Dynamic vacuolation in skeletal muscle fibres after fatigue

Vacuoles develop after fatiguing stimulation in frog skeletal muscle fibres. Experiments on isolated Xenopus muscle fibres show that this vacuolation is a dynamic process that reaches its maximum about 20 min after the end of fatiguing stimulation and then recedes. Fatigue-induced vacuoles originate from the t-tubular system. Recent data indicate that vacuoles are formed because of lactate accumulation in the t-tubules resulting in increased osmotic pressure and subsequent water influx. There is no obligatory connection between the presence of vacuoles and force depression, which is another common feature during the recovery from fatigue. Nevertheless, extensive vacuolation may exaggerate this force depression.     

Calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.     

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.     

A domain peptide of the cardiac ryanodine receptor regulates channel sensitivity to luminal Ca<Superscript>2+</Superscript> via cytoplasmic Ca<Superscript>2+</Superscript> sites

The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca²⁺. A synthetic peptide corresponding to part of the central domain (DPc10:²⁴⁶⁰G–P²⁴⁹⁵) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca²⁺ as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca²⁺ and a large increase in the magnitude of luminal Ca²⁺ activation. The increase in the luminal Ca²⁺ response appeared reliant on the luminal-to-cytoplasmic Ca²⁺ flux in the channel, indicating that luminal Ca²⁺ was activating the RyR2 via its cytoplasmic Ca²⁺ sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca²⁺ sensing sites. The results were fitted by the luminal-triggered Ca²⁺ feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca²⁺-activation mechanism.     

Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3-containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0. 7 micrometers. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 micrometer, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.     

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse

Summary. Taurine as well as tauret (retinyliden taurine) levels were measured in locust Locusta migratoria compound eyes. HPLC measurements revealed relatively low taurine levels (1.9 ± 0.16 mM) in dark-adapted eyes. Glutamate, aspartate and glycine levels were 2.0 ± 0.2, 2.7 ± 0.4 and 3.0 ± 0.37 mM, respectively, while GABA was present only in trace amounts. After about 4 h of light adaptation at 1500–2000 lx, amino acid levels in the compound eye were as follows: taurine, 1.8 ± 0.17 mM; glutamate, no change at 2.1 ± 0.2 mM; aspartate sharply increased to 4.7 ± 0.7 mM; glycine slightly decreased to 2.8 ± 0.3 mM; and GABA trace levels. In the compound eye of locust Locusta migratoria, the existence of endogenous tauret in micro-molar range was established. In the dark, levels were several times higher compared with compound eye after light adaptation 1500 lx for 3 h, as estimated by TLC in combination with spectral measurements. Existence of tauret in compound eye is of special interest because in the compound eye, rhodopsin regeneration is based on photoregeneration.     

Calcium efflux during the cold-induced contraction of mammalian striated muscle fibres

The efflux of ⁴⁵Ca from mammalian slow twitch muscle fibres has been studied to provide a measure of the concentration of free Ca²⁺ in the sarcoplasm. The kinetically complex early phases of washout of the isotope are succeeded by a prolonged slower phase which exhibits first-order kinetics. This later phase is accelerated by caffeine, by preventing oxidative phosphorylation and also during an isometric contraction, whether this contraction is produced by lowering the temperature or by electrical stimulation. The local anaesthetic tetracaine abolishes the contraction caused by cold and in this case the rate constant for efflux is progressively lowered as the temperature is reduced (Q₁₀ value of 2.3). The removal of external Na⁺ and Ca²⁺ reduces the efflux rate constant. Caffeine, sodium removal and the inhibition of oxidative phosphorylation, all potentiate the cold contraction and the associated extra ⁴⁵Ca efflux. Ca removal causes the cold contraction to become phasic. It appears that caffeine, sodium removal, the inhibition of oxidative phosphorylation and a decrease in temperature to below 10°C are all treatments which, like electrical stimulation, increase the sarcoplasmic free calcium concentration to varying degrees.     

The I4895T mutation in the type 1 ryanodine receptor induces fiber-type specific alterations in skeletal muscle that mimic premature aging

The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation-contraction uncoupling. We characterized age-dependent and fiber-type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca(2+) release in heterozygous Ryr1(I4895T/WT) (IT/+) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically evoked Ca(2+) release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT/+ mice. Structural changes are strongly fiber-type specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT/+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.     

