The stiffness of the single myosin motor (epsilon) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T0) modulated by changing either [Ca2+] or temperature. The hs compliance is 43.0+/-0.8 nm MPa-1 in isometric contraction at saturating [Ca2+], whereas in rigor it is 28.2+/-1.1 nm MPa-1. The equivalent compliance of myofilaments is 21.0+/-3.3 nm MPa-1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5+/-1.7 kPa nm-1 in isometric contraction at saturating [Ca2+] (e0), and in rigor (er) it rises to 138.9+/-21.2 kPa nm-1. Epsilon, calculated from er and the lattice molecular dimensions, is 1.21+/-0.18 pN nm-1. epsilon estimated, using a thermodynamic approach, from the relation of T0 at saturating [Ca2+] versus the reciprocal of absolute temperature is 1.25+/-0.14 pN nm-1, similar to that estimated for fibers in rigor. Consequently, the ratio e0/er (0.33+/-0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e0 and er increase by approximately 40%. Epsilon becomes approximately 1.7 pN nm-1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.

Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle, including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rate of this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mm amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.

Sphingosine 1-phosphate (S1P) activates a subset of plasma membrane receptors of the endothelial differentiation gene family (EdgRs) in many cell types. In C2C12 myoblasts, exogenous S1P elicits Ca2+ transients by activating voltage-independent plasma membrane Ca2+ channels and intracellular Ca2+-release channels. In this study, we investigated the effects of exogenous S1P on voltage-dependent L-type Ca2+ channels in skeletal muscle fibers from adult mice. To this end, intramembrane charge movements (ICM) and L-type Ca2+ current (I(Ca)) were measured in single cut fibers using the double Vaseline-gap technique. Our data showed that submicromolar concentrations of S1P (100 nM) caused a approximately 10-mV negative shift of the voltage threshold and transition voltages of q(gamma) and q(h) components of ICM, and of I(Ca) activation and inactivation. Biochemical studies showed that EdgRs are expressed in skeletal muscles. The involvement of EdgRs in the above S1P effects was tested with suramin, a specific inhibitor of Edg-3Rs. Suramin (200 microM) significantly reduced, by approximately 90%, the effects of S1P on ICM and I(Ca), suggesting that most of S1P action occurred via Edg-3Rs. Moreover, SIP at concentration above 10 microM elicited intracellular Ca2+ transients in muscle fibers loaded with the fluorescent Ca2+ dye Fluo-3, as detected by confocal laser scanning microscopy.

Intramembrane charge movements, IICM, were measured in rat skeletal muscle fibres in response to voltage steps from a −90 mV holding potential to a wide test voltage range (−85 to 30 mV), using a double Vaseline‐gap voltage‐clamp technique. Solutions were designed to minimise ionic currents. Ca2+ current was blocked by adding Cd2+ (0.8 mm) to the external solution. In a subset of experiments Cd2+ was omitted to determine which components of the charge movement best correlated with L‐type Ca2+ channel gating. Detailed kinetic analysis of IICM identified two major groups of charges. The first two components, designated Qa and Qb, were the only charges moved by small depolarising steps. The second group of components, Qc and Qd, showed a more positive voltage threshold, −35.6 ± 2.0 mV, (n= 6) in external solution with Cd2+, and −41.1 ± 2.0 mV (n= 12) in external solution without Cd2+. Notably, in external solution without Cd2+ the voltage threshold of Ca2+ current, ICa, activation had a similar value, being −38.1 ± 2.4 mV. The sum of three Boltzmann functions, Q1, Q2 and Q3, showing progressively more positive transition voltages, could be fitted to charge versus voltage, QICM‐V, plots. The three Boltzmann terms identified three charge components: Q1 described the shallow voltage‐dependent Qa and Qb charges, Q2 and Q3 described the steep voltage‐dependent Qc and Qd charges. In external solution without Cd2+ the charge kinetics changed: a slow decaying phase was replaced by a pronounced delayed hump. Moreover, the transition voltages of the individual steady‐state charge components were shifted towards negative potentials (from 6.3 to 8.2 mV). Nevertheless, the overall charge and steepness factors were conserved. In conclusion, these experiments allowed a clear separation of four components of intramembrane charge movements in rat skeletal muscle, showing that there are no fundamental differences with respect to charge movement components between amphibian and mammalian twitch muscle. Moreover, Qc and Qd charge are correlated with L‐type Ca2+ channel gating.

The dihydropyridine receptors (DHPRs)/L-type Ca 2+ channels of skeletal muscle are coupled with ryanodine receptors/Ca 2+ release channels (RyRs/CRCs) located in the sarcoplasmic reticulum (SR). The DHPR is the voltage sensor for excitation-contraction (EC) coupling and the charge movement component q γ has been implicated as the signal linking DHPR voltage sensing to Ca 2+ release from the coupled RyR. Recently, a new charge component, q h , has been described and related to L-type Ca 2+ channel gating. Evidence has also been provided that the coupled RyR/CRC can modulate DHPR functions via a retrograde signal. Our aim was to investigate whether the newly described q h is also involved in the reciprocal interaction or cross-talk between DHPR/L-type Ca 2+ channel and RyR/CRC. To this end we interfered with DHPR/L-type Ca 2+ channel function using nifedipine and 1-alkanols (heptanol and octanol), and with RyR/CRC function using ryanodine and ruthenium red (RR). Intramembrane charge movement (ICM) and L-type Ca 2+ current (I Ca ) were measured in single cut fibres of the frog using the double-Vaseline-gap technique. Our records showed that nifedipine reduced the amount of q γ and q h moved by ∼90% and ∼55%, respectively, whereas 1-alkanols completely abolished them. Ryanodine and RR shifted the transition voltages of q γ and q h and of the maximal conductance of I Ca by ∼4−9 mV towards positive potentials. All these interventions spared q β . These results support the hypothesis that only q γ; and q h arise from the movement of charged particles within the DHPR/L-type Ca 2+ channel and that these charge components together with I Ca are affected by a retrograde signal from RyR/CRC.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349–357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

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Amrinone is a bipyridine drug with characteristic effects on the force-velocity relationship of fast skeletal muscle. Here we combined in vitro motility assays, transient biochemical kinetics and optical tweezers studies to elucidate the mechanisms underlying the drug effects. Amrinone (1-2 mM) reduced the sliding velocity of heavy meromyosin (HMM) propelled actin filaments by 31.0 5 2.5% (n ¼ 15) at different ionic strengths of the assay solution (20 -160 mM). The drug also reduced (by 2 -18%) the sliding velocity of actin filaments propelled by subfragment 1 (S1). Stopped-flow studies of myofibrils, acto-HMM and acto-S1 showed no amrinone-induced reduction in the rate of MgATP induced actomyosin dissociation and optical tweezers studies detected no changes in the working stroke length. In contrast, the ADP affinity of acto-HMM (but not acto-S1) was increased about two-fold by 1 mM amrinone. Our results are consistent with inhibition of a strain-dependent MgADP-release step as the basis for amrinone induced reduction in sliding velocity. Modeling suggests that such an effect may also account for most other amrinone-induced changes of the force-velocity relationship of muscle (e.g. in isometric force and in shape of the force-velocity curve). Moreover, the results point to the possible importance of cooperative interactions between the two myosin heads in muscle contraction.