Contractile protein interactions in smooth muscle.

Smooth muscle tone and 'holding economy' depend on the rate constants governing the cross-bridge cycle. Thus, calcium activation via calmodulin-dependent myosin light chain phosphorylation may determine the apparent rate constant ('f') at which cross-bridges enter the force-generating state, forming actin-attached, strongly bound cross-bridges. This phosphorylation of the light chain may be inhibited in skinned fibers by a peptide mimic of the calmodulin recognition site of the myosin light chain kinase (RS 20) that relaxes smooth muscle. In smooth muscle, the apparent cross-bridge detachment rate constant ('g') also seems to be variable, a low constant allowing for a high holding economy and low shortening velocity in the 'latch state'. It may also account for force maintenance at low levels of myosin phosphorylation. Additionally, cross-bridge attachment may, however, be also controlled by other regulatory proteins such as calponin and caldesmon.     

Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction.

The effect of Ca2+ upon the rate constant of force redevelopment following a period of isotonic shortening with immediate restretch to the starting sarcomere length was studied in rabbit psoas fibers at 5 degrees C. Control experiments support the assumption that the rate constant of force redevelopment represents isometric cross-bridge turnover kinetics (fapp + gapp), where fapp and gapp are the rate constants characterizing the transitions from the non-force-generating states to the force-generating states and back to the non-force-generating states, respectively. Parallel measurements of the rate constant of force redevelopment and of force, stiffness, and fiber ATPase during isometric contraction allow the effect of Ca2+ upon fapp and gapp to be determined. Analysis reveals that Ca2+ has a marked effect upon fapp, while gapp remains approximately unchanged. Furthermore, in the range above 25-30% of maximum Ca2+ activation, regulation of force, stiffness, and ATPase is mediated through changes in fapp. Below this range, however, it cannot be ruled out that, in addition, cross-bridges are also switched in and out of the turnover process ("recruitment"). As a consequence of regulation through turnover kinetics, both Ca2+ sensitivity and the slope of force-pCa (-log[Ca2+]) relations are shown to be affected by the ratio fapp/gapp, which may represent an important mechanism of modulation of contractile function in addition to modulation through changes within the regulatory protein system.     

Fraction of myosin cross-bridges bound to actin in active muscle fibers: estimation by fluorescence anisotropy measurements.

Time-resolved and steady-state fluorescence anisotropy measurements from fluorescence-labeled myosin cross-bridges in single glycerinated skeletal muscle fibers in rigor, relaxed, MgADP-induced, and contracting states have been made in order to estimate the fraction of actin-bound cross-bridges in active muscle. When the plane of polarization of the excitation light is perpendicular to the fiber axis and its propagation vector has a component parallel to this axis, actin-bound cross-bridge states, such as rigor and MgADP-induced, have time-zero and steady-state anisotropies that are substantially lower than has the relaxed state. This difference provides a means of determining the fraction of cross-bridges bound to actin in active isometric fibers, by comparing the fluorescence anisotropy from active fibers with the anisotropy from bound and unbound cross-bridges in static states. By assuming that the active cross-bridges are either bound (in the manner of rigor or MgADP-induced states) or relaxed, we estimate that greater than 80% of the cross-bridges are actin-bound in active isometric fibers.     

Phosphorylation and thiophosphorylation by myosin light chain kinase: different effects on mechanical properties of chemically skinned ventricular fibers from the pig

The influence of myosin light chain phosphorylation (treatment with myosin light chain kinase = MLCK, calmodulin and ATP) and thiophosphorylation (incubation with MLCK, calmodulin and ATP gamma S) on the maximal shortening velocity (Vmax) and Ca2+ sensitivity of chemically-skinned ventricular fibers from the pig has been studied. Vmax was determined by the slack-test method and by extrapolation of the force-velocity relation by the isotonic quick release method. Vmax was 1.53 muscle length/s (L/s) and 1.94 L/s using the force-velocity relation and the slack-test, respectively. Phosphorylation increased the Ca2+ sensitivity for isometric force development of skinned fibers but had no influence on Vmax. Thiophosphorylation decreased Vmax but had no influence on Ca2+ sensitivity. Phosphorylation pattern of the myosin light chains of the skinned fibers was studied using [gamma-32P]ATP or [gamma-P35S]ATP (250 muCi each) and autoradiography. Incubation of skinned fibers with labeled ATP led to a phosphate incorporation into the 18-kDa myosin light chain (MPLC or regulatory light chain) while incubation with labeled ATP gamma S led to an incorporation of thiophosphate into the 28-kDa myosin light chain (alkali light chain) and tropomyosin. We suggest that the difference in mechanical behavior between phosphorylated and thiophosphorylated skinned fibers are due to differences in the phosphorylation profiles of myofibrillar regulatory proteins.     

