Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: implications for regulation of actin-myosin interaction.

Myosin light chain phosphorylation in permeable skeletal muscle fibers increases isometric force and the rate of force production at submaximal levels of calcium activation; myosin light chain phosphorylation may underlie the increased rate and extent of force production associated with isometric twitch potentiation in intact fibers. To understand the mechanism by which myosin light chain phosphorylation manifests these effects, we have measured isometric force, isometric stiffness, rate of isometric force redevelopment after isotonic shortening, and isometric ATPase activity in permeabilized rabbit psoas muscle fibers. These measurements were made in the presence and absence of myosin light chain phosphorylation over a range of calcium concentrations that caused various levels of activation. The results were analyzed with a two-state cross-bridge cycle model as suggested by Brenner [Brenner, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3265-3269]. The results indicate that myosin light chain phosphorylation exerts its effect on force generation and the isometric rate of force redevelopment in striated muscle through a single mechanism, namely, by increasing the rate constant describing the transition from non-force-generating cross-bridges to force-generating states (fapp). gapp, the reverse rate constant, is unaffected by phosphorylation as are the number of cycling cross-bridges. Since both calcium and myosin light chain phosphorylation increase fapp, the possibility is considered that modulation of fapp may represent a general mechanism for regulating force in actin-myosin systems.     

Quercetin inhibits Ca2+ uptake but not Ca2+ release by sarcoplasmic reticulum in skinned muscle fibers.

Quercetin inhibited Ca2+-dependent ATP hydrolysis, ATP-dependent Ca2+ uptake, chelator-induced [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] Ca2+ release, and ATP synthesis coupled to Ca2+ release in isolated vesicles of sarcoplasmic reticulum. Use of this inhibitor permitted evaluation of whether Ca2+ release from sarcoplasmic reticulum in situ occurs through a reversal of the uptake pathway. Release of Ca2+ from the sarcoplasmic reticulum of skinned muscle fibers can be detected by the measurement of tension in the fiber. If the sarcoplasmic reticulum of these preparations is first allowed to accumulate Ca2+, tension development may be induced by the addition of Ca2+ itself or of caffeine to the bathing medium or by depolarization with Cl-. The presence of quercetin during the loading phase inhibited Ca2+ uptake by sarcoplasmic reticulum in situ. When quercetin was added together with initiators of tension development, however, the rate of tension development was enhanced 4- to 7-fold and the relaxation rate of the fibers was greatly inhibited. These results suggest that quercetin had no effect on Ca2+ release in skinned fiber; its effect on Ca2+ reuptake could account for the apparent enhancement of the release rate and for the prolonged relaxation time. These observations rule out reversal of the Ca2+ pump as the mechanism of Ca2+ release in situ.     

Insulin and glucose 6-phosphate stimulation of Ca2+ uptake by skinned muscle fibers.

Calcium uptake by skinned muscle fibers is stimulated by physiological concentrations of insulin. These fibers, which lack a functional plasma membrane, are permeable to macromolecules but retain extensive portions of their sarcolemma in the form of transverse tubules intercalated between the myofibrils. They have an active sarcoplasmic reticulum that removes 45Ca2+ from solution at concentrations below the threshold that initiates contraction (less than 1 microM). The Ca2+ uptake activity is stimulated by insulin, presumably in response to its binding to those receptors located in the transverse tubules. Addition of glucose 6-phosphate, whose intracellular concentration increases in response to insulin, also stimulates Ca2+ uptake, a unique property of this preparation. These data indicate that insulin and glucose 6-phosphate act in concert to stimulate the sarcoplasmic reticulum. The resulting decrease in myoplasmic Ca2+ and the increase in glucose 6-phosphate would serve to mediate some of the anabolic effects of the hormone.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Evidence for cross-bridge order in contraction of glycerinated skeletal muscle.

