Altered Contractility of Skeletal Muscle in Mice Deficient in Titin’s M-Band Region

We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca²⁺ handling, as indicated by analysis of Ca²⁺ transients in intact myofibers and expression of Ca²⁺-handling proteins, but can be explained by the reduced myofilament Ca²⁺ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca²⁺ sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.     

A mathematical model of airway and pulmonary arteriole smooth muscle

Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca²⁺), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca²⁺ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca²⁺ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca²⁺ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca²⁺ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca²⁺-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.     

Impact of detubulation on force and kinetics of cardiac muscle contraction

Action potential–driven Ca²⁺ currents from the transverse tubules (t-tubules) trigger synchronous Ca²⁺ release from the sarcoplasmic reticulum of cardiomyocytes. Loss of t-tubules has been reported in cardiac diseases, including heart failure, but the effect of uncoupling t-tubules from the sarcolemma on cardiac muscle mechanics remains largely unknown. We dissected intact rat right ventricular trabeculae and compared force, sarcomere length, and intracellular Ca²⁺ in control trabeculae with trabeculae in which the t-tubules were uncoupled from the plasma membrane by formamide-induced osmotic shock (detubulation). We verified disconnection of a consistent fraction of t-tubules from the sarcolemma by two-photon fluorescence imaging of FM4-64–labeled membranes and by the absence of tubular action potential, which was recorded by random access multiphoton microscopy in combination with a voltage-sensitive dye (Di-4-AN(F)EPPTEA). Detubulation reduced the amplitude and prolonged the duration of Ca²⁺ transients, leading to slower kinetics of force generation and relaxation and reduced twitch tension (1 Hz, 30°C, 1.5 mM [Ca²⁺]_o). No mechanical changes were observed in rat left atrial trabeculae after formamide shock, consistent with the lack of t-tubules in rodent atrial myocytes. Detubulation diminished the rate-dependent increase of Ca²⁺-transient amplitude and twitch force. However, maximal twitch tension at high [Ca²⁺]_o or in post-rest potentiated beats was unaffected, although contraction kinetics were slower. The ryanodine receptor (RyR)2 Ca-sensitizing agent caffeine (200 µM), which increases the velocity of transverse Ca²⁺ release propagation in detubulated cardiomyocytes, rescued the depressed contractile force and the slower twitch kinetics of detubulated trabeculae, with negligible effects in controls. We conclude that partial loss of t-tubules leads to myocardial contractile abnormalities that can be rescued by enhancing and accelerating the propagation of Ca²⁺-induced Ca²⁺ release to orphan RyR2 clusters.     

The heart in Duchenne muscular dystrophy: early detection of contractile performance alteration

Progressive cardiomyopathy is a major cause of death in Duchenne muscular dystrophy (DMD) patients. Coupling between Ca²⁺ handling and contractile properties in dystrophic hearts is poorly understood. It is also not clear whether developing cardiac failure is dominated by alterations in Ca²⁺ pathways or more related to the contractile apparatus. We simultaneously recorded force and Ca²⁺ transients in field‐stimulated papillary muscles from young (10–14 weeks) wild‐type (wt) and dystrophic mdx mice. Force amplitudes were fivefold reduced in mdx muscles despite only 30 % reduction in fura‐2 ratio amplitudes. This indicated mechanisms other than systolic Ca²⁺ to additionally account for force decrements in mdx muscles. pCa‐force relations revealed decreased mdx myofibrillar Ca²⁺ sensitivity. ‘In vitro’ motility assays, studied in mdx hearts here for the first time, showed significantly slower sliding velocities. mdx MLC/MHC isoforms were not grossly altered. Dystrophic hearts showed echocardiography signs of early ventricular wall hypertrophy with a significantly enlarged end‐diastolic diameter ‘in vivo’. However, fractional shortening was still comparable to wt mice. Changes in the contractile apparatus satisfactorily explained force drop in mdx hearts. We give first evidence of early hypertrophy in mdx mice and possible mechanisms for already functional impairment of cardiac muscle in DMD.     

Regulation analysis of contractile ATPase flux in skeletal muscle

The [Ca²⁺] regulation of contractile ATPase flux, J _p, in skeletal muscle was analysed by computation of the Response R ^Jp _Ca2+ for a 10 Hz range of electrical stimulation frequencies. Results of our analysis of the kinetic controls in ATP free energy metabolism in a network model of contracting muscle (J.A.L. Jeneson, H.V. Westerhoff and M.J. Kushmerick (2000) Am. J. Physiol. 279, C813–C832) formed the basis for the computations of R ^Jp _Ca2+. We found that neural regulation of sustained force generation via simple [Ca²⁺]_cyto frequency encoding in the network was robust for frequencies up to 2 Hz. Above 2 Hz, however, this regulation design broke down because of a shift in contractile ATPase flux control from the Ca²⁺-sensitive contractile filaments to mitochondria with low Ca²⁺ sensitivity. The role of glyco(geno)lytic ATP production at high contraction workloads is discussed in the context of this result     

