The composition and calcium transport activity of the sarcoplasmic reticulum from goats with and without heritable myotonia.

Studies were conducted on purified sarcoplasmic reticulum isolated from myotonic goats, an animal model of heritable myotonia. When compared to sarcoplasmic reticulum from normal goats, fragmented sarcoplasmic reticulum from the myotonic goat had (1) increased levels of calcium, (2) increased rates of calcium uptake and efflux, (3) an increased sialic acid content, and (4) an increased content of saturated fatty acids. These differences support the concept of a structural and functional defect as a basis for the abnormal contraction-relaxation characteristics of myotonia.     

Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity.

The effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca2+-ATPase inhibitor, on isometric tension were studied in response to prolonged steady-state depolarization induced by a rapid change in extracellular potassium concentration (potassium contractures) in frog semitendinosus muscle fibres. Cyclopiazonic acid (1-10 microM) enhanced the amplitude and time-course of relaxation of 146 mM potassium contracture. In the presence of cyclopiazonic acid 0.5 microM, the relationship between the amplitude of potassium contractures and the membrane potential shifted to more negative potentials, whereas the steady-state inactivation curve was unchanged. These observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal muscle fibres implies that the amplitude and slow relaxation of tension during prolonged steady-state depolarization may be expected to depend not only on inactivation of the process regulating calcium release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump calcium.     

Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity

The effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, on isometric tension were studied in response to prolonged steady-state depolarization induced by a rapid change in extracellular potassium concentration (potassium contractures) in frog semitendinosus muscle fibres. Cyclopiazonic acid (1–10 μM ) enhanced the amplitude and time-course of relaxation of 146 m M potassium contracture. In the presence of cyclopiazonic acid 0.5 μM , the relationship between the amplitude of potassium contractures and the membrane potential shifted to more negative potentials, whereas the steady-state inactivation curve was unchanged. These observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal muscle fibres implies that the amplitude and slow relaxation of tension during prolonged steady-state depolarization may be expected to depend not only on inactivation of the process regulating calcium release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump calcium.     

Development of the excitation-contraction coupling apparatus in skeletal muscle: peripheral and internal calcium release units are formed sequentially

Summary The development of calcium release units and of transverse tubules has been studied in skeletal muscle fibres from embryonal and newborn chicken. Three constituents of calcium release units: the tetrads, the feet and an internal protein directly associated with junctional surface of the sarcoplasmic reticulum are visualized by various electron microscope techniques. Evidence in the literature indicates that the three components correspond to the voltage sensors, the sarcoplasmic reticulum calcium release channels and the calcium binding protein calsequestrin respectively.We recognize two stages at which important events in membrane morphogenesis occur. The first stage coincides with early myofibrillogenesis (starting at approximately embryonal day E5.5), and it involves the assembly of calcium release units at the periphery of the muscle fibre in which feet and the internal protein are identified. Groups of tetrads also are present at very early stages and their disposition indicates a relation to the feet of peripheral couplings. Thus three major components of the excitation-contraction coupling pathway are in place as soon as myofibrils develop. The density of groups of tetrads in the surface membrane of primary and secondary fibres is similar, despite differences in developmental stages. The second stage involves the formation of a complex transverse tubule network and of internal sarcoplasmic reticulum-transverse tubule junctions, while peripheral couplings disappear. This stage starts abruptly (between E15 and E16) and simultaneously in primary and secondary fibres. It coincides with the myotube-to-myofibre transition. The two stages are separated by a relatively long intervening period (between E9 and E16). During the latter part of this period some primitive transverse tubules appear, and form junctions with the sarcoplasmic reticulum, but they remain strictly located at the periphery of the fibre and are not numerous. Finally, after the second stage there is a prolonged (up to 4 weeks) period of maturation, during which density of free sarcoplasmic reticulum increases, triads acquire a location at the A-I junction and fibre type differences appear. We conclude that a system for calcium uptake, storage and release exists at the periphery of the myotube during early myogenesis. The complexity of the system and its ability to deliver calcium through the entire fibre develop in parallel to the formation of myofibrils.     

Cytochemical localization of lysosomal enzyme activity in normal and ischemic dog myocardium.

