accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1

When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.     

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.     

Magnesium effects on activation of skinned fibers from striated muscle

The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.     

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.     

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.     

Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice

Dystrophin is absent in muscle fibers of patients with Duchenne muscular dystrophy (DMD) and in muscle fibers from the mdx mouse, an animal model of DMD. Disrupted excitation-contraction (E-C) coupling has been postulated to be a functional consequence of the lack of dystrophin, although the evidence for this is not entirely clear. We used mechanically skinned fibers (with a sealed transverse tubular system) prepared from fast extensor digitorum longus muscles of wild-type control and dystrophic mdx mice to test the hypothesis that dystrophin deficiency would affect the depolarization-induced contractile response (DICR) and sarcoplasmic reticulum (SR) function. DICR was similar in muscle fibers from mdx and control mice, indicating normal voltage regulation of Ca2+ release. Nevertheless, rundown of DICR (<50% of initial) was reached more rapidly in fibers from mdx than control mice [control: 32 +/- 5 depolarizations (n = 14 fibers) vs. mdx: 18 +/- 1 depolarizations (n = 7) before rundown, P < 0.05]. The repriming rate for DICRs was decreased in fibers from mdx mice, with lower submaximal DICR observed after 5, 10, and 20 s of repriming compared with fibers from control mice (P < 0.05). SR Ca2+ reloading was not different in fibers from control and mdx mice, and no difference was observed in SR Ca2+ leak. Caffeine (2-7 mM)-induced contraction was diminished in fibers from mdx mice compared with control (P < 0.05), indicating depressed SR Ca2+ release channel activity. Our findings indicate that fast fibers from mdx mice exhibit some impairment in the events mediating E-C coupling and SR Ca2+ release channel activity.     

Human skeletal sarcoplasmic reticulum Ca2+ uptake and muscle function with aging and strength training.

This study investigated the adaptations of skeletal muscle sarcoplasmic reticulum (SR) Ca2+ uptake, relaxation, and fiber types in young (YW) and elderly women (EW) to high-resistance training. Seventeen YW (18-32 yr) and 11 EW (64-79 yr) were assessed for 1) electrically evoked relaxation time and rate of the quadriceps femoris; and 2) maximal rates of SR Ca2+ uptake and Ca2+-ATPase activity and relative fiber-type areas, analyzed from muscle biopsies of the vastus lateralis. EW had significantly slower relaxation rates and times, decreased SR Ca2+ uptake and Ca2+-ATPase activity, and a larger relative type I fiber area than did YW. A subgroup of 9 young (YWT) and 10 elderly women (EWT) performed 12 wