Calcium-independent activation of skinned cardiac muscle by secophalloidin

Thin filament regulation of muscle contraction is believed to be mediated by both Ca2+ and strongly bound myosin cross-bridges. We found that secophalloidin (SPH, 5-8 mM) activates cross-bridge cycling without Ca2+ causing isometric force comparable to that induced by Ca2+. At saturated [SPH], Ca2+ further increased force by 20%. SPH-induced force was reversible upon washing with a relaxing solution. However, there was more than 30% irreversible loss in subsequent Ca2+-activated force. We hypothesize that SPH activates muscle via strongly bound cross-bridges. SPH-activated contraction provides a new model for studying the role of Ca2+ and cross-bridges in muscle regulation.     

Secophalloidin and phalloidin-(S)-sulfoxide as contraction modifiers for comparative study of skeletal and cardiac muscles

Phalloidin, a toxic product of the mushroom Amanita phalloides, binds specifically to F-actin resulting in strong stabilization of F-actin structure (for review, see; Wieland, 1986). Binding to a specific site on the muscle thin filament F-actin, phalloidin modifies contraction in a tissue specific manner. Phalloidin induced changes depend on functionally important parameters (thin filament activation, cross-bridge kinetics), indicating changes in essential steps of the contractile mechanism. Moreover, there is a different action with different phalloidin derivatives. Such properties make phallotoxins (phalloidin and its derivatives) powerful modifiers for muscle research (for review, see: Bukatina, 1996). Phalloidin-induced changes vary qualitatively with muscle types. In all types of skinned skeletal muscle preparations that have been studied (fast and slow muscles from evolutionarily distant animals), the most general effect of phalloidin is to cause a decrease in tension (Bukatina, Morozov, 1979; Alievskaya et al., 1987; Bukatina et al., 1993). In mammalian skeletal muscles, this decrease in tension may be followed by a slowly developing increase in tension. The resulting tension may considerably exceed the tension before phalloidin administration. In contrast, skinned cardiac muscle responds to phalloidin only by increasing isometric tension from the onset of the response. Moreover, the phalloidin response is completed in approximately one-tenth the time in cardiac muscle that it takes in skeletal muscle. These phalloidin effects in cardiac muscle result in an enhanced Ca2+ responsiveness (Boels, Pfitzer, 1992) with an increase in both the force at maximum Ca2+ activation and the Ca2+ sensitivity (Bukatina et al., 1995).     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction.

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).     

A pH-sensitive interaction of troponin I with troponin C coupled with strongly binding cross-bridges in cardiac myofilament activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca(2+) regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca(2+) regulation.     

Influence of phallotoxins and metal ions on the rate of proteolysis of actin

The rate of proteolytic degradation of rabbit skeletal muscle actin by trypsin, alpha-chymotrypsin, and, mainly, subtilisin was followed by dual wavelength spectroscopy at 285 nm by reference at 320 nm. Phalloidin and phallacidin, two toxic bicyclic heptapeptides from the mushroom Amanita phalloides, protect F-actin against degradation by the proteolytic enzymes. G-actin, which does not combine with phalloidin when maintained in the monomeric state by working at low ionic strength, and bovine serum albumin, which also has no affinity to the toxin, are hydrolyzed at the same rates in the presence or absence of phalloidin. The proteolysis of F-actin is distinctly retarded by KCl alone, i.e., without phalloidin, whereas Mg2+ or Ca2+ as sole cations permit a rather high rate of hydrolysis. An even faster degradation of F-actin by subtilisin is observed in the presence of Mg2+ plus cytochalasin B. Adenosine diphosphate and triphosphate have no influence on the rate of the enzymatic degradation. The S sulfoxide of phalloidin, the nontoxic diastereomer of the toxic R form, exerts only a limited inhibitory effect on the enzymatic hydrolysis; secophalloidin, another nontoxic derivative, which does not bind to F-actin has practically no effect.     

The role of troponins in muscle contraction

Troponin (Tn) is the sarcomeric Ca2+ regulator for striated (skeletal and cardiac) muscle contraction. On binding Ca2+ Tn transmits information via structural changes throughout the actin-tropomyosin filaments, activating myosin ATPase activity and muscle contraction. Although the Tn-mediated regulation of striated muscle contraction is now well understood, the role of different Tn isoforms in these processes is the subject of intensive investigations. This review addresses the physiological significance of the multiple Tn isoforms in skeletal and cardiac muscles as well as their role in the regulation of contraction.     

BDM compared with P(i) and low Ca2+ in the cross-bridge reaction initiated by flash photolysis of caged ATP.