The effect of ischemia on the integrity of myocardial lysosomes was observed 3 1/2 and 24 hours after the production of infarcts in 20 anesthetized closed-chest dogs by electrically induced thrombosis of the left anterior descending coronary artery. Biopsies from normal, marginal and infarcted areas were fixed and incubated to localize the lysosomal enzymes acid phosphatase and aryl sulphatase. Reaction product in normal cells was localized in small circular or oblong profiles between bundles of myofilaments and adjacent to mitochondria. In addition, curvilinear, membrane-bound profiles containing reaction product were found in close apposition to transverse tubules and near the free margins of the myocardial cells. Thus the distribution of elements of the sarcoplasmic reticulum. Additional reaction product was also seen in residual bodies, on myelin figures, and in the few conventional appearing spherical lysosomes. Little or no acid phosphatase or aryl sulphatase reaction product was seen in the sarcoplasmic reticulum of infarcted myocardium. The degree of cellular degeneration correlated with disappearance of enzyme activity from the sarcoplasmic reticulum and included disruption of membranes and loss of mitochondrial matrix and erosion of I but not A bands. Marginal areas showed variable amounts of cellular degeneration. Separation of myofilament bundles and loss of glycogen correlated with the localized disappearance of acid phosphatase and aryl sulphatase activity in marginal tissue. Disruption of mitochondrial and erosion of I bands correlated with extensive loss of these enzymes. The data suggest that degeneration of myocardial cells following ischemic injury is associated with release of endogenous lysosomal enzymes from the sarcoplasmic reticulum.     

The effects of changes to action potential duration on the calcium content of the sarcoplasmic reticulum in isolated guinea-pig ventricular myocytes

. We have estimated sarcoplasmic reticulum calcium content using rapid application of caffeine on voltage clamped, isolated guinea-pig ventricular myocytes. Caffeine induces the release of calcium from the sarcoplasmic reticulum and this calcium is extruded from the cells by the sarcolemmal Na/Ca exchange. Integrating the inward Na/Ca exchange current thus allows estimations of sarcoplasmic reticulum calcium content. Ventricular myocytes were stimulated to reach new steady-states by action potential voltage clamps of varying duration. Once contractile steady-state had been reached caffeine was rapidly applied in place of the next action potential and sarcoplasmic reticulum calcium content measured. Prolonging the action potential duration increased sarcoplasmic reticulum calcium content and vice-versa. This calcium loading may underlie the positive inotropic effect of increased action potential duration. Received: 11 July 1996 / Received after revision: 15 October 1996 / Accepted: 26 November 1996     

Structure and molecular organisation of the sarcoplasmic reticulum of skeletal muscle fibers

Activation of muscle contraction is a rapid event that is initiated by depolarization of the plasma membrane and transverse (T) tubules, which following transduction in the interior of the muscle cell, activate the release of calcium from the sarcoplasmic reticulum (SR). Pioneer studies using electron microscopy defined the organization of the sarcoplasmic reticulum and the details of the junctions between sarcoplasmic reticulum and T tubules, which are essential for translating the electrical signal on the plasma membrane to calcium release from the sarcoplasmic reticulum. Molecular biology and biochemistry studies have revealed the presence of several proteins located on the sarcoplasmic reticulum, some of which participate together with the ryanodine receptors to the assembly of a large multi-protein complex, while others, like the calcium pumps, have independent localization and activities. As a whole, the current view of this system contemplates the existence of a high level of structural organization in the sarcoplasmic reticulum with respect to the localization of ryanodine receptors and other proteins. In this review we shall summarize studies on the expression and possible functional significance of the ryanodine receptor type 3 in mammalian skeletal muscles and recent studies aimed to dissect the mechanisms that establish the organization of the SR in striated muscles.     

Freeze-fractured sarcoplasmic reticulum in adult and embryonic fast and slow muscles

Summary There is evidence to suggest that 8 nm calcium transport particles in the sarcoplasmic reticulum are involved in the regulation of twitch properties in adult muscles. We have studied ultrastructural characteristics of the sarcoplasmic reticulum in relation to previously defined physiological changes that take place in the normal course of development. The fast twitch posterior latissimus dorsi (PLD) and the slow tonic anterior latissimus dorsi (ALD) of the chicken were compared using the procedure of freeze-fracture. In the adult PLD, the sarcoplasmic reticulum was composed of longitudinal tubules, which gave rise to fenestrated cisternae at the centre of the H band and to terminal cisternae that form triads regularly at each A-I junction. In most of the fibres (85%), 8 nm intramembrane particles were closely packed in the concave fracture face (P-face). In the ALD, a tubular network with an open circular pattern extended the entire length of the A band and usually throughout the I band as well. Dyads or triads, which were infrequent, were often oriented obliquely. The density of intramembrane particles was low in the majority of the fibres, but there was a significant minority population (30%) in which particle density was relatively high. At 10 daysin ovo, when speed of contraction in both the ALD and PLD is slow, there was a circular configuration of sarcoplasmic reticulum components in both muscles, and particle density was low. Surprisingly, at 18 daysin ovo, when the rate of tension development and relaxation have reached nearly adult values in the fast PLD, this muscle, like the ALD, continued to exhibit a circular arrangement of sarcoplasmic reticulum tubules. The density of P-face particles, although greater than at 10 days, was still low relative to the adult PLD. Estimated values for the 18-day PLD were similar to those calculated for the adult slow muscle. Our observations, along with those of other investigators, suggest that abundant intramembrane particles may be related to the fast twitch properties of the adult PLD. However, they indicate that neither the pattern of membranes typical of the adult fast muscle nor the high content of calcium transport particles is required for the differentiation of fast twitch characteristics.     

Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery

45Ca electron microscopic autoradiography was used to examine the effects of buffer composition and temperature on the distribution of calcium in rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the temperature of the bathing solution (25 degrees C or 34 degrees C). Krebs buffer at 34 degrees C gave the highest relative activity in the plasma membrane, sarcoplasmic reticulum, and mitochondria. Buffer and temperature had little effect on the relative activity of the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45Ca in Krebs buffer at 34 degrees C. The results show that sarcoplasmic reticulum and plasma membrane are the primary sites of calcium accumulation during influx into these cells. Although the amount of 45Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275 nm of the plasma membrane) is similar to the membrane alone and is lower than the sarcoplasmic reticulum alone.     

Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle

Summary Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP₃) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP₃ receptors is unknown. InsP₃ receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP₃ receptor labelling was predominatly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP₃ receptor. In aorta (tonic muscle) the InsP₃ receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP₃ and caffeine, but not cyclic ADP-ribose, released intracellular Ca²⁺ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP₃ receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.     

Ultrastructural localization of calcium in normal and abnormal skeletal muscle.

Calcium was demonstrated ultrastructurally as a fine black reaction product with unbuffered 2% saturated potassium pyroantimonate, pH 9.4. In comparison with normal muscle, there was increased precipitate in degenerating skeletal muscle fibers and some degenerating-regenerating fibers occurring in pathologic human muscle, regardless of disease entity, and in experimentally injured rat muscle. The pathologically increased calcium was mainly within the sarcoplasmic reticulum and mitochondria. Both structures could be completely blackened. Nuclear calcium was also increased, the precipitates being localized as circular profiles within the nucleoli and heterochromatin as well as being associated with the nuclear envelope. Myofibrillar calcium was only modestly increased. When normal rat muscle was preincubated in 136 mM calcium-enhanced Hanks' medium, calcium accumulated in the muscle fibers--it was especially heavy in the mitochondria and sarcoplasmic reticulum and appeared identical with the pathologic human and rat muscle fibers. Preincubation of normal rat muscle in 0.1 M acetate buffer (pH 4.65) before calcium loading augmented myofibrillar staining, mainly in the H-zone of the A-bands excluding the M-zone and in broad irregular N1, N2, and "N3" lines of the I-bands. EMMA-4 electron probe microanalysis and EGTA (ethylene glycolbis (beta-aminoethyl ether)N,N'-tetraacetic acid) chelation prior to staining confirmed that the precipitate in the several loci was calcium antimonate. It is proposed that in skeletal muscle fibers injured by various pathologic processes, a breach of the plasmalemma barrier to calcium occurs as a very early abnormality. Extracellular calcium would then pour into the aqueous sarcoplasm of the muscle fiber, from which it would be withdrawn by and accumulated with the still active organelles normally having a great avidity for uptake of this ion, especially the mitochondria and sarcoplasmic reticulum. The resultant organellar calcification would impair function and damage the structure of proteins and phospholipids.     

Relative capabilities of sarcoplasmic reticulum in fast and slow mammalian skeletal muscles.

1. The calcium uptake capabilities of the sarcoplasmic reticulum (SR) of the fat-twitch muscles extensor digitorum longus (EDL) and tibialis anterior (TA) of the rat and the extensor digitorum longus of the cat have been compared with the same capabilities of the slow-twitch soleus muscles of the rat and cat. 2. For the ra the Vmax values of sarcoplasmic reticulum from tibialis anterior, extensor digitorum longus and from soleus muscles were 50, 51, and 10 micronmole Ca2+/g per minute, respectively. 3. For the extensor digitorum longus and soleus muscles of the cat the Vmax values were 34 and 5-6 micronmole Ca2+/g per minute, respectively. 4. These data were compared with mechanical data as reported in the literature for the same muscles. The relative calcium uptake capabilities of sarcoplasmic reticulum from slow and fast muscles corresponded closely to the relative rates of relaxation of these muscles.     