The linear dichroism of iodoacetylrhodamine labels attached to the single reactive thiol groups of myosin heads was measured to determine the spatial orientation of myosin cross-bridges in single glycerinated skeletal muscle fibers. We have shown previously that in rigor the chromophoric labels are well ordered and assume an orientation nearly perpendicular to the fiber axis; in the presence of MgADP, a large fraction of probe remains well ordered but the probe attitude assumes a more slanted orientation; in relaxed muscle, the probe order is largely lost, implying a high degree of cross-bridge disorder. In this paper, we report that during isometric contraction a large fraction of the probe shows a high degree of order, suggesting the attachment of approximately equal to 65% of the cross-bridges to actin. These ordered cross-bridges have a probe attitude similar to that of the MgADP-induced static state and hence are in a mechanical state quite distinct from rigor.     

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.     

Dissociation of action potentials from contraction in single crab muscle fibers.

In single crab fibers (Callinectes danae) bathed in Ca2+-free media, Ba2+ action potentials did not elicit tension. In contrast, Sr2+ spikes evoked twitches similar in amplitude to those accompanying the control Ca2+ spikes. Tension development in these fibers, therefore, depends on the ionic species carrying the inward current during membrane excitation. The Ca2" or Sr2+ influx appears insufficient to evoke the observed twitch tensions, and it seems necessary to postulate mobilization of an intracellular source of Ca1+. Procaine, which suppresses release of Ca2+ from sarcoplasmic reticulum, depressed twitch tension but did not reduce the overshoot or duration of Ca2+ or Sr2+ spikes. This finding is compatible with the suggestion that the contractions results from release of Ca2+ from the sarcoplasmic reticulum initiated by signals given by the influx of Ca2+ or Sr2+, but not Ba2+.     

Alpha-adrenergic modification of the Ca2+ transient and contraction in single rat cardiomyocytes.

Intracellular Ca2+ transients and contraction were measured simultaneously in single rat cardiomyocytes loaded with the fluorescent Ca2+ indicator fura-2, using a recently described high-speed digital imaging method (O'Rourke et al., 1990, Am J Physiol 259: H230-H242). In cardiomyocytes electrically-stimulated at 1 Hertz, alpha-adrenoceptor activation in the presence of beta-adrenoceptor blockade resulted in enhanced cell shortening associated with an increase in the amplitude of the cytosolic Ca2+ transient. Both effects developed in parallel over a 10-min time period and occurred without a change in the half-times for decay of Ca2+ or relaxation of the cell. To determine if the increase in contractility was proportional to the increase in peak cytosolic Ca2+, the effect of raising extracellular Ca2+ ([Ca2+]o) from 0.5 to 3 mM was examined in the absence and presence of alpha-adrenoceptor activation. At [Ca2+]o concentrations up to 1 mM, alpha-adrenoceptor-mediated effects on contraction were directly correlated with changes in peak cytosolic Ca2+ and resembled the effect of raising [Ca2+]o alone. In 2 and 3 mM [Ca2+]o, peak cytosolic Ca2+ approached a maximal level and alpha-adrenoceptor activation induced a slight enhancement in the extent of shortening in the absence of a detectable alteration of the Ca2+ transient. In contrast, under similar conditions, beta-adrenergic effects on shortening never exceeded those of alpha-adrenoceptor activation, although much higher peak cytosolic Ca2+ concentrations were achieved at high [Ca2+]o. The results suggest that the mechanism underlying the positive inotropic effect of alpha-adrenergic stimulation in rat ventricular cells is primarily dependent on an enhancement of the cytosolic Ca2+ transient, although there is also an increase in the myofibrillar response to intracellular Ca2+ under the condition of high extracellular Ca2+.     