Using flash photolysis of caged ATP in skinned muscle fibers from rat psoas, we examined the inhibitory effects of 2,3-butanedione monoxime (BDM) on the contraction kinetics and the rate of ATP hydrolysis of the cross-bridges at approximately 10 degrees C. The hydrolysis rate was estimated from the stiffness records. The effects of BDM were compared with those of orthophosphate (P(i)) and of reduction in [Ca2+] (low Ca2+), and it was found that i) BDM and low Ca2+ inhibited ATPase activity to the same extent as they inhibited the steady tension, whereas P(i) inhibited ATPase activity much less than tension; ii) BDM and P(i) decreased tension per stiffness during the steady contraction more than did low Ca2+; iii) neither BDM nor low Ca2+ affected the initial relaxation of the fiber on release of ATP, but P(i) slightly slowed it; and iv) BDM hardly influenced the rate of contraction development after relaxation, although P(i) and low Ca2+ accelerated it. We concluded that BDM inhibits the Ca(2+)-regulated attachment of the cross-bridges and force-generation of the attached cross-bridges.     

Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C

The trigger Ca2+-binding sites in troponin C, those which initiate muscle contraction, are thought to be the first two of four potential sites (sites I-IV). In cardiac troponin C, the first Ca2+-binding site is inactive, and initiation of contraction in cardiac muscle appears to involve only the second site. To study this phenomenon and associated Ca2+-dependent protein conformational changes in cardiac troponin C, the cDNA for the chicken protein was incorporated into a bacterial expression plasmid to allow site-specific mutagenesis. Ca2+-binding site I was activated by deletion of Val-28 and conversion of amino acids 29-32 to those found at the first four positions in the active site I of fast skeletal troponin C. In a series of proteins, Ca2+-binding site II was inactivated by mutation of amino acids Asp-65, Asp-67, and Gly-70. All mutated proteins exhibited the predicted calcium-binding characteristics. The single mutation of converting Asp-65 to Ala was sufficient to inactivate site II. Ca2+-dependent conformational changes in the normal and mutated proteins were monitored by labeling with a sulfhydryl-specific fluorescent dye. Activation of Ca2+-binding site I or inactivation of site II, eliminated the large Ca2+-dependent increase in fluorescence seen in the wild type protein and there was, instead, a Ca2+-dependent decrease in fluorescence. All mutant proteins could associate with troponin I and troponin T to form a troponin complex. Activation of Ca2+-binding site I changed the characteristics of contraction in skinned slow skeletal muscle fibers such that the response to Ca2+ was more cooperative. Inactivation of Ca2+-binding site II abolished Ca2+-dependent contraction in skinned muscle fibers. The data provide a direct demonstration that Ca2+-binding site II in cardiac troponin C is essential for triggering muscle contraction and support the hypothesis that site I functions to modify the characteristics of contraction.     

Smooth muscle contraction: mechanochemical formulation for homogeneous finite strains

Chemical kinetics of smooth muscle contraction affect mechanical properties of organs that function under finite strains. In an effort to gain further insight into organ physiology, we formulate a mechanochemical finite strain model by considering the interaction between mechanical and biochemical components of cell function during activation. We propose a new constitutive framework and use a mechanochemical device that consists of two parallel elements: (i) spring for the cell stiffness; (ii) contractile element for the sarcomere. We use a multiplicative decomposition of cell elongation into filament contraction and cross-bridge deformation, and suggest that the free energy be a function of stretches, four variables (free unphosphorylated myosin, phosphorylated cross-bridges, phosphorylated and dephosphorylated cross-bridges attached to actin), chemical state variable driven by Ca2+-concentration, and temperature. The derived constitutive laws are thermodynamically consistent. Assuming isothermal conditions, we specialize the mechanical phase such that we recover the linear model of Yang et al. [2003a. The myogenic response in isolated rat cerebrovascular arteries: smooth muscle cell. Med. Eng. Phys. 25, 691-709]. The chemical phase is also specialized so that the linearized chemical evolution law leads to the four-state model of Hai and Murphy [1988. Cross-bridge phosphorylation and regulation of latch state in smooth muscle. Am. J. Physiol. 254, C99-C106]. One numerical example shows typical mechanochemical effects and the efficiency of the proposed approach. We discuss related parameter identification, and illustrate the dependence of muscle contraction (Ca2+-concentration) on active stress and related stretch. Mechanochemical models of this kind serve the mathematical basis for analyzing coupled processes such as the dependency of tissue properties on the chemical kinetics of smooth muscle.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.     

Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures

Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.     

