(125)I]-N-[(3-azido-5-iodo)benzyl]dantrolene and [(125)I]-N-[[3-iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl]dantrolene: photoaffinity probes specific for the physiological Ca(2+) release from sarcoplasmic reticulum of skeletal muscle

In order to capture and identify key molecules that regulate the release of Ca(2+) from the sarcoplasmic reticulum (SR) of skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a highly selective inhibitory effect on physiological Ca(2+) release (PCR) without affecting Ca(2+)-induced Ca(2+) release (CICR). Successful realization of the sharp discrimination between PCR and CICR has led to the creation of [(125)I]GIF-0082 and [(125)I]GIF-0276, which were synthesized by substituting a stannyl group with (125)I in the corresponding phenylstannane precursors.     

Design of dantrolene-derived probes for radioisotope-free photoaffinity labeling of proteins involved in the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle

Bifunctional dantrolene derivatives have been synthesized as probes for radioisotope-free photoaffinity labeling with the aim of elucidating the molecular mechanism of skeletal muscle contraction. GIF-0430 and GIF-0665 are aromatic azido-functionalized derivatives that were designed to selectively inhibit physiological Ca2+ release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without a strong effect on Ca2+-induced Ca2+ release (CICR). These photoaffinity probes consist of either an azidomethyl or an ethynyl group, respectively, which could function as a tag for introduction of an optional detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation. Actually, the former probe worked to photolabel its target proteins specifically as confirmed by subsequent fluorescent visualization.     

Changes in the electromechanical activity in the course of tetanic contraction

The functioning of excitation-contraction coupling during tetanic contraction was investigated on frog skeletal muscle. The effect of the calcium release blocker dantrolene was tested on electrically evoked twitches and tetanic contractions. It was shown that the first: developmental stage of tetanus is inhibited by dantrolene as well as a twitch contraction, and does not influenced by calcium-free medium. This substantiates it as based on "voltage dependent Ca-release" (VDCR) mechanism of activation, when depolarization directly opens the rhyanodin receptor calcium channels. The next stage: the long lasting plateau of tetanic contraction, is directly dependent on external Ca2+ entry and also inhibited by dantrolene, and therefore may be described as "calcium-induced Ca-release" (CICR) activation mechanism. It is proposed that such change in ECC mechanism taking place during tetanic contraction, can occur also in conditions of natural muscle activity, because of its rhythmical nature.     

Partial inhibition of skeletal muscle contraction by dantrolene sodium and its modification with perchlorate and Bay K 8644.

The effects of dantrolene sodium (DAN) on the dihydropyridine receptor (DHPR) of the transverse (T) tubule voltage sensor (Ca2+ channel) was studied with single fibers from bullfrog toe muscle. Perchlorate (ClO4-), which acts selectively on the DHPR, overcame DAN-induced inhibition of twitch tension. Bay K 8644, a DHPR agonist, slowed the rate of twitch inhibition by DAN. DAN inhibited twitch tension to a greater extent in Ca(2+)-free solution than in Ringer solution or solution containing Zn2+, whereas twitch inhibition by DAN was less in caffeine-containing solution than in the control. The effects of DAN on Zn(2+)- and caffeine-treated fibers and on fibers in Ca(2+)-free solution suggest that DAN must act near the voltage sensor of the T tubule. However, differences in net twitch inhibition by DAN between control fibers and fibers potentiated by ClO4- or Bay K 8644 suggest that DAN does not bind to the same site as these potentiating agents do. The role of myoplasmic Ca2+ in DAN-induced inhibition of twitch and the effects of DAN on the mechanical threshold and membrane potential in skeletal muscle are discussed.     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.     

Effects of agonists and antagonists of rhyanodine receptors on potassium contractures in twitch and tonic frog skeletal muscle fibers

A comparative analysis of the effects of the concentrations of Ca2+ in external medium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of rhyanodine-sensitive Ca2+ channels of carcoplasmic reticulum on the characteristics of potassium contracture in frog twitch and tonic skeletal muscles has been performed. It was shown that the duration of contracture in tonic muscles is not restricted by the presence of Ca2+, as distinct from twitch muscles. Dandrolene does not practically affect the contractile responses of tonic fibres, and the concentration of cresol eliciting the contracture for tonic fibres is substantially higher (1 mM) than for twitch fibers (0.25 mM). In twitch fibers, the potassium contracture activated in the presence of cresol is comparable in amplitude and dynamics with the contracture under control conditions, and in tonic fibers a summing of responses without relaxation after the washing of excessive potassium is observed. This suggests that, in twitch fibers, the influx of Ca2+ can directly create the concentration sufficient for the maintenance of contraction, and in tonic fibers its involvement is mediated through the Ca(2+)-dependent activation of the beta-isoform of rhyanodine-sensitive channels.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

The off rate of Ca(2+) from troponin C is regulated by force-generating cross bridges in skeletal muscle.