Effect of ionic strength on length-dependent Ca(2+) activation in skinned cardiac muscle

The length-dependence of myofilament Ca(2+) sensitivity is considered to be an important component of the steep force-length relationship in cardiac muscle (Frank-Starling relation). Recent studies suggest that Ca(2+) sensitivity is a function of the number of strong-binding cross-bridge interactions formed at a given sarcomere length. However, the length-dependent step in the thin filament activation process is still unknown. This study was designed to test the hypothesis that sarcomere length influences the transition of the thin filament from the unattached (blocked) state to the weakly bound (closed) state. This hypothesis was tested by determining the length-dependence of Ca(2+) sensitivity as a function of ionic strength in skinned bovine ventricular muscle. Previous studies have shown that reduction in ionic strength below a critical level, in the absence of Ca(2+), shifts the thin filament to the closed state. In this study normal Ca(2+) regulation was maintained at low ionic strength but the length-dependence of Ca(2+) sensitivity and the length-dependence of Ca(2+) binding were eliminated. These results are consistent with the hypothesis that the transition from the blocked to the closed state is a function of filament geometry as well as Ca(2+) and ionic strength.     

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.     

Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

The synthesis of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) and of acetylcholine receptors (AcChoR) by cultured rat muscle fibers is influenced strongly by the level of muscle contractile activity. If fibers are grown in the presence of tetrodotoxin (TTX) to block spontaneous contraction, the total amount of AcChoE decreases markedly, as does the percentage of AcChoE assembled as the collagen-tailed presumed synaptic form of the enzyme. Under these conditions, however, the number of AcChoR increases. We demonstrate here that each effect of TTX can be prevented by treating the muscle cells with the calcium ionophore A23187. Thus, cells treated with A23187 and TTX have 30- to 40-fold higher levels of collagen-tailed AcChoE and lower levels of AcChoR by a factor of 4-5 than do cells grown in TTX alone. These results suggest that an increase in muscle cytoplasmic Ca2+ mediates the known effects of muscle contraction on these cholinergic macromolecules.     

Effectof MgATP on stiffness measured at two frequencies in Ca2+-activated muscle fibers.

The stiffness of skinned crayfish single muscle fibers was continuously monitored at two frequencies. The length of the fibers was oscillated by the sum of two sine waves (5 Hz and 100 Hz) of small amplitudes. In saline containing saturating amounts of Ca2+, the stiffness ratio (5 Hz:100Hz) was constant as the MgATP (substrate) concentration was raised from 0 to 2 mu M, then it decreased with a further increment in MgATP. The systematic decrease in the stiffness ratio in MgATP above 2 mu M indicates the presence of faster transitions in the cross-bridge cycle. This dependence of the stiffness ratio on MgATP is predictable if we use the two-state model of A. F. Huxley (1957) with a modification, in which MgATP promotes the dissociation of the attached cross-bridges.     

Effect of Ca2+ on the ciliary beat frequency of skinned dog tracheal epithelium

Beat frequency of dog tracheal ciliated epithelium was measured using a profile projector and a photomultiplier. The preparation, treated with 50 micrograms/ml of saponin for 15 min, lost osmotic behavior and the ciliary beat came to depend on externally applied MgATP2- indicating that ciliated epithelium is skinned with saponin. Beat frequency of skinned cilia did not increase with Ca2+ in 0.1 mM MgATP2- with no ATP-regenerating system. Under 4 mM Mg ATP2- the beat frequency increased with an increase in Ca2+ from 0.3 to 10 microM, although a marked beat continued in the virtual absence of Ca2+ sensitivity and maximum beat frequency increased with the addition of 2.4 microM calmodulin. The effect of calmodulin inhibitor (W-7) on skinned preparations was somewhat weaker than that on intact ones. We concluded that Ca2+, within the physiological range of concentrations, directly activated the ciliary proteins and increased the ciliary beat frequency. The addition of calmodulin augments the effect of Ca2+ but the basal beat frequency is not Ca2+ dependent.     

Different actions of cardioprotective agents on mitochondrial Ca2+ regulation in a Ca2+ paradox-induced Ca2+ overload.

