Malignant hyperthermia and excitation–contraction coupling

Malignant hyperthermia (MH) is a state of elevated skeletal muscle metabolism that may occur during general anaesthesia in genetically pre‐disposed individuals. Malignant hyperthermia results from altered control of sarcoplasmic reticulum (SR) Ca²⁺ release. Mutations have been identified in MH‐susceptible (MHS) individuals in two key proteins of excitation–contraction (EC) coupling, the Ca²⁺ release channel of the SR, ryanodine receptor type 1 (RyR1) and the α1‐subunit of the dihydropyridine receptor (DHPR, L‐type Ca²⁺ channel). During EC coupling, the DHPR senses the plasma membrane depolarization and transmits the information to the ryanodine receptor (RyR). As a consequence, Ca²⁺ is released from the terminal cisternae of the SR. One of the human MH‐mutations of RyR1 (Arg614Cys) is also found at the homologous location in the RyR of swine (Arg615Cys). This animal model permits the investigation of physiological consequences of the homozygously expressed mutant release channel. Of particular interest is the question of whether voltage‐controlled release of Ca²⁺ is altered by MH‐mutations in the absence of MH‐triggering substances. This question has recently been addressed in this laboratory by studying Ca²⁺ release under voltage clamp conditions in both isolated human skeletal muscle fibres and porcine myotubes.     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

Isolation of a Ca2+-releasing factor from caffeine-treated skeletal muscle fibres and its effect on Ca2+ release from sarcoplasmic reticulum

Summary In the presence of 2mm caffeine, skeletal muscles of the frog exert small irregular oscillations of single sarcomeres. A factor, released from these oscillating muscles, was partially purified, and its activity was tested on skinned fibres and isolated vesicles of the sarcoplasmic reticulum (SR). Purification was achieved in three steps by gel filtration and reversed phase chromatography, and the active compound of the released material was shown probably to be a small peptide. In skinned fibres, the purified factor evoked repetitive contractions and subthreshold sarcomeric oscillations. In heavy SR vesicles passively loaded with⁴⁵Ca²⁺, the factor induced a small but significant increase in the⁴⁵Ca²⁺ efflux rate. At the single channel level, the open probability of the SR Ca²⁺ release channel increased when the factor was added to the cytoplasmic side of the channel. The results reveal that the released factor potentiates Ca²⁺ release from the SR by increasing the open time of the Ca²⁺ release channel.     

Effects of tetracaine and procaine on skinned muscle fibres depend on free calcium

Summary The local anaesthetics, tetracaine and procaine have previously been found to block, induce or potentiate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle depending on the preparation, experimental conditions and design. We now show that low concentrations of tetracaine and procaine block SR Ca²⁺ release whereas high concentrations induce release from the SR of amphibian and mammalian skinned fibres. Both actions depend on pCa, such that a shift in pCa can alter their effect from blocking to releasing Ca²⁺. In skinned fibres with Ca²⁺-loaded SR, tetracaine (1mm) produced a tonic contraction with a time to half-peak of 15–20 s and a magnitude reaching 80% of maximum force. Ca²⁺ release by tetracaine or procaine occured at pCa 6.5 and was not blocked by Ruthenium Red (RR) (25 m). This action of tetracaine was attributed to SR Ca²⁺ release rather than to a displacement of bound Ca²⁺ because fibres lacking a functional SR due to pre-treatment with quercetin (100 m), A 23187 (100 g ml^–1) or Triton X-100 (1%) did not contract after additions of tetracaine. Lower concentrations of tetracaine (0.5mm) and procaine (10mm) blocked contractions due to caffeine (at pCa 6.73), sulphydryl oxidizing agents, or Ca²⁺-induced Ca²⁺ release (CICR). The inhibition of CICR as a function of pCa was difficult to measure quantitatively since lowering pCa to elicit CICR twitches was sufficient to initiate tetracaine-induced tonic contractions.Experiments with isolated SR vesicles showed that 1mm tetracaine inhibited CICR, over a wide range of pCa but 3–5mm tetracaine induced rapid Ca²⁺ release. The opposite effects of tetracaine and procaine depend mostly on their concentration in SR vesicles and/or pCa in skinned fibres. Blockade of release seems to occur via the CICR pathway, and induction of release through an increase in SR membrane permeability.Abbreviations SR sarcoplasmic reticulum - HEPES N-2-hydroxy-ethylpiperazine-N¹-2-ethanesulphonic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N¹,-N¹-tetraacetic acid - CICR Ca²⁺-induced Ca²⁺ release - MOPS morpholinopropane sulphonic acid - RR Ruthenium Red     

Effect of bovine serum albumin on the calcium release channel of sarcoplasmic reticulum from rabbit skeletal muscle