Characteristics of skeletal muscle calsequestrin: comparison of mammalian,amphibian and avian muscles

Summary Calsequestrin was identified in the isolated sarcoplasmic reticulum from skeletal muscle of three mammalian species (man, rat and rabbit) and from frog and chicken muscle, using electrophoretic and immunoblot techniques. It was further characterized in sarcoplasmic reticulum protein mixtures and at several stages of purification, following extraction with EDTA.We found extensive similarities in apparent molecular weight values, Stains All staining properties and in Cleveland's peptide maps, between mammalian calsequestrins, and no detectable difference within a species between fast and slow muscle. Human calsequestrin, with an apparent molecular weight of 60 000 when measured at alkaline pH and of 41 000 when measured at neutral pH, appears to be the smallest in size. Frog calsequestrin, although weakly cross-reactive with rabbit calsequestrin and having a relatively higher apparent molecular weight at alkaline pH (72 000), shares several significant properties with mammalian calsequestrins. It bound calcium with a high capacity (1300 nmol per mg protein), it contained about 32% acidic amino acid residues and focused at closely similar pI values. We observed the formation of a complex with Stains All absorbing maximally at 535 nm, rather than at 600 nm, and an even more marked shift in apparent molecular weight at neutral pH.We found distinct differences in the case of chicken calsequestrin, in addition to those previously reported. It is a highly acidic, calcium-precipitable protein, but its amino acid composition is contradistinguished by a higher ratio of glutamate to aspartate and its rate of electrophoretic mobility is minimally affected by changes in pH. It stained deep bluish with Stains All after gel electrophoresis and yielded a protein-dye complex in aqueous solution, absorbing maximally at 560 nm, and finally, it bound fluorescent Concanavalin A.     

Relationship between sarcoplasmic reticulum volume and calcium capacity in skinned frog skeletal muscle fibres

Semitendinosus fibres from Rana pipiens were examined in the electron microscope. When the aqueous solutions in which the fibres were prepared contained no sucrose, the sarcoplasmic reticulum was markedly swollen and possibly fragmented. When as little as 50 mM sucrose was included in the aqueous solution, the sarcoplasmic reticulum retained the flattened appearance characteristic of intact fibres. The degree of flattening was not substantially different with concentrations of sucrose ranging from 50 to 300 mM, suggesting that the flattened volume may be determined by structural elements. In all the fibre segments, the regularity of the hexagonal filament lattice was disrupted and the disruption was not affected by sucrose. Radioactive calcium uptake was measured in the presence and absence of 200 mM sucrose. The capacity of the sarcoplasmic reticulum (SR) was depressed by about 30% by the sucrose but the initial uptake was not significantly affected. This finding suggests that most of the calcium within the SR is bound and not free in solution.     

Cardiac cell membrane repolarization is required for onset of mechanical restitution in papillary muscle

In 10 voltage clamped ferret papillary muscles at 37 degrees C (single sucrose gap), the duration of resting (diastolic, holding) potential was varied in order to define the mechanical restitution process. Following a period of steady state voltage clamp depolarizations to +20 mV, a single test depolarization clamp of 200 or 500 ms duration was introduced. Then, the following period at resting (holding) potential was varied. All the mechanical restitution curves for the 500 ms clamps were delayed by 300 ms compared with the 200 ms clamps. When mechanical restitution was plotted as the relationship between contractile force and test electrical diastolic interval, all processes started from zero interval (i.e. the time of repolarization). Variation of diastolic holding potential between -70 mV and -40 mV did not affect the starting time, but the final force values at full restitution were approached faster and were higher for -70 mV than for -40 mV. There was an inverse relationship between force and second inward current during mechanical restitution after an initial phase of restitution of current. Mechanical restitution is postulated to be due to passage of contractile calcium with time from an uptake to a release compartment within the sarcoplasmic reticulum. Thus the rise of contractile force with increasing test cycle duration should have been independent of whether a 200 or 500 ms depolarization was used. In order to accommodate the discrepancy, we postulate either that (1) sarcoplasmic reticulum calcium release channels require sarcolemmal repolarization to begin to be reactivated or (2) that trigger calcium (calcium induced calcium release from the sarcoplasmic reticulum) is derived from the sarcolemma.     

Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle

The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca--Pi precipitation- induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mm--50 mm range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca--Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mm myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 m s–1 while the amount of Pi loaded appeared to saturate at around 3.5 mm (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca--Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mm inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mm) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca--Pi solubility product is exceeded in the sarcoplasmic reticulum lumen     

Cytological studies of muscle dedifferentiation and differentiation during limb regeneration of the newt Triturus

During limb regeneration of the newt Triturus, muscle first dedifferentiates giving rise to mesenchymal cells which, subsequently, differentiate to reform normal muscle. In dedifferentiating muscle, myofibrils and elements of the sarcoplasmic reticulum decrease gradually in number. Myofilaments become less distinct and seem to be supplanted by zones of amorphous material. Large masses of glycogen accumulate in dedifferentiating cells and mitochondria show a transient enlargement. In the later stages of dedifferentiation, glycogen decreases in amount while cytoplasmic ribosomes and cisternae of rough-surfaced endoplasmic reticulum appear. Mesenchymal cells are formed by disappearance of all traces of myofilaments, by further elaboration of rough endoplasmic reticulum, and increase in number of Golgi complexes. During the earliest stages of differentiation, muscle has less endoplasmic reticulum and many more free ribosomes which occur in clusters or linear chains. Thin (?70 A) and thick (?135 A) filaments appear in the cytoplasm, often in association with ribosomal olusters, and shortly thereafter aggregate into small fibrils. In early fibrils, the thin filaments overlap the thick to produce I and A bands and the free ends of the thin filaments converge and cross to mark the site of the Z line. Fibrils enlarge by addition of filaments along their sides and at their ends. In larger fibrils, the H band and M line are apparent. Initially, the transverse tubular system develops by the confluence of vesicular inpocketings of the surface plasma membrane. The smooth sarcoplasmic reticulum is continuous with rough-surfaced membranous cisternae. Some of the smooth tubules, probably sarcoplasmic reticulum, contain intracisternal masses of dense granular material and are situated adjacent to the Z line. When dense material occurs in the membranous structures, material of similar density and texture appears within the adjacent Z line. Normal muscle fibers are formed by fusion of myoblasts, increase in number and size of myofibrils, organization of transverse tubules and sarcoplasmic reticulum into triads, loss of ribosomes, and appearance of glycogen.     

Changes in the functioning of the electromechanical connection during tetanic contraction

The functioning of the electromechanical connection during tetanic contraction in frog skeletal muscle was studied. Analysis using caffeine, calcium-free medium, the ryanodine receptor blocker dantrolene, and the Ca-ATPase inhibitor thapsigargin showed that the initial increase in tetanus, as in twitch contractions, did not require the presence of calcium ions in the surrounding medium, which is in agreement with published data. Contraction was accompanied by activation of potential-dependent release of calcium from the sarcoplasmic reticulum. In contrast, the secondary rise phase and/or the duration of the tetanus plateau were critically dependent on the present of Ca²⁺ in the surrounding medium. Given that contraction in this situation was inhibited by dantrolene, activation of prolonged contraction was also mediated by calcium released from the sarcoplasmic reticulum, though ryanodine receptors were now activated not by changes in the membrane potential but by the influx of external calcium. Thus, external calcium plays a significant role in the formation of prolonged contractile responses, providing for longer-lasting maintenance of power in contracted muscles. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 11, pp. 1288–1298, November 2005.     

Contractile proteins and sarcoplasmic reticulum in physiologic cardiac hypertrophy

To study effects of physiologic hypertrophy on contractile protein ATPases and sarcoplasmic reticulum, hypertrophy was caused in female Wistar rats by a chronic swimming program. Nonhypertrophied hearts of female control sedentary rats and rats made to run on a treadmill program were also examined. The swimming program, but not the running program, resulted in a significant increase in heart weight. Actomyosin ATPase activity was also increased by 15% in the hearts of swimmers but not runners. Similar increases were observed for Ca2+-activated myosin ATPase activity and actin-activated ATPase of myosin. Sarcoplasmic reticulum from the hearts of swimmers showed increased calcium binding and calcium uptake as a function of time and of calcium concentration. Sarcoplasmic reticulum ATPase activities were not altered by hypertrophy. These findings in physiologic hypertrophy contrast with those of pathologic hypertrophy in which ATPase activity of contractile proteins and calcium binding and uptake of sarcoplasmic reticulum have generally been found to be depressed.     

Na+-Ca2+ exchange induces low Na+contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca2+-ATPase

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.