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The effects of D-600 phenylalkylamine calcium antagonist on the excitation-contraction coupling of intact skeletal muscle fibers were analyzed in frog single phasic tibialis anterior muscle fibers. Analysis of isolated sacroplasmic reticulum (SR) from frog single phasic muscle fibers indicated the role of D-600 in inhibiting Ca2+ uptake. However, the phenylalkylamine calcium antagonist did not alter caffeine-induced Ca2+ release from isolated rabbit junctional SR indicating the inhibitory effect of D-600 on Ca2+-adenosine triphosphatase.

Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum [Ca.sup.2+]-ATPase. Am J Physiol Cell Physiol 279: C166-C172, 2000.--Experiments were performed to determine whether the organic [Ca.sup.2+] channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) [Ca.sup.2+] uptake by directly inhibiting the light SR [Ca.sup.2+]-ATPase. We have previously shown that at 10 [micro]M, D-600 inhibits LSR ATP-dependent [Ca.sup.2+] uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR [Ca.sup.2+]-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas [Ca.sup.2+] binding and translocation are associated with the transmembrane domain (18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermal inactivation experiments using the SR [Ca.sup.2+]-ATPase demonstrated that D-600 decreased the thermal stability of [C.sup.a2+] transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 [micro]M did not have any leaking effect of [Ca.sup.2+] on the [Ca.sup.2+]-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the [Ca.sup.2+]-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the [Ca.sup.2+] blocker D-600 inhibits the SR [Ca.sup.2+] pump specifically by interacting with the transmembrane [Ca.sup.2+]-binding domain of the [Ca.sup.2+]-ATPase. calcium channel blocker; thermal denaturation profiles; transmembrane domain

We used four antibodies to regions of obscurin isoforms A and B, encoded by the obscurin gene, to investigate the location of these proteins in skeletal myofibers at resting and stretched lengths. Obscurin A ( approximately 800 kDa) which was recognized by antibodies generated to the N-terminal, Rho-GEF, and the non-modular C-terminal domain that lacks the kinase-like domains, localizes at the level of the M-band. Obscurin B ( approximately 900 kDa) which has the N-terminal, Rho-GEF, and the C-terminal kinase-like domains, localizes at the level of the A/I junction. Additional isoforms, which lack one or more of these epitopes, are present at the Z-disk and Z/I junction.

In skeletal muscle, contractile force can be transmitted laterally between the z-disks and M-lines of neighboring myofibrils, across the sarcolemma, and through the extracellular matrix to the tendon. Here we examine the data that support this model and the sarcolemmal properties and structures, termed "costameres," that are consistent with it.

This study was designed to investigate the effects on single skeletal muscle fibers of a novel thienylhydrazone, referred to as LASSBio-294, which is a bioisoster of pyridazinone compounds that inhibit the cyclic AMP-specific phosphodiesterase (PDE) 4. Twitch and fatigue were analyzed in single skeletal muscle fibers isolated from either the semitendinous or the tibialis anterior muscles dissected from the frog Rana pipiens. LASSBio-294 (12.5-100 microM) increased twitch tension, accelerated the maximal rate of tension decay during relaxation, and had very little effect in the maximal rate of tension development of muscle fibers directly stimulated at < or =30 Hz. The positive inotropic effect of LASSBio-294 developed slowly, reaching its maximum at 40 min and was inversely proportional to the frequency of stimulation, becoming negligible at 60 and 90 Hz. The concentration-response relationship for LASSBio-294-induced potentiation of twitch tension was bell-shaped, with maximal effect occurring at 25 microM. In addition, LASSBio-294 reduced development of fatigue induced by tetanic stimulation of the muscle fibers and reduced the time needed for 80% prefatigue tension recovery after fatigue had developed to 50% of the maximal pretetanic force. These effects of LASSBio-294 can be fully explained by stimulation of the sarcoplasmic reticulum Ca2+ pump and could be ascribed to an increase in cellular levels of cyclic AMP due to PDE inhibition. The novel thienylhydrazone LASSBio-294 may be useful for treatment of patients suffering from conditions in which muscle fatigue is a debilitating symptom (e.g., chronic heart failure).