The effect of bovine serum albumin (BSA) on the activity of the calcium release channel of the sarcoplasmic reticulum from rabbit skeletal muscle was investigated using both tension recording from skinned fibres and electrophysiological recording of unitary channel currents from planar lipid membranes. BSA had no effect on the Ca²⁺ affinity of the contractile proteins, elicited no tension per se in Ca²⁺-loaded skinned fibres, but potentiated caffeine-induced tension. Maximum potentiation was observed with 0.05–0.5% BSA. BSA (0.1%) had no detectable effect on the basal activity of the Ca²⁺-release channel incorporated in lipid bilayer. However, channel stimulation elicited by either caffeine (2 mM ) or ATP (60 μM ) was further enhanced by BSA (0.1%), as indicated by significant increases in P_o, the open probability of the channel. These results suggest that BSA can modulate the response of the skeletal muscle SR Ca²⁺-release channel to different activators such as caffeine and ATP.     

Trifluoperazine: a rynodine receptor agonist

Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca²⁺ binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca²⁺ release in ventricular myocytes. We explored TFP actions on cardiac SR Ca²⁺ release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca²⁺ release and over time depleted SR Ca²⁺ content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca²⁺ binding) increased the open probability (Po) of CSQ-free channels with an EC₅₀ of 5.2 μM or 8.9 μM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.     

Voltage dependence of membrane charge movement and calcium release in frog skeletal muscle fibres

Summary Voltage dependent membrane charge movement (gating current) and the release of Ca²⁺ from intracellular stores have been measured simultaneously in intact frog skeletal muscle fibres. Charge movement was measured using the three microelectrode voltage clamp technique. Ca²⁺ release was measured using the metallochromic indicator dye arsenazo III. Fibres were bathed in 2.3×hypertonic solutions to prevent contraction. Rb⁺, tetraethylammonium and tetrodotoxin (TTX) were used to eliminate voltage-dependent ionic currents. The maximum rate of Ca²⁺ release from the sarcoplasmic reticulum in response to voltage-clamp step depolarizations to 0 mV was calculated using the dye-related parameters of model 2 of Bayloret al. (1983) and a method described in the Appendix for calculating a scaling factor (1+p) that accounts for the additional Ca²⁺ buffering power of the indicator dye. The estimates of the maximum rate of Ca²⁺ release at 5–6° C ranged from 3 to 19 µM ms^–1 in the 17 fibres examined. The mean value was 8.9±1.1 µM ms^–1 (S.E.M.) The maximum rate of Ca²⁺ release was linearly related to the magnitude of the nonlinear membrane change moved during suprathreshold depolarizing steps. The voltage dependence of charge movement and the maximum rate of Ca²⁺ releases were nearly identical at 6° C. The voltage-dependence of the delay between the test step and the onset of Ca²⁺ release could be adequately described by an equation having the same functional form as the voltage dependence of nonlinear charge movement. The relationship between the test pulse voltage and the delay was shifted to more negative voltages and to shorter delays as the temperature was raised from 6° C to 15° C. The inactivation of Ca²⁺ release was found to occur at more negative holding voltages and to be more steeply voltage dependent than the immobilization of nonlinear membrane charge movement. The above data are discussed using the hypothetical coupler model of excitation-contraction coupling (Milediet al., 1983b) applied to the specific case in which each mobile charge group controls the gating of one Ca²⁺ release site in the sarcoplasmic reticulum.     

Caffeine- and Ca2+-induced mechanical oscillations in isolated skeletal muscle fibres of the frog

Summary Isometric force and subthreshold sarcomeric oscillations were studied in isolated muscle fibres of the frog. In intact muscle fibres, caffeine in a concentration of 2mm caused a subthreshold oscillatory activation of single sarcomeres, called sarcomeric oscillations. They occurred independently of membrane potential and were blocked by agents which directly interfere with Ca²⁺ release from the sarcoplasmic reticulum (SR). In skinned muscle fibres, sarcomeric oscillations were also induced by the Ca²⁺ ion itself (pCa = 6.1). When the free EGTA concentration of the bathing solutions was reduced, fibres responded with long lasting oscillations of force. Both types of oscillations were blocked when the membranes of the SR were solubilized by detergent. The results reveal that caffeine- and Ca²⁺-induced oscillations in skeletal muscle fibres are triggered by a cyclic release of Ca²⁺ ions from the SR. It is suggested that they interfere with the process of Ca²⁺-induced Ca²⁺ release:     

FPL-64176 alters both charge movement and Ca2+ release properties in amphibian muscle fibres

