Trifluoperazine and the rapid, Ca2+-triggered damage of skeletal and cardiac muscle

[Ca2+]i was raised experimentally in mammalian and amphibian skeletal and cardiac muscles by A23187, DNP, anoxia or the Ca2+ -paradox. Trifluoperazine (TFP) at 10(-5) M failed to protect against the characteristic and rapid damage triggered by elevated [Ca2+]i in any of the preparations. It is concluded that calmodulin is not implicated in this rapid ultrastructural damage. TFP alone also causes identical patterns of damage. It may be acting to raise [Ca2+]i in skeletal and cardiac muscle cells.     

Calcium-related defects in cardiac and skeletal muscles of dystrophic mice

Ca2+ ATPase and calcium binding proteins were studied in cardiac and skeletal muscles of normal and dystrophic mice. In normal and dystrophic mice, Ca2+ ATPase was quite reduced in cardiac muscle compared to skeletal muscle and was, unlike skeletal muscle, insensitive to orthovanadate. Ca2+ ATPase in skeletal muscle of dystrophic mice was reduced as compared to normal mice. In both cases (normal and dystrophic), calcium binding proteins were the same (identical molecular weight). The effect of 2 drugs (Polymixine B and Bepridil) which decrease protein bound calcium was studied: the muscle proteins of dystrophic mice did not present the same sensitivity to Bepridil as controls. These findings suggest the existence of a calcium-related defect in skeletal and cardiac muscle of dystrophic mice.     

Increased calcium influx in dystrophic muscle

We examined pathways which might result in the elevated resting free calcium [( Ca2+]i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes. We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Ca2+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Ca2+]i levels. Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]i levels were similar, suggesting that calcium-sequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2+]i. The defect appears to be specific for calcium since resting free sodium levels and sodium influx rates in the absence of Na+/K(+)-ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes. These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca2+]i in fibers and myotubes. These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+]i in dystrophic muscle.     

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.     

Ryanodine receptor channelopathies

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.     

Cytotoxicity of phenazine methosulphate on skeletal muscle. The role of the sarcoplasmic reticulum in initiating myofilament damage

Phenazine methosulphate (PMS) or ferricyanide caused ultrastructural damage, including sarcolemma folds and swelling of the sarcoplasmic reticulum (SR), in amphibian skeletal muscle which corresponds with that triggered by a rise in [Ca]i and which, it is suggested, is caused by the activation of NAD(P)H oxidases at the sarcolemma (where it causes sarcolemma folding) and SR (where it causes myofilament damage). PMS also caused SR swelling and more limited damage in chemically-skinned muscle at zero [Ca]. In contrast with the oxygen paradox of cardiac muscle, there is no evidence for the production of oxygen radicals since no protection was provided by N2, mannitol, desferrioxamine or alpha-tocopherol, nor was the cell damage produced by an influx of Ca across the sarcolemma.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Comparative studies on the role of calcium in triggering subcellular damage in cardiac muscle

Isolated cardiac muscle strips from amphibians and mammals, together with isolated frog hearts, have been used as model systems for studying the action of elevated [Ca2+]i in promoting severe damage. A23187 and caffeine are believed to cause a rise in [Ca2+]i. Elevated [Ca2+]i causes characteristic damage which has been categorized and includes hypercontraction, Z-line damage and myofilament dissolution. The damage closely resembles that described in the isolated mammalian heart and in skeletal muscle preparations when [Ca2+]i is raised dramatically. Damage can therefore be triggered by releasing Ca2+ from intracellular sites, as distinct from increasing Ca2+ entry (as in the Ca2+-paradox). DNP and ruthenium red also cause identical damage and the results suggest that whilst the fall in pHi associated with ischaemia is probably the consequence of Ca2+/2H+ exchange at the mitochondria, coupled with ATP hydrolysis, lowered pHi by mitochondrial action is probably not the only cause of myofilament dissolution. Damage is not prevented by pretreatment with leupeptin, an inhibitor of Ca2+-activated neutral proteases, and it is concluded that the latter are probably not implicated in rapid and dramatic damage. The possible involvement of lysosomal enzymes in damage triggered by high [Ca2+]i is discussed.     

Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump.

Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.     

Oxidative stress and muscular dystrophy

Oxidative stress may be the fundamental basis of many of the structural, functional and biochemical changes characteristic of the inherited muscular dystrophies in animals and humans. The presence of by-products of oxidative damage, and the compensatory increases in cellular antioxidants, both indicate oxidative stress may be occurring in dystrophic muscle. Changes in the proportions and metabolism of cellular lipids, abnormal functions of cellular membranes, altered activity of membrane-bound enzymes such as the SR Ca2+-ATPase, disturbances in cellular protein turnover and energy production and a variety of other changes all indicate that these inherited muscular dystrophies appear more like the results of oxidative stress to muscle than any other type of underlying muscle disturbance. Particular details of these altered characteristics of dystrophic muscle, in combination with current knowledge on the processes of oxidative damage to cells, may provide some insight into the underlying biochemical defect responsible for the disease, as well as direct research towards the ultimate goal of an effective treatment.     

DISEASED MUSCLE CELLS IN CULTURE

(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster. (2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells. (3) Myoblasts obtained from dy^2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy^2J spinal cord neurones on the differentiation of normal, dy^2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder. (4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non-fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue-culture studies suggest that the dy mouse mutation is a myopathic disorder. (5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well-differentiated myotubes with cross-striations. mdg myotubes have normal electro-physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation-contraction coupling. (6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti-proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi-directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy. (7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue-culture studies have not been made of hamster skeletal muscle. (8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl-cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid-maltase deficiency but phosphorylase activity re-appeared in myotubes cultured from biopsies of phosphorylase-deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes. (9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.     

Free and bound intracellular calmodulin measurements in cardiac myocytes

Calmodulin (CaM) is a ubiquitous Ca2+ binding protein and Ca2+-CaM activates many cellular targets and functions. While much of CaM is thought to be protein bound, quantitative data in cardiac myocytes is lacking regarding CaM location, [CaM]free and CaM redistribution during changes in [Ca2+]i. Here, we demonstrated that in adult rabbit cardiac myocytes, CaM is highly concentrated at Z-lines (confirmed by Di-8-ANEPPS staining of transverse tubules) using three different approaches: immunocytochemistry (endogenous CaM), Alexa Fluor 488 conjugate CaM (F-CaM) in both permeabilized cells (exogenous CaM) and in patch clamped intact cells (via pipette dialysis). Using 100 nM [CaM]free we washed F-CaM into permeabilized myocytes and saw a two-phase (fast and slow) CaM binding curve with a plateau after 40 min of F-CaM wash-in. We also measured myocyte [CaM]free using two modified null-point titration methods, finding [CaM]free to be 50-75 nM (which is only 1% of total [CaM]). Higher [Ca2+]i increased CaM binding especially in the nucleus and at Z-lines and significantly slowed F-CaM dissociation rate when F-CaM was washed out of permeabilized myocytes. Additionally, in both permeabilized and intact myocytes, CaM moved into the nucleus when [Ca2+]i was elevated, and this was reversible. We conclude that [CaM]free is very low in myocytes even at resting [Ca2+]i, indicating intense competition of CaM targets for free CaM. Bound CaM is relatively concentrated at Z-lines at rest but translocates significantly to the nucleus upon elevation of [Ca2+]i, which may influence activation of different targets and cellular functions.     

Free radicals: a potential pathogenic mechanism in inherited muscular dystrophy

Despite years of intensive work, the biochemical defect responsible for the pathogenesis of inherited muscular dystrophy has not been identified either in humans or animal models. This review examines evidence in support of the hypothesis that free radicals may be responsible for muscle degeneration in this disorder. A variety of cellular abnormalities noted in dystrophic muscles can be accounted for by free radical mediated damage. In addition, chemical by-products associated with free radical damage are found in dystrophic muscle tissue from humans and animals with this disease. Various enzymatic antioxidant systems can be enhanced as a normal cellular response to oxidative stress, and such changes are seen both in dystrophic muscle cells and certain other tissues of dystrophic animals. An increased level of free radical damage would follow from either: enhanced production of free radical species, or a deficient component of the cellular antioxidant system, such as vitamin E. The free radical hypothesis of muscular dystrophy can account for data supporting several alternative theories of the pathogenesis of this disease, as well as other observations which have not previously been explained.     