Regulation of contraction and relaxation in arterial smooth muscle.

Intracellular calcium concentration ([Ca2+]i)-dependent activation of myosin light chain kinase and its phosphorylation of the 20-kd light chain of myosin is generally considered the primary mechanism responsible for regulation of contractile force in arterial smooth muscle. However, recent data suggest that the relation between [Ca2+]i and myosin light chain phosphorylation is variable and depends on the form of stimulation. The dependence of myosin phosphorylation on [Ca2+]i has been termed the "[Ca2+]i sensitivity of phosphorylation." The [Ca2+]i sensitivity of phosphorylation is "high" when relatively small increases in [Ca2+]i induce a large increase in myosin phosphorylation. Conversely, the [Ca2+]i sensitivity of phosphorylation is "low" when relatively large increases in [Ca2+]i are required to induce a small increase in myosin phosphorylation. There are two proposed mechanisms for changes in the [Ca2+]i sensitivity of phosphorylation: Ca(2+)-dependent decreases in the [Ca2+]i sensitivity of phosphorylation induced by phosphorylation of myosin light chain kinase by Ca(2+)-calmodulin protein kinase II and agonist-dependent increases in the [Ca2+]i sensitivity of phosphorylation by inhibition of a myosin light chain phosphatase. I will review the proposed mechanisms responsible for the regulation of [Ca2+]i and the [Ca2+]i sensitivity of phosphorylation in arterial smooth muscle.     

Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C

Beta-adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during beta-adrenergic stimulation.     

Muscle fiber types and function.

A major recent advance in the field of muscle fiber types has been the discovery that myogenic factors regulate fiber phenotypic properties. Myogenic influences occur in limb and trunk (somitic) muscle, but are particularly strong in jaw-closing muscles and extraocular muscles that express some unique myofibrillar proteins. In somitic muscles, a variant of fast fiber has been discovered, making four types of fibers: I, IIA, IIX, and IIB. These fibers express different isoforms of myofibrillar and other proteins. The speed and power of the four types of fibers are distinct and are controlled principally by their myosin heavy chains, which modulate the two regulatory steps in the crossbridge cycle, one controlling the rate of development of force, the other controlling the maximal velocity of shortening. Fast fibers have a higher threshold for Ca(2+)-activated force and a steeper force-pCa relation than do slow fibers. This difference is largely attributable to the cooperativity in the attachment of crossbridges and to the difference in Ca2+ binding capacity of their troponin C. Ca2+ initiates force development in muscle by increasing the rate of attachment of crossbridges. The phosphorylation of myosin light chain enhances this action. This effect of phosphorylation underlies the phenomenon of posttetanic potentiation of the isometric twitch in fast fibers.     

Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.     

Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibres

Summary We investigated the influence of myosin P-light chain phosphorylation by Ca²⁺-calmodulin dependent myosin light chain kinase (MLCK) on the sensitivity of the tension-pCa relation and maximum unloaded shortening velocity (v _max) of chemically skinned heart fibres of the pig.Submaximum Ca²⁺ stimulation (pCa 5.5) induced 20±5% of the isometric tension achieved at maximum Ca²⁺ activation (pCa 4.3).MLCK-induced myosin P-light chain phosphorylation increased the isometric force development at pCa 5.5 by 40% whereas maximum tension at pCa 4.3 was not affected.Unloaded shortening velocity (v _max) was not altered by myosin P-light chain phosphorylation either at maximum or at submaximum Ca²⁺ concentration, being c. 1.2 muscle length/s at pCa 5.5 and 2.2 muscle length/s at pCa 4.3.The MLCK-induced increase of the myosin P-light chain phosphorylation level was evaluated by determination of³²P-incorporation. Two phosphorylatable myosin P-light chains could be demonstrated.     

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.     