A number of recent reports have suggested that ryanodine receptor (RyR)-Ca²⁺ release channels are gated by tubular depolarization in skeletal muscle through their direct coupling to intramembrane dihydropyridine receptor (DHPR)-voltage sensors. The q charge movement, which is inhibited by DHPR antagonists, is often regarded as the electrical signature for the voltage sensing process, yet pharmacological modifications of the RyR produce reciprocal upstream kinetic effects on an otherwise conserved q charge. This study investigates the effect of DHPR-specific agonists upon intramembrane charge and the release of intracellularly stored Ca²⁺. We empirically demonstrate kinetic effects of FPL-64176 upon charge movements that closely resemble the consequences of previous interventions directed instead at the RyR. Increases in extracellular FPL-64176 concentration from 10 to 40 M converted delayed q transients to monotonic decays indistinguishable from the exponential q current component. Yet total steady-state intramembrane charge and the steepness of its dependence upon test potential closely resembled previous reports from untreated fibres. These changes accompanied an appearance of transient cytosolic [Ca²⁺] elevations in confocal line-scans in fluo-3-loaded fibres studied in 10 mM K⁺ and 40, but not 10 M, FPL-64176 that resembled elementary Ca²⁺ release events (sparks). Pharmacological manipulations of the DHPR whose effects on intramembrane charge resembled those from manoeuvres directed at the RyR can thus produce downstream effects upon Ca²⁺ release.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Decrease in sodium–calcium exchange and calcium currents in diabetic rat ventricular myocytes

This study was designed in order to gain insight into possible changes in the inward sodium–calcium exchange current (I_Na–Ca) and the L -type calcium current (I_Ca), in ventricular myocytes isolated from streptozotocin-induced diabetic rats. Recordings were made using the nystatin-perforated patch technique which minimizes interference with the normal intracellular Ca²⁺ buffering mechanisms. The averaged I_Na–Ca current density elicited by Ca²⁺ current was smaller in diabetic than in normal myocytes at all potentials tested. I_Na–Ca activated by rapid application of caffeine was significantly reduced and the decay phase was prolonged. The density of I_Ca was also significantly reduced by diabetes in the range of test potentials between –10 and +50 mV. In addition, the fast time constant of I_Ca inactivation, which represents mainly the sarcoplasmic reticulum (SR) Ca²⁺ release-induced inactivation, was significantly higher in diabetic than in normal myocytes. The decrease in I_Ca, which is the main source of trigger Ca²⁺ for SR Ca²⁺ release, may explain the significantly lowered peak systolic [Ca²⁺]_i previously shown in diabetic myocytes. As activation of I_Ca is essential for subsequent stimulation of I_Na–Ca, reduced I_Ca may contribute to decreasing activation of the Na⁺–Ca²⁺ exchanger.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+

Aim: We have investigated the influence of Ca²⁺ ions on the basic biophysical properties of T‐type calcium channels. Methods: The Ca_v3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca²⁺ or Na⁺ ions were used as charge carriers. The intracellular Ca²⁺ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 μm Ca²⁺ into the non‐buffered intracellular solution. Various combinations of extra‐ and intracellular solutions yielded high, intermediate or low intracellular Ca²⁺ levels. Results: The amplitude of the calcium current was independent of intracellular Ca²⁺ concentrations. High levels of intracellular Ca²⁺ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca²⁺ by Na⁺ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca²⁺ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ?100 mV for the Ca²⁺ current, but did not reach steady state even at ?150 mV for the Na⁺ current. Conclusions: Ca²⁺ ions facilitate transitions of Ca_v3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor.     

Physiology and pathophysiology of excitation–contraction coupling: the functional role of ryanodine receptor

Calcium (Ca²⁺) release from intracellular stores plays a key role in the regulation of skeletal muscle contraction. The type 1 ryanodine receptors (RyR1) is the major Ca²⁺ release channel on the sarcoplasmic reticulum (SR) of myocytes in skeletal muscle and is required for excitation–contraction (E–C) coupling. This article explores the role of RyR1 in skeletal muscle physiology and pathophysiology.     

Activity of cardiac L-type Ca2+ channels is sensitive to cytoplasmic calcium

Ca²⁺ -induced inactivation of L-type Ca²⁺ channels is proposed as an important negative feedback mechanism regulating Ca²⁺ entry. Here, for the first time, evidence for modification of heart L-type Ca²⁺ channel activity by cytoplasmic calcium is provided from excised insideout membrane patches. Ba²⁺ currents through cardiac L-type Ca²⁺ channels exhibited only modest inactivation in the absence of cytoplasmic Ca²⁺. Elevation of cytoplasmic Ca²⁺ to micromolar concentrations strikingly affected L-type Ca²⁺ channel activity as evaluated from ensemble average Ba²⁺ currents. Inactivation was markedly increased concomitant with a reduction of peak inward current, which was almost completely eliminated at about 15 M cytoplasmic Ca²⁺ concentration. Half maximal suppression of Ba²⁺ currents was observed at 2.3 M Ca²⁺. The observed modifications of L-type Ca²⁺ channel activity show that cytoplasmic Ca²⁺ induces channel closure. Below 4 M Ca²⁺, channels can be reversibly reactivated during repetitive depolarizations, while at high Ca²⁺ concentrations (15 M) most Ca²⁺ channels reside in a closed state. This may allow for a delicate regulation of Ca²⁺ entry, and consequently of heart contraction.     