Effect of myosin cross-bridge interaction with actin on the Ca2(+)-binding properties of troponin C in fast skeletal myofibrils

Ca2+ binding to fast skeletal muscle troponin C reincorporated into troponin C-depleted (CDTA-treated) myofibrils has been measured directly by using 45Ca and indirectly by using a fluorescent probe. Direct Ca2(+)-binding measurements have shown that the Ca2+ affinity of the low-affinity sites is enhanced in the absence of ATP and conversely reduced when myosin is selectively extracted from myofibrils, compared to the Ca2+ affinity in the presence of ATP. Fluorescence intensity changes of a dansylaziridine label at the Met-25 residue of troponin C have shown the same Ca2(+)-sensitivity whether or not ATP is present, while much lower Ca2(+)-sensitivity is seen in the myosin-extracted myofibrils. Since the Met-25 residue is in the amino terminal side alpha-helix of Ca2(+)-binding site I and far from Ca2(+)-binding site II in the primary structure, Ca2+ binding to site II has been evaluated by assuming that the fluorescence change monitors Ca2+ binding to site I alone. Ca2+ binding to site II thus estimated has shown high positive cooperativity only in the presence of ATP and has been found to be nearly proportional to the activation of myofibrillar ATPase, suggesting that Ca2(+)-binding site II is directly involved in the activation of myofibrillar ATPase activity. On the other hand, Ca2(+)-binding site I has been suggested to regulate the interaction of weakly binding cross-bridges with the thin filament, since the fluorescence change in the presence of ATP is saturated at the free Ca2+ concentration required for the activation of myofibrillar ATPase.     

Activating efficiency of Ca2+ and cross-bridges as measured by phosphate analog release

To assess the activating efficiency of Ca2+ and cross-bridges, the release rates of phosphate analogs from skinned fibers were estimated from the recovery of contractility and that of stiffness. Estimations were performed based on the assumptions that contractility was indicative of the population of analog-free myosin heads and that stiffness reflected the population of formed cross-bridges. Aluminofluoride (AlFx) and orthovanadate (Vi) were used as phosphate analogs with mechanically skinned fibers from rabbit psoas muscle. The use of the analogs enabled the functional assessment of activation level in the total absence of ATP. Fibers loaded with the analogs gradually recovered contractility and stiffness in normal plain rigor solution. The addition of Ca2+ to the plain rigor solution significantly accelerated their recovery, whereas ADP had no appreciable effect. ATP plus Ca2+(contracting condition) accelerated the recovery by several tens of times. These results indicate that the cross-bridges formed during contraction have prominent activating efficiency, which is indispensable to attain full activation. A comparison between the activating efficiency evaluated from stiffness and that from contractility suggested that Ca2+ is more potent in accelerating the binding of actin to analog-bound myosin heads whereas cross-bridges mainly accelerate the subsequent analog-releasing step.     

Effects of Ca2+ on the kinetics of phosphate release in skeletal muscle.

The process of phosphate dissociation during the muscle cross-bridge cycle has been investigated by photoliberation of inorganic phosphate (Pi) within skinned fibers of rabbit psoas muscle. This permitted a test of the idea that Ca2+ controls muscle contraction by regulating the Pi release step of the cycle. Photoliberation of Pi from structurally distinct "caged" Pi precursors initiated a rapid tension decline of up to 12% of active tension, and this was followed by a slower tension decline. The apparent rate constant of the fast phase, kPi, depended on both [Pi] and [Ca2+], whereas the slow phase generally occurred at 2-4 s-1. At maximal Ca2+, kPi increased in a nonlinear manner from 43 +/- 2 s-1 to 118 +/- 7 s-1, as Pi was raised from 0.9 to 12 mM. This was analyzed in terms of a three-state kinetic model in which a force-generating transition is coupled to Pi dissociation from the cross-bridge. As Ca(2+)-activated tension was reduced from maximal (Pmax) to 0.1 Pmax, (i) kPi decreased by up to 2.5-fold, (ii) the relative amplitude of the rapid phase increased 2-fold, and (iii) the relative amplitude of the slow phase increased about 6-fold. Changes in the rapid phase are compatible with Ca2+ influencing an apparent equilibrium constant for the force-generating transition. By comparison, kPi was faster than the rate constant of tension redevelopment, ktr, and was influenced less by Ca2+. Ca2+ effects on the caged Pi transient cannot account for the large effects of Ca2+ on actomyosin ATPase rates or cross-bridge cycling kinetics but may be a manifestation of reciprocal interactions between the thin filament and force-generating cross-bridges, and may represent Ca2+ regulation of the distribution of cross-bridges between non-force-and force-generating states.     

Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.     

