to actin and on Ca²⁺ binding to sites I and II it would switch to the site in the N-terminal domain of TnC. Detachment of TnI from actin gives rise to further events in thin filament regulation.     

What does TnCDANZ fluorescence reveal about the thin filament state?

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnC_DANZ) increases upon activation with Ca²⁺. This fluorescence enhancement is due to Ca²⁺ binding to the Ca²⁺-specific binding sites of TnC_DANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca²⁺ binding to TnC_DANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnC_DANZ fluorescence. Weakly bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnC_DANZ fluorescence. Strongly attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.     

Regulating the contraction of insect flight muscle

The rapid movement of the wings in small insects is powered by the indirect flight muscles. These muscles are capable of contracting at up to 1,000?Hz because they are activated mechanically by stretching. The mechanism is so efficient that it is also used in larger insects like the waterbug, Lethocerus. The oscillatory activity of the muscles occurs a low concentration of Ca(2+), which stays constant as the muscles contract and relax. Activation by stretch requires particular isoforms of tropomyosin and the troponin complex on the thin filament. We compare the tropomyosin and troponin of Lethocerus and Drosophila with that of vertebrates. The characteristics of the flight muscle regulatory proteins suggest ways in which stretch-activation works. There is evidence for bridges between troponin on thin filaments and myosin crossbridges on the thick filaments. Recent X-ray fibre diffraction results suggest that a pull on the bridges activates the thin filament by shifting tropomyosin from a blocking position on actin. The troponin bridges are likely to contain extended sequences of tropomyosin or troponin I (TnI). Flight muscle has two isoforms of TnC with different Ca(2+)-binding properties: F1 TnC is needed for stretch-activation and F2 TnC for isometric contractions. In this review, we describe the structural changes in both isoforms on binding Ca(2+) and TnI, and discuss how the steric model of muscle regulation can apply to insect flight muscle.     

Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV

 This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2–4-) in which the Ca²⁺-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca²⁺ binding at both Ca²⁺-binding sites of barnacle TnC did not prevent the incorporation of BTnC2–4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg²⁺ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca²⁺-binding sites. Competition experiments between BTnC2–4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC₂ indicate that BTnC2–4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC₂. These results indicate that disabling sites II and IV changes the affinity of BTnC2–4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament. Received: 30 September 1998 / Received after revision: 12 February 1999 / Accepted: 15 February 1999     

Series elastic properties of skinned muscle fibres in contraction and rigor

Summary Isometric tension of skinned fibres from the frog semitendinosus muscle is sigmoidally related to Ca²⁺ concentration betweenpCa 7 and 6. Stiffness measurements showed that the Ca²⁺-activated tension may be due to recruitment of attached cross-bridges. In the absence of ATP (rigor solution) the skinned fibre develops a rigor tension which reaches about 80–110% of the maximum Ca²⁺-activated tension.However, stiffness measurements showed that in rigor many more cross-bridges are attached to actin at any one moment than in contraction. It was concluded that the force per cross-bridge is 37% smaller in rigor than in contraction.     

Modulation of actomyosin motor function by 1-hexanol

This study examines the effects of 1-hexanol as a perturbing agent on actomyosin ATPase and its related functions in the concentration range between 0 and 20 mM. In this range the denaturation of myosin subfragment 1 (S1), as measured by the inactivation rate of its K-EDTA-ATPase, and depolymerization of F-actin were insignificant. Major findings showed that hexanol had the following effects which were fully reversible, (a) a marked activation of S1 MgATPase (approximately 10-fold at 20 mM) without greatly affecting the enhancement of tryptophan fluorescence by formation of S1.ADP.Pi intermediate and the rate of ADP release from S1.ADP; (b) an inhibition of the maximum actin-activated ATPase activity; (c) an increase in the affinity of S1 for actin in the presence of ATP and a decrease in the presence of ADP or the absence of nucleotide; (d) a reduction in the sliding velocity of actin filaments in in vitro motility assays with myosin, and (e) a decrease in isometric tension of single skinned muscle fibers. Thus, the effects of hexanol on actomyosin interaction are distinct for the weak and strong binding states, consistent with a change in the hydrophobic interaction in the interface between myosin and actin accompanying the transition from the weak to the strong binding state. Hexanol also accelerates the Pi release from S1.ADP.Pi, which is the transition step from the weak to the strong binding state. The fact that hexanol accelerates Pi release suggests that this alcohol perturbs the S1.ADP.Pi conformation. We speculate that this intermediate-specific structural perturbation is related to the inhibition of the maximum actin-activated ATPase, in vitro motility, and isometric tension.     