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

 To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca²⁺ on the Ca²⁺ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca²⁺ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca²⁺ overload, induced by elevation of extracellular [Ca²⁺], Ca²⁺ sparks represented single populations of events. During Ca²⁺ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca²⁺ sparks, however, was not altered. Changes in the properties of Ca²⁺ sparks were accompanied by only an ≈ 30% increase in the SR Ca²⁺ content, as determined by emptying the intracellular Ca²⁺ stores using caffeine. When single Ca²⁺ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca²⁺ (≈ 100 nM) and ATP (3 mM), elevation of Ca²⁺ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (P _o). This potentiation of P _o was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca²⁺ was greater at low levels of cytoplasmic [Ca²⁺] than at high levels of cytoplasmic [Ca²⁺], and no effect of luminal Ca²⁺ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca²⁺ in the absence of ATP. Our results suggest that SR Ca²⁺ release channels are modulated by SR intraluminal Ca²⁺. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca²⁺ release in cardiac myocytes Received: 15 May 1996 / Received after revision: 5 June 1996 / Accepted: 8 July 1996     

Discrimination of Ca2+-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities

Summary We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca²⁺-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca²⁺-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca²⁺-dependent ATPase activity, in spite of having little or no Ca²⁺-induced isometric tension. The residual ATPase activity is ascribed to the Ca²⁺-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca²⁺-dependence of the ATPase activity is very similar to that of Ca²⁺-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca²⁺-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca²⁺-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca²⁺-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca²⁺-ATPase of SR. Ca²⁺-ATPase activity of SR in skinned fibres was inhibited completely by 10 m CPA and held to one-half by about 0.2 m. This effect was only partially reversible. CPA at 10 m or higher concentrations showed Ca²⁺-sensitizing action on myofibrils, which was readily reversible. CPA at 3 m inhibited almost completely the Ca²⁺-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.     

Effects of the PKA inhibitor H-89 on excitation–contraction coupling in skinned and intact skeletal muscle fibres

This study investigated the effects of the protein kinase A (PKA) inhibitor, H-89, in mechanically-skinned muscle fibres and intact muscle fibres, in order to determine whether PKA phosphorylation is essential for normal excitation–contraction (E–C) coupling. In skinned EDL fibres of the rat, force responses to depolarization (by ion substitution) were inhibited only slightly by 10M H-89, a concentration more than sufficient to fully inhibit PKA. Staurosporine (1 M), a potent non-specific kinase inhibitor, also had little if any effect on depolarization-induced responses. At 1–2 M, H-89 significantly slowed the repriming rate in rat skinned fibres, most likely due to it deleteriously affecting the T-system potential. With 100 M H-89, the force response to depolarization by ion substitution was completely abolished. This inhibitory effect was reversed by washout of H-89 and was not due to block of the Ca²⁺ release channel in the sarcoplasmic reticulum (SR). In intact single fibres of the flexor digitorum longus (FDB) muscle of the mouse, 1–3 M H-89 had no noticeable effect on action-potential-mediated Ca²⁺ transients. Higher concentrations (4–10 M) caused Ca²⁺ transient failure in fibres stimulated at 20 Hz in a manner indicative of action-potential failure. At 10–100 M, H-89 also inhibited net Ca²⁺ uptake by the SR and affected the Ca²⁺-sensitivity of the contractile apparatus in rat skinned fibres. All such effects were proportionately greater in toad muscle fibres. These results do not support the hypothesis that phosphorylation is essential for the Ca²⁺ release channel to open in response to voltage-sensor activation in skeletal muscle fibres.     

Ryanodine modification of RyR1 retrogradely affects L-type Ca2+ channel gating in skeletal muscle

In skeletal muscle, there is bidirectional signalling between the L-type Ca²⁺ channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca²⁺ release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of “orthograde signalling” (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca²⁺ release from the SR. “Retrograde coupling” is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 μM, 1 h at 37°C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a ~5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated myotubes were also shifted to more hyperpolarizing potentials (~13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca²⁺, CaM, CaMK, redox potential) may also affect DHPR gating.