Cardiac ryanodine receptor is absent in type I slow skeletal muscle fibers: immunochemical and ryanodine binding studies

Cardiac ryanodine receptor was purified from canine ventricle as a single polypeptide of Mr 400,000 by a stepwise sucrose density gradient centrifugation and heparin-Sepharose CL-4B column chromatography. The [3H]ryanodine binding capacity (Bmax) was 60-fold enriched from cardiac microsomes without a change in affinity for [3H]ryanodine. The purity of the final preparation was determined to be greater than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified preparation as an antigen, we produced six monoclonal antibodies which immunoprecipitated the cardiac ryanodine receptor. Three of these antibodies recognized the cardiac receptor on immunoblot analysis. In contrast, no protein in the microsomes isolated from Type I (slow) or Type II (fast) skeletal muscles was recognized by these antibodies. The [3H]ryanodine binding to cardiac and skeletal muscle microsomes was dependent on free Ca2+ concentration. In skeletal muscle microsomes, the [3H]ryanodine binding was remarkably enhanced by the addition of ATP or KCl and inhibited by high free Ca2+, whereas it was less sensitive to these agents in cardiac microsomes. All of these results clearly demonstrate that the cardiac ryanodine receptor is different from the skeletal muscle receptors and is not present even in Type I (slow) skeletal muscle fibers, in which cardiac isoforms of some of the muscle proteins are constitutively expressed.     

The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation

The effect of changes in temperature on the calcium sensitivity of tension development was examined in permeabilized cellular preparations of rat ventricle and rabbit psoas muscle. Maximum force and Ca2+ sensitivity of force development increased with temperature in both muscle types. Cardiac muscle was more sensitive to changes in temperature than skeletal muscle in the range 10-15 degrees C. It was postulated that the level of thin filament activation may be decreased by cooling. To investigate this possibility, troponin C (TnC) was partially extracted from both muscle types, thus decreasing the level of thin filament activation independent of temperature and, at least in skeletal muscle fibers, decreasing cooperative activation of the thin filament as well. TnC extraction from cardiac muscle reduced the calcium sensitivity of tension less than did extraction of TnC from skeletal muscle. In skeletal muscle the midpoint shift of the tension-pCa curve with altered temperature was greater after TnC extraction than in control fibers. Calcium sensitivity of tension development was proportional to the maximum tension generated in cardiac or skeletal muscle under all conditions studied. Based on these results, we conclude that (a) maximum tension-generating capability and calcium sensitivity of tension development are related, perhaps causally, in fast skeletal and cardiac muscles, and (b) thin filament activation is less cooperative in cardiac muscle than in skeletal muscle, which explains the differential sensitivity of the two fiber types to temperature and TnC extraction. Reducing thin filament cooperativity in skeletal muscle by TnC extraction results in a response to temperature similar to that of control cardiac cells. This study provides evidence that force levels in striated muscle influence the calcium binding affinity of TnC.     

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.     

The calcium-ryanodine receptor complex of skeletal and cardiac muscle

[3H]Ryanodine binds with high affinity to saturable and Ca2+-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle. Ruthenium red, known to interfere with Ca2+-induced Ca2+ release from SR vesicles, inhibits [3H]ryanodine specific binding in both skeletal and cardiac preparations whereas Mg2+, Ba2+, Cd2+ and La3+ selectively inhibit the skeletal preparation. The toxicological relevance of the [3H]ryanodine binding site is established by the correlation of binding inhibition with toxicity for seven ryanoids including two botanical insecticides. These findings provide direct evidence for Ca2+-ryanodine receptor complexes that may play a role in excitation-contraction coupling.     

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.