In excitable cells, the concentration of intracellular free Mg2+ ([Mg2+]i) is several hundred times lower than expected if Mg2+ ions were at electrochemical equilibrium. Since Mg2+ is a permeant ion across the plasmalemma, it must be constantly extruded. An ATP-dependent Na/Mg exchanger has been proposed as the sole mechanism responsible for Mg2+ extrusion. However, this hypothesis fails to explain numerous observations including the fact that K+ and Cl- appear to be involved in Mg2+ transport. Until now three main limitations have hampered the studies of plasmalemmal Mg2+ transport: i) 28Mg, the only useful radioactive isotope of Mg2+, has a short half-life and is difficult to obtain; ii) squid giant axons, the ideal preparation to carry out transport studies under "zero-trans" conditions, are only available during the summer months; and iii) the ionic fluxes mediated by the Mg2+ transporter are very small and difficult to measure. The purpose of this manuscript is to review how these limitations have been recently overcame and to propose a novel hypothesis for the plasmalemmal Mg2+ transporter in squid axons and barnacle muscle cells. Overcoming the limitations for studying the plasmalemmal Mg2+ transporter has been possible as a result of the following findings: i) the Mg2+ exchanger can operate in "reverse", thus extracellular Mg2+-dependent ionic fluxes (e.g., Na+ efflux) can be utilized to measure its activity; ii) internally perfused, voltage-clamped barnacle muscle cells which are available all year long can be used in addition to squid axons; and iii) phosphoinositides (e.g., PIP2) produce an 8-fold increase in the ionic fluxes mediated by the Mg2+ exchanger. The hypothesis that we postulate is that, in squid giant axons and barnacle muscle cells, a 2Na+2K+2Cl:1Mg exchanger is responsible for transporting Mg2+ across the plasmalemma and for maintaining [Mg2+]i under steady-state conditions.

A central working hypothesis in our laboratory is that deficient cellular cyclic AMP concentrations may be responsible, at least in part, for striated muscle dysfunction, both cardiac and skeletal, in heart failure. These results suggest that therapy aimed at restoring cyclic AMP to normal levels may be effective with regard to improving systolic and diastolic function in the heart and may decrease the development of fatigue in skeletal muscle of patients with failure. The use of cyclic AMP-dependent drugs in clinical practice has been limited by side effects associated with raising total cellular content of this cyclic nucleotide. However, evidence suggesting that separate pools of cyclic AMP may exist within the cell raises the possibility that those pools associated with excitation/contraction coupling could serve as more specific therapeutic targets.

Deficient myocardial cyclic AMP concentrations contribute to abnormal Ca2+ handling and systolic and diastolic dysfunction in chronic heart failure (CHF). We tested the hypothesis that decreased cyclic AMP in skeletal muscle of animals with failure may contribute to the weakness and easy fatiguability also common in patients with CHF. We compared intracellular Ca2+ signaling and contractility in skeletal muscle preparations from rats 6 weeks after myocardial infarction-induced CHF versus sham-operated controls. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control and CHF rats. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches. Treatment with 2mM dibutyryl cyclic AMP returned tension and Ca2+ towards normal levels. There was no evidence of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling that could be reversed with cyclic AMP supplementation as previously reported for the heart. This suggests that deficient cyclic AMP levels may contribute to both cardiac and skeletal muscle dysfunction in CHF.

We have examined the spatial distribution of [Ca2+]i during tetanic stimulation in frog skeletal muscle cells using a fluorescence imaging method. We have found a completely unexpected pattern of Ca release: Ca is released forming gradients composed of spots of very significant and slow fluctuations of calcium release. Our findings could be explained if the calcium release process in skeletal muscle is influenced significantly by [Ca2+]i, such as in cardiac muscle, and suggests that the SR/Ca release control can include the established voltage-dependent plus a cardiac-like process of calcium-induced Ca release and a Ca release inhibition by Ca.

The bioluminescent calcium indicator aequorin was loaded into bundles of skeletal muscle fibers from the rat extensor digitorum longus by macroinjection, a technique previously applied only to cardiac muscle. After loading, the amplitude and time course of the twitch returned to control values, indicating lack of damage to the fibers. Individual light signals (i.e., calcium transients) were recorded during each twitch or tetanus without the need for signal averaging. The calcium transients obtained were qualitatively and quantitatively similar to those reported previously with microinjection of aequorin. Our data suggest that macroinjection may be the method of choice for loading aequorin into mammalian skeletal muscle.