The effects of dissociation of force-generating cross bridges on intracellular Ca(2+), pCa-force, and pCa-ATPase relationships were investigated in mouse skeletal muscle. Mechanical length perturbations were used to dissociate force-generating cross bridges in either intact or skinned fibers. In intact muscle, an impulse stretch or release, a continuous length vibration, a nonoverlap stretch, or an unloaded shortening during a twitch caused a transient increase in intracellular Ca(2+) compared with that in isometric controls and resulted in deactivation of the muscle. In skinned fibers, sinusoidal length vibrations shifted pCa-force and pCa-actomyosin ATPase rate relationships to higher Ca(2+) concentrations and caused actomyosin ATPase rate to decrease at submaximal Ca(2+) and increase at maximal Ca(2+) activation. These results suggest that dissociation of force-generating cross bridges during a twitch causes the off rate of Ca(2+) from troponin C to increase (a decrease in the Ca(2+) affinity of troponin C), thus decreasing the Ca(2+) sensitivity and resulting in the deactivation of the muscle. The results also suggest that the Fenn effect only exists at maximal but not submaximal force-activating Ca(2+) concentrations.     

Cardiac-type EC-coupling in dysgenic myotubes restored with Ca2+ channel subunit isoforms alpha1C and alpha1D does not correlate with current density

Ca(2+)-induced Ca(2+)-release (CICR)-the mechanism of cardiac excitation-contraction (EC) coupling-also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca(2+) release, by reconstituting dysgenic myotubes with the cardiac Ca(2+) channel alpha(1C) (Ca(V)1.2) subunit. Ca(2+) influx through alpha(1C) provides the trigger for opening the sarcoplasmic reticulum Ca(2+) release channels. Here we show that also the Ca(2+) channel alpha(1D) isoform (Ca(V)1.3) can restore cardiac-type EC-coupling. GFP-alpha(1D) expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca(2+) transients with the same efficiency as GFP-alpha(1C) despite threefold smaller Ca(2+) currents. In contrast, GFP-alpha(1A), which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca(2+) transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca(2+) channels and less on their current size. Combined patch-clamp/fluo-4 Ca(2+) recordings revealed that the induction of Ca(2+) transients and their maximal amplitudes are independent of the different current densities of GFP-alpha(1C) and GFP-alpha(1D). These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca(2+) gradients in the triad junctions.     

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.     

In vivo monitoring of Ca(2+) uptake into mitochondria of mouse skeletal muscle during contraction

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca(2+) handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca(2+) transients reliably during the contraction-relaxation cycle. Using two-photon microscopy and genetically expressed "cameleon" Ca(2+) sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca(2+) transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca(2+) during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca(2+) increase is delayed by a few milliseconds compared with the cytosolic Ca(2+) rise and occurs both during a single twitch and upon tetanic contraction.     

The inhibitory effects of dantrolene on action potential-induced calcium transients in cultured rat dorsal root ganglion neurons

We investigated the actions of dantrolene Ca(2+)-induced on Ca(2+)-release (CICR) evoked by action potentials in cultured rat sensory neurons. The effect of dantrolene on action potential after-depolarization and voltage-activated calcium currents was studied in cultured neonatal rat dorsal root ganglion cells (DRG) using the whole-cell patch-clamp technique. Depolarizing current injection evoked action potentials and depolarizing after-potentials, which are activated as a result of CICR following a single action potential in some cells. The type of after-potentials was determined by inducing action potentials from the resting membrane potential. Extracellular application of dantrolene (10 microM) abolished after-depolarizations without affecting action potential properties. Furthermore, dantrolene significantly reduced repetitive action potentials after depolarizing current injection into these neurons, but had no significant effect on the steady-state current voltage relationship of calcium currents in these neurons. We conclude that dantrolene inhibits the induction of action potential after depolarizations by inhibiting CICR in cultured rat sensory neurons.     

Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction

The steps that couple depolarization of the cardiac cell membrane to initiation of contraction remain controversial. Depolarization triggers a rise in intracellular free Ca(2+) which activates contractile myofilaments. Most of this Ca(2+) is released from the sarcoplasmic reticulum (SR). Two fundamentally different mechanisms have been proposed for SR Ca(2+) release: Ca(2+)-induced Ca(2+) release (CICR) and a voltage-sensitive release mechanism (VSRM). Both mechanisms operate in the same cell and may contribute to contraction. CICR couples the release of SR Ca(2+) closely to the magnitude of the L-type Ca(2+) current. In contrast, the VSRM is graded by membrane potential rather than Ca(2+) current. The electrophysiological and pharmacological characteristics of the VSRM are strikingly different from CICR. Furthermore, the VSRM is strongly modulated by phosphorylation and provides a new regulatory mechanism for cardiac contraction. The VSRM is depressed in heart failure and may play an important role in contractile dysfunction. This review explores the operation and characteristics of the VSRM and CICR and discusses the impact of the VSRM on our understanding of cardiac excitation-contraction coupling.     

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.