Regulation of cardiac contractile proteins. Correlations between physiology and biochemistry

Modulation of the functional properties of the contractile proteins of mammalian heart muscle plays a significant role in the response of the heart to beta-adrenergic stimulation. The most well understood modification is a change in the concentration of calcium ions that is required to activate the contractile system. By means of a cAMP-sensitive phosphorylation of the inhibitory subunit of troponin (TNI), the threshold concentration for activation can be increased as much as 5-fold without changing the maximum calcium-activated force. The protein kinase involved in this regulation is located in the sarcolemma. Cholinergic stimulation causes a dephosphorylation of TNI by a cGMP-sensitive phosphatase. The concentration of calcium ions required to activate contraction also decreases as muscle length increases. This response of the contractile proteins does not involve phosphorylation of TNI. Regulation of the maximum calcium-activated force can take place by a cAMP-sensitive reaction involving a different protein kinase that is located inside the cell. This mechanism involves at least two sequential reactions, one a cAMP-controlled phosphorylation of a protein bound to an intracellular membrane to release an active factor, and the second, an interaction between the active factor and the contractile proteins to enhance the capacity for generating force in the presence of calcium. Phosphorylation of the light chain of myosin is produced by a calmodulin-regulated kinase. The light chain of myosin is partially phosphorylated in the intact heart, but beta-adrenergic stimulation of the heart does not increase the decrease of phosphorylation in parallel with the increase in contractility.     

Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution.

Crossbridge models of muscle contraction based on biochemical studies predict that there may be a relationship between the rate-limiting step in the actomyosin ATPase cycle in vitro and the rate of force development in vivo. In the present study, we measured the rate of force redevelopment in skinned rabbit muscle fibers following unloaded isotonic shortening and a rapid restretch. For comparison, ATPase activity was measured under identical conditions, using myosin subfragment-1 chemically crosslinked to actin. We found that the time course of force redevelopment is well fitted by a single exponential function, implying that force redevelopment is a first-order process, described by a single rate constant. The magnitude of this rate constant is in close agreement with the rate constant necessary to simulate the experimental force-velocity relation on the basis of a crossbridge model of the type proposed by A. F. Huxley in 1957. In addition, the observed close correlation between the rate constant for force redevelopment and the maximal actin-activated actomyosin ATPase rate under a variety of conditions suggests that the step that determines the rate of force generation in the crossbridge cycle may be the physiological equivalent of the rate-limiting step in the actomyosin ATPase cycle in solution.     

Modulation of crossbridge kinetics by myosin isoenzymes in skinned human heart fibers

Skinned fibers from the normal human heart with the beta-myosin heavy chain (ventricular fibers) revealed both a higher force generation per cross section and a higher Ca2+ sensitivity than skinned fibers with the alpha-myosin heavy chain (atrial fibers). The relation between isometric ATPase activity and isometric tension of atrial fibers was higher than that of ventricular fibers. Since the ATPase-tension relation equals the rate constant for the transition from force-generating into non-force-generating crossbridge states (g(app)), myosin heavy chain isoenzymes seem to have different crossbridge turnover kinetics. Modulation of g(app) by myosin heavy chain isoenzymes could explain the different contractile behavior of atrial and ventricular fibers. g(app) was independent of Ca2+.     

Reverse actin sliding triggers strong myosin binding that moves tropomyosin

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca²⁺ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca²⁺], and stretch activation, at lower [Ca²⁺], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored “actin target zones.” Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca²⁺] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca²⁺], Vi-“paralyzed” fibers produce force substantially above passive response at pCa ~ 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding “brakes” by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.     

Rapid dissociation and reassociation of actomyosin cross-bridges during force generation: a newly observed facet of cross-bridge action in muscle.

The force response of skinned fibers of the rabbit psoas muscle to stretches (and releases) was studied. At physiological ionic strength and low experimental temperature (5 degrees C) the force response to stretches apparently is affected neither by cross-bridges that occupy weak-binding states nor by transitions among various attached force-generating states. Plots of force vs. imposed length change (T plots) recorded during stretches suggest that cross-bridges even in force-generating states dissociate and reassociate rapidly from and to actin as had previously been proposed [Brenner, B. (1986) Basic Res. Cardiol. 81, 1-15]. Plots of fiber stiffness vs. speed of imposed length changes (stiffness-speed relations) imply rate constants for dissociation (k-) in the force-generating states ranging from 50 to 1000 s-1, while the rate constant for reassociation (k+) has to be at least an order of magnitude larger (high actin affinity). Rapidly reversible actin interaction of cross-bridges in force-generating states provides a mechanism for rapid detachment of force-generating cross-bridges during high-speed shortening which, in contrast with the hypothesis of A. F. Huxley [(1957) Prog. Biophys. 7, 255-318], and related cross-bridge models, does not require completion of the ATP-hydrolysis cycle and thus may account for the unexpectedly low ATPase activity during high-speed shortening.     

Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.     

Parallel inhibition of active force and relaxed fiber stiffness in skeletal muscle by caldesmon: implications for the pathway to force generation.

In recent hypotheses on muscle contraction, myosin cross-bridges cycle between two types of actin-bound configuration. These two configurations differ greatly in the stability of their actin-myosin complexes ("weak-binding" vs. "strong-binding"), and force generation or movement is the result of structural changes associated with the transition from the weak-binding (preforce generating) configuration to strong-binding (force producing) configuration [cf. Eisenberg, E. & Hill, T. L. (1985) Science 227, 999-1006]. Specifically, in this concept, the main force-generating states are only accessible after initial cross-bridge attachment in a weak-binding configuration. It has been shown that strong and weak cross-bridge attachment can occur in muscle fibers [Brenner, B., Schoenberg, M., Chalovich, J. M., Greene, L. E. & Eisenberg, E. (1982) Proc. Natl. Acad. Sci. USA 79, 7288-7291]. However, there has been no evidence that attachment in the weak-binding states represents an essential step leading to force generation. It is shown here that caldesmon can be used to selectively inhibit attachment of weak-binding cross-bridges in skeletal muscle. Such inhibition causes a parallel decrease in active force, while the kinetics of cross-bridge turnover are unchanged by this procedure. This suggests that (i) cross-bridge attachment in the weak-binding states is specific and (ii) force production can only occur after cross-bridges have first attached to actin in a weakly bound, nonforce-generating configuration.     

Calcium control of smooth muscle contractility

Ca2+ is a primary second messenger that binds to an intracellular receptor protein, calmodulin. Increases in cytosolic Ca2+ concentration mediated by activation of cell surface receptors result in the formation of a Ca2+ calmodulin complex that regulates many Ca2+-dependent cellular processes. In smooth muscle, Ca2+/calmodulin activates myosin light chain kinase, which phosphorylates the regulatory light chain of myosin. This phosphorylation reaction increases the actin-activated MgATPase activity of myosin and is associated with increases in contractile properties, including force, stiffness, and maximal shortening velocity. These biochemical and biomechanical responses occur rapidly (seconds) in response to physiological stimulation involving neurotransmitter activation of smooth muscle cells. Thus, the Ca2+-dependent phosphorylation of the myosin light chain is a primary event in activation of smooth muscle contraction.     

High force development and crossbridge attachment in smooth muscle from swine carotid arteries

In experiments designed to achieve maximal activation, the active force/cell cross-sectional area in tissues prepared from the swine carotid media was 6.7 +/- 0.3 (sd) X 10(5) N/m5. This value exceeds that reported for other vertebrate muscle cells and is striking because of the low smooth muscle myosin content. The hypothesis that high force generation may, in part, reflect an increase in the crossbridge duty cycle, i.e., the fraction of the cycle during which force is generated, was tested by determining the rate of force redevelopment after a step shortening and the ration of the load-bearing capacity of the contractile system to the developed stress during the course of isometric contractions. Maximal crossbridge cycling rates estimated by the rate of force redevelopment occurred 30 seconds after the onset of a high K+-induced contraction, and decreased thereafter, although the load-bearing capacity or maximum active stress was maintained. These results from isometric experiments support the hypothesis and provide further evidence that attached, non-cycling crossbridges contribute to force maintenance in tonically contracting arterial smooth muscle.     

Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle

We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-Pi) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2(+)-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 100 microns in diameter and 4 mm long, were mounted for isometric measurements and immersed in calcium-EGTA buffers. Fibers preincubated with anti-MLCK Fab in relaxing solution (Ca2+ less than 1 nM) for 75 minutes developed about 25% of the isometric force of a parallel control contraction when transferred to contracting solution (Ca2+ = 0.5 microM). When added to contracting solution at the peak of a contracture, anti-MLCK Fab elicited a relaxation that was complete in about 120 minutes despite the presence of Ca2+. No significant effect on isometric force was observed when fibers were incubated with another affinity-purified mouse Fab raised against the Fc region of human IgG (control Fab).(ABSTRACT TRUNCATED AT 250 WORDS)