BACKGROUND: Mitochondrial Ca2+ overload is a major cause of irreversible cell injury during various metabolic stresses. The protective effects of various agents that affect mitochondrial function against Ca2+ overload during Ca2+ paradox were investigated in rat ventricular myocytes. METHODS AND RESULTS: On Ca2+ repletion following Ca2+ depletion, [Ca2+]i increased rapidly, and 90 of 210 cells (43%) died. In viable cells, the increase in [Ca2+]i was lower than in dead cells. KB-R7943 prevented the increase in [Ca2+]i, and completely inhibited cell death. Ruthenium red (RuR), diazoxide (Dz) or cyclosporin A (CsA) prevented cell death (15%, 26% and 17%, respectively; p < 0.05), and the protective effect of Dz was abolished by 5-hydroxydecanoate. These agents did not reduce the increase in [Ca2+]i in viable cells or the rate of initial increase in [Ca2+]i in all cells. RuR and Dz decreased [Ca2+]m in skinned myocytes, but CsA did not affect [Ca2+]m. Dz reduced NADH fluorescence, whereas RuR and CsA did not. CONCLUSIONS: The protective effects of RuR and Dz could be ascribed to altered Ca2+ regulation by decreasing [Ca2+]m, and Dz could have an additional effect on oxidative phosphorylation. The protective effect of CsA could be directly associated with the mitochondrial permeability transition pore.     

Altered regulation of cardiac contraction and relaxation by Ca2+ in heart failure

Myocardial contractile dysfunction in congestive heart failure is characterized by a decrease in force developed and retardation of relaxation. These alterations are mainly due to those in intracellular Ca(2 +) transients (CaT) . CaT are regulated by a number of functional proteins, including sarcolemmal L-type Ca(2 +) channels, Na(+)/Ca(2 +) exchanger and Ca(2 +) ATPase, sarcoplasmic reticulum Ca(2 +) ATPase (SERCA 2 ) , phospholamban and ryanodine receptors, and mitochondrial Ca(2 +) uniporter. Changes in expression and function of these regulatory proteins that occur in the course of increasing severity of heart failure are responsible for the characteristic changes in force development and relaxation observed under pathophysiological conditions in congestive heart failure.     

X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers.

Biochemical data, stiffness measurements, and equatorial x-ray diffraction patterns provide evidence that actomyosin cross-bridges form in relaxed skinned rabbit fibers at low ionic strength (20 mM). In the present study we examined the structure of these cross-bridges by using two-dimensional x-ray diffraction. In contrast to rigor cross-bridges, which significantly weaken the myosin-based reflections characteristic of relaxed fibers at 120 mM ionic strength (notably the 86-A and 108-A layer lines and the 72-A and 143-A meridionals), the formation of low ionic strength cross-bridges produced only small changes in these reflections. In addition, these cross-bridges did not produce the additional intensity on the 59-A actin-based layer line near the meridian that is associated with rigor cross-bridges. However, the formation of low ionic strength cross-bridges caused the 215-A meridional reflection to decrease in intensity, as is also the case when rigor cross-bridges are formed. These observations show that the structure of the low ionic strength cross-bridge is significantly different from that of the rigor cross-bridge, and they raise the possibility that contractile force may be generated by a transition between these two actomyosin configurations.     

Tension relaxation induced by pulse photolysis of caged ATP in partially crosslinked fibers from rabbit psoas muscle.

Muscle contractile force is thought to be generated by ATP-induced conformational changes in myosin crossbridges. In the present study, we investigated the response to ATP binding of force-bearing, attached cross-bridges. For this investigation, skinned fibers, in which myosin heads were in part covalently crosslinked to thin filaments with a zero-length crosslinker, were prepared. Caged ATP [the P3-1-(2-nitro)phenylethyl ester of ATP] was then pulse-photolyzed in these crosslinked fibers, which retained ATP-induced "rigor" tension, and then the subsequent tension changes were followed at 14-16 degrees C and ionic strengths of 0.1-2 M. A rapid tension decrease was observed after the photolysis in the partially crosslinked fibers. The rate of the decrease was not any different from that in the uncrosslinked fibers compared at ionic strength of 0.2 M. This and other results thus indicate a kinetic similarity in the crosslinked and uncrosslinked crossbridges in response to ATP binding. These findings also suggest that ATP-induced structural changes take place in the attached crossbridges at a rate similar to that of the ATP-induced dissociation of crossbridges from thin filaments.     

Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase.

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.