Theoretical and experimental investigation of calcium-contraction coupling in airway smooth muscle

We investigated theoretically and experimentally the Ca²⁺-contraction coupling in rat tracheal smooth muscle. [Ca²⁺]_i, isometric contraction and myosin light chain (MLC) phosphorylation were measured in response to 1 mM carbachol. Theoretical modeling consisted in coupling a model of Ca²⁺-dependent MLC kinase (MLCK) activation with a four-state model of smooth muscle contractile apparatus. Stimulation resulted in a short-time contraction obtained within 1 min, followed by a long-time contraction up to the maximal force obtained in 30 min. ML-7 and Wortmannin (MLCK inhibitors) abolished the contraction. Chelerythrine (PKC inhibitor) did not change the short-time, but reduced the long-time contraction. [Ca²⁺ _i responses of isolated myocytes recorded during the first 90 s consisted in a fast peak, followed by a plateau phase and, in 28% of the cells, superimposed Ca²⁺ oscillations. MLC phosphorylation was maximal at 5 s and then decreased whereas isometric contraction followed a Hill-shaped curve. The model properly predicts the time course of MLC phosphorylation and force of the short-time response. With oscillating Ca²⁺ signal, the predicted force does not oscillate. According to the model, the amplitude of the plateau and the frequency of oscillations encode for the amplitude of force, whereas the peak encodes for force velocity. The long-time phase of the contraction, associated with a second increase in MLC phosphorylation, may be explained, at least partially, by MLC phosphatase (MLCP) inhibition, possibly via PKC inhibition.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Reiji Natori, Setsuro Ebashi, and excitation-contraction coupling

The achievements of Natori and Ebashi, which greatly contributed to the progress in studies of excitation–contraction coupling, were reviewed. Natori succeeded in removing the cell membrane of an isolated fiber of skeletal muscle to prepare a skinned fiber, which still responded to an electrical stimulation with propagated contraction. Skinned fibers showed elastic extensibility beyond the elastic limit of intact muscle fibers. Based on this elasticity Natori predicted the presence of an elastic components, later found as connectin. Skinned fibers, an excellent experimental system, contributed greatly to the progress in subsequent studies. Ebashi showed that the essential principle of the relaxing factor was not the ATP-regenerating enzymes as generally thought, but a particulate fraction with MgATPase. Then he clearly showed that a minute amount of Ca²⁺ is necessary for the contractile reaction of actomyosin, and that the relaxing factor strongly accumulates Ca²⁺ in the presence of ATP and causes relaxation by the removal of Ca²⁺. He further discovered that the Ca²⁺-induced regulation of the contractile reaction of the myosin–actin system requires the presence of tropomyosin and a new protein, troponin. Troponin binds to a specific site on tropomyosin, which in turn binds to actin in the thin filament. Troponin is the Ca²⁺-receptive protein, and changes in troponin molecules upon Ca²⁺ binding is transmitted to actin through tropomyosin to regulate the actin–myosin interaction. Through these findings, the excitation was connected by Ca²⁺ with the contraction.     

Accelerated communication the direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca²⁺ release, and verapamil, a Ca²⁺ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED₅₀ was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca²⁺-release from intracellular calcium stores in the contractile response to GRP.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses¹. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation². This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency⁴. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively⁵. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.Contraction of smooth muscle is generally regulated by the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) plus sensitivity to Ca^2+, of the contractile elements in response to changes in the environment surrounding the cell⁶. [Ca²⁺]_i is determined by the combination of the movement of Ca²⁺ through plasma membrane ligand or voltage gated Ca²⁺ channels and the release and uptake of Ca²⁺ from internal stores. Cytosolic Ca²⁺ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction⁸. However, the sensitivity of this pathway to Ca²⁺ can be regulated by the MLC phosphatase (MLCP)⁹. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.Here, we present a method to evaluate changes in [Ca²⁺]_i over time in isolated, perfused lymphatics in order to study Ca²⁺-dependent and Ca²⁺-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca²⁺]_i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca²⁺]_i was determined by loading lymphatics with the ratiometric, Ca²⁺-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.     