Investigation of a genetically engineered mutant of barnacle troponin C containing a central helix deletion

To examine the importance of the central alpha-helix of troponin C (TnC) we have bacterially expressed one of the isoforms of barnacle TnC (BTnC2), BTnCWT, but without the aspartate residue at position 80 in the central helix (BTnC80-). This manipulation is expected to produce an approximately 100 degrees axial rotation of the C-domain with respect to the N-domain, and a net charge change of -1. BTnC80- mutant was able to restore force to TnC-depleted skinned barnacle myofibrillar bundles to a greater extent than wild-type protein (approximately = 170%). Competition experiments between BTnC80- and BTnC2-4-, a mutant lacking both of the calcium-specific sites (sites II and IV), shows that deletion of a single amino acid in the central helix results in a protein with increased affinity for the thin filament and one that is bound preferentially compared to BTnC2-4- when at equimolar concentrations.     

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 comp are 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 μm kg−1 muscle tissue of BTnC1 plus BTnC2 compared to about 91 μm kg−1 of TnC in rabbit psoas muscle fibres. Extraction of BTnC is achieved using similar low ionic strength, low divalent ion Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-Mg2+ binding sites that are thought to be involved in the Mg2+-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 Ca2+-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). Ca2+-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 fluoresence decrease paralleled the increase in force with increas ing 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 fluoresence. 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 Ca2+-activation and rigor with RTnCDanz exchanged into rabbit psoas skinned muscle fibres. The data support a similar model for Ca2+-activation of force in barnacle muscle and in rabbit psoas skeletal muscle fibres This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of pH on spontaneous tension oscillation in skinned bovine cardiac muscle

Skinned cardiac muscle preparations exhibit spontaneous tension oscillations (spontaneous oscillatory contractions; SPOCs) in the absence of Ca²⁺, and in the presence of MgATP, MgADP and inorganic phosphate (Pi; ADP-SPOC). Similar oscillations occur in the presence of sub-micromolar concentrations of Ca²⁺ under normal activating conditions without MgADP and Pi (Ca-SPOC). In the study presented here, we investigated the effects of pH on both types of SPOC in skinned bovine cardiac ventricular muscle. First, a decrease in pH increased the MgADP concentration required to induce the half-maximal isometric tension that is obtained in the absence of Ca²⁺ and in the presence of MgATP (ADP-contraction). The inhibitory effect of Pi on ADP-contractions was not affected by pH. Second, ADP-SPOCs occurred upon the addition of Pi to the solution that resulted in ADP-contraction, and the relative amplitude and the period of the tension oscillation in the presence of 2 mM MgATP, 10 mM MgADP and 10 mM Pi were unchanged under all pH conditions examined (6.6, 7.0, 7.4). On the contrary, the relative amplitude and the period of the Ca-SPOCs were markedly diminished at pH 6.6. Finally, we constructed state diagrams showing the effects of pH on SPOC conditions. The state diagram shows that SPOCs occur less frequently under acidic conditions than at neutral pH. We suggest that the intermediate state of crossbridges that is required for SPOCs is more difficult to attain at a low pH. Received: 14 September 1998 / Received after revision: 23 February 1999 / Accepted: 10 March 1999