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles

Ca²⁺ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca²⁺, 2) calmodulin, and 3) Ca²⁺/calmodulin-dependent phosphorylation. Bell-shaped Ca²⁺-dependence, of Ca²⁺ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca²⁺ release channel could be regulated by [Ca²⁺ _o] . The time- and concentration-dependent inhibition of Ca ²⁺ release from skeletal SR by calmodulin was also studied using passively-Ca²⁺ loaded SR vesicles. Up to 50% of Ca ²⁺ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca²⁺/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca²⁺ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca²⁺-loaded SR vesicles. Approximately 80% of the initial rates of Ca²⁺-induced Ca²⁺ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)     

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation-contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are "leaky." RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

The influence of amino-reactive substances on contraction threshold of frog skeletal muscle

Summary The action of the amino-reactive substances pyridoxal phosphate, 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid and 2,4,6-trinitrobenzene sulfonic acid on the contraction threshold, taken as parameter for the initiation of contraction, was investigated in fibers of the sartorius muscle of the frog. The contraction threshold was shifted by 1 to 11 mV tomore negative potentials with 1 to 20mm PDP. Similar shifts from 2 to 17 mV were produced by 0.66 to 20mm SITS. The threshold shift was only partially reversible. The shift of the contraction threshold obtained with 2mm SITS was nearly constant at different [Ca²⁺]_o and [Mg²⁺]_o from 1.5 to 50mm with a tendency to increase at higher divalent cation concentration. TNBS had no effect on the contraction threshold.The action of PDP and SITS on the contraction threshold was successfully described by the surface charge model used earlier to explain the effect of lanthanum, neuraminidase and ruthenium red on the contraction threshold (M. Dörrscheidt-Käfer,Pfluegers Arch. 380:171–179, 181–187, 1979;J. Membrane Biol. 62:95–103, 1981). Here it was assumed that PDP and SITS bind to positive fixed charges on the surface of the T-tubular wall. This results in a shift of the calculated surface potential to more negative values which is thought to account for the measured shift of the contraction threshold.     

Thin filament flexibility and its role in muscle contraction

Contemporary experimental methods do not allow unequivocal determination of molecular structural events during muscle contraction. To analyze existing contradictions, an original computer program has been developed. This program reconstructs the hexagonal lattice of a sarcomere for different states of muscle and finds the most realistic structure by comparing the calculated Fourier spectrum with the actual diffraction pattern. Previously, the new approach allowed reconstructing the actual structure of a myosin filament from mammalian striated muscle (http://zope.ibib.waw.pl/pspk). In this work, the thin filament is reconstructed for three states: relaxed, activated, and contracting. The good fit between the calculated Fourier spectra and the actual diffraction patterns taken from the literature suggests that the thin filament owing to its flexibility may play an active role in muscle contraction, as myosin cross-bridges do.     

Effect of deuterium oxide on contraction characteristics and ATPase activity in glycerinated single rabbit skeletal muscle fibers

We studied the effect of deuterium oxide (D₂O) on contraction characteristics and ATPase activity of single glycerinated muscle fibers of rabbit psoas. D₂O increased the maximum isometric force P₀ by about 20%, while the force versus stiffness relation did not change appreciably. The maximum shortening velocity under zero load V_max did not change appreciably in D₂O, so that the force-velocity (P-V) curve was scaled depending on the value of P₀. The Mg-ATPase activity of the fibers during generation of steady isometric force P₀ was reduced by about 50% in D₂O. Based on the Huxley contraction model, these results can be accounted for in terms of D₂O-induced changes in the rate constants f₁ and g₁ for making and breaking actin-myosin linkages in the isometric condition, in such a way that f₁/(f₁+g₁) increases by about 20%, while (f₁+g₁) remains unchanged. The D₂O effect at the molecular level is discussed in connection with biochemical studies on actomyosin ATPase.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Simulation of calcium sparks in cut skeletal muscle fibers of the frog

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca²⁺ (1.25 up to 11.25 mM) to force generation at 25 °C (acclimation temperature), 15 and 35 °C. The post-rest tension and force–frequency relationship were conducted with and without 10 μM ryanodine in the bathing medium. Increments in extracellular Ca²⁺ resulted in increases in twitch force development only at 35 °C. A significant post-rest potentiation was recorded for the control preparations at 25 °C (100% to 119.8 ± 4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 °C (100% to 97.6 ± 1.5%). At 35 °C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6 ± 4.6%) while at 15 °C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 °C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.