Ionic interventions that alter the association of troponin C C-domain with the thin filaments of vertebrate striated muscle

The regulatory complex of vertebrate skeletal muscle integrates information about cross-bridge binding, divalent cations and other intracellular ionic conditions to control activation of muscle contraction. Relatively little is known about the role of the troponin C (TnC) C-domain in the absence of Ca2+. Here, we use a standardized condition for measuring isometric tension in rabbit psoas skinned fibers to track TnC attachment and detachment in the absence of Ca2+ under different conditions of ionic strength, pH and MgATP. In the presence of MgATP and Mg2+, TnC detaches more readily and has a 1.5- to 2-fold lower affinity for the intact thin filament at pH 8 and 250 mM K+ than at pH 6 or in 30 mM K+; changes in affinity are fully reversible. The response to ionic strength is lost when Mg2+ and MgATP are absent, whereas the response to pH persists, suggesting that weaker electrostatic TnC-TnI-TnT interactions can be overridden by strongly bound cross-bridges. In solution, titration of a fluorescent C-domain mutant (F154W TnC) with Mg2+ reveals no significant changes in Mg2+ affinity with pH or ionic strength, suggesting that these parameters influence TnC binding by acting directly on electrostatic forces between TnC and TnI rather than by changing Mg2+ binding to C-domain sites III and IV.     

Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.     

Evaluation of the cross-bridge-dependent change in the Ca2+ affinity of troponin C in aequorin-injected ferret ventricular muscles

Ca2+ affinity of cardiac troponin C (TnC) is regulated by the active cross-bridges (downstream-dependent mechanism). In the present study, we showed one of the methods to evaluate the downstream-dependent change in the Ca2+ affinity of TnC during contraction using the aequorin-injected ferret papillary muscle. For this purpose, the tension-dependent change in the extra-Ca2+ (a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) in response to a quick length reduction) was measured under various conditions. We examined whether the regression line between the magnitude of tension reduction and the magnitude of the normalized extra-Ca2+ (the extra-Ca2+ was divided by [Ca2+]i immediately before length change) (the normalized extra-Ca2+-tension relation) in twitch contraction can be used for the estimation of the downstream-dependent change in the Ca2+ affinity of TnC. The normalized extra-Ca2+-tension relation became shallow by EMD 57033 (EMD) (one of the Ca2+ sensitizers) and by an increase in Ca2+ concentration in the solution ([Ca2+]o) in a concentration-dependent manner. However, 2,3-butanedione monoxime (BDM) (one of the desensitizers) antagonized the effects of EMD and higher [Ca2+]o in a concentration-dependent manner. These effects of EMD and BDM were also observed in the normalized extra-Ca2+-tension relation in tetanic contraction. The normalized extra-Ca2+-tension relation became steep by shortening the initial muscle length before contraction in tetanic contraction. Length-tension relation in twitch contraction was significantly shifted upward by higher [Ca2+]o and EMD, but BDM showed the opposite effects on them in a concentration-dependent manner. Thus, the downstream-dependent change in the Ca2+ affinity of TnC which physiologically functions in intact cardiac muscle can be evaluated using the normalized extra-Ca2+-tension relation.     

Role of Ca2+ and cross-bridges in skeletal muscle thin filament activation probed with Ca2+ sensitizers

Thin filament regulation of contraction is thought to involve the binding of two activating ligands: Ca2+ and strongly bound cross-bridges. The specific cross-bridge states required to promote thin filament activation have not been identified. This study examines the relationship between cross-bridge cycling and thin filament activation by comparing the results of kinetic experiments using the Ca2+ sensitizers caffeine and bepridil. In single skinned rat soleus fibers, 30 mM caffeine produced a leftward shift in the tension-pCa relation from 6.03 +/- 0.03 to 6.51 +/- 0.03 pCa units and lowered the maximum tension to 0.60 +/- 0.01 of the control tension. In addition, the rate of tension redevelopment (ktr) was decreased from 3.51 +/- 0.12 s-1 to 2.70 +/- 0.19 s-1, and Vmax decreased from 1.24 +/- 0.07 to 0.64 +/- 0.02 M.L./s. Bepridil produced a similar shift in the tension-pCa curves but had no effect on the kinetics. Thus bepridil increases the Ca2+ sensitivity through direct effects on TnC, whereas caffeine has significant effects on the cross-bridge interaction. Interestingly, caffeine also produced a significant increase in stiffness under relaxing conditions (pCa 9.0), indicating that caffeine induces some strongly bound cross-bridges, even in the absence of Ca2+. The results are interpreted in terms of a model integrating cross-bridge cycling with a three-state thin-filament activation model. Significantly, strongly bound, non-tension-producing cross-bridges were essential to modeling of complete activation of the thin filament.