Rho Kinase Enhances Contractions of Rat Mesenteric Collecting Lymphatics

The mechanisms that control phasic and tonic contractions of lymphatic vessels are poorly understood. We hypothesized that rho kinase ROCK, previously shown to increase calcium (Ca²⁺) sensitivity in vascular smooth muscle, enhances lymphatic contractile activity in a similar fashion. Contractions of isolated rat mesenteric lymphatic vessels were observed at a luminal pressure of 2 cm H₂O in a 37°C bath. The expression of ROCK in isolated rat mesenteric lymphatic vessels was assessed by Western blotting and confocal microscopy. The role of ROCK in contractile function was tested using two specific yet structurally distinct inhibitors: H1152 (0.1–10 μM) and Y-27632 (0.5–50 μM). In addition, lymphatics were transfected with constitutively active (ca)-ROCK protein (2 μg/ml) to assess gain of contractile function. Vessel diameter and the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i) were simultaneously measured in a subset of isolated lymphatics loaded with the Ca²⁺-sensing dye fura-2. The results show expression of both the ROCK1 and ROCK2 isoforms in lymphatic vessels. Inhibition of ROCK increased lymphatic end diastolic diameter and end systolic diameter in a concentration-dependent manner. Significant reductions in lymphatic tone and contraction amplitude were observed after treatment 1–10 μM H1152 or 25–50 μM Y-27632. H1152 (10 μM) also significantly reduced contraction frequency. Transient increases in [Ca²⁺]_i preceded each phasic contraction, however this pattern was disrupted by either 10 μM H1152 or 50 μM Y-27632 in the majority of lymphatics studied. The significant decrease in tone caused by H1152 or Y-27632 was not associated with a significant change in the basal [Ca²⁺]_i between transients. Transfection with ca-ROCK protein enhanced lymphatic tone, but was not associated with a significant change in basal [Ca²⁺]_i. Our data suggest that ROCK mediates normal tonic constriction and influences phasic contractions in lymphatics. We propose that ROCK modulates Ca²⁺ sensitivity of contractile proteins in lymphatics.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

Calmodulin and the regulation of smooth muscle contraction

Calmodulin, the ubiquitous and multifunctional Ca²⁺-binding protein, mediates many of the regulatory effects of Ca²⁺, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca²⁺]_i transientvia the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca²⁺/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca²⁺. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca²⁺/calmodulin or indirectly by phosphorylation catalysed by Ca²⁺/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca²⁺ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca²⁺/calmodulin, e.g. the sarcolemmal Ca²⁺ pump and the ryanodine receptor/Ca²⁺ release channel, and other proteins which indirectly regulate [Ca²⁺]_i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.     

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse,a model of Huntington’s disease

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca²⁺ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca²⁺ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca²⁺ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca²⁺ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca²⁺ removal from the myoplasm and Ca²⁺ release flux from the sarcoplasmic reticulum were characterized using a Ca²⁺ binding and transport model, which indicated a significant reduction in slow Ca²⁺ removal activity and Ca²⁺ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca²⁺ channel conductance. These results indicate profound changes of Ca²⁺ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.     

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae)

Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca²⁺ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca²⁺ in toad fibers, without affecting the rate of decay. The difference in entire Ca²⁺ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K⁺], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K⁺] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K⁺] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property. Accepted: 27 August 1998     

Length-dependent deactivation of ventricular trabeculae in the bivalve, Spisula solidissima

Shortening-deactivation has been identified and characterized in ventricular trabeculae of the bivalve, Spisula solidissima (Heterodonta, Mactridae). This muscle had ultrastructural similarities to vertebrate smooth muscle. Deactivation was defined as the fraction of maximal force lost during a contraction when a muscle is shortened rapidly (by a quick-release, QR) to a known length, relative to a control isometric contraction at that same length. The magnitude of deactivation was dependent on the size of the release and the point at which the release was applied during the cycle of contraction. QR/quick-stretch (QS) perturbations at the same point during the contraction resulted in negligible deactivation. The magnitude of deactivation was independent of shortening rate. Deactivation was attenuated by applying caffeine (100 μM) and blocked with high extracellular Ca²⁺ (56 mM). The Ca²⁺ ionophore, A23187 (10 μM), augmented deactivation as did the positive inotrope serotonin (100 nM). Treatment with ryanodine (5 μM) had no significant effect on deactivation. These results suggest that a reduction in Ca²⁺ at the contractile element and/or sequestration of Ca²⁺ may occur during shortening. Deactivation may minimize the magnitude of work done during active shortening of bivalve cardiac muscle, particularly against the low afterload exhibited in the bivalve peripheral circulatory system. Intracellular Ca²⁺ fluxes during sudden length perturbations may explain the effect of stretch on action potential duration in the bivalve heart, as shown previously.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.     

A Role for the Tyrosine Kinase Pyk2 in Depolarization-induced Contraction of Vascular Smooth Muscle

Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC₂₀). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca²⁺-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K⁺-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC₂₀ phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K⁺, Pyk2 autophosphorylation, and LC₂₀ phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca²⁺, elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca²⁺ channel blocker nifedipine inhibited peak and sustained K⁺-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K⁺-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca²⁺ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC₂₀ phosphorylation then accounts for the tonic contractile response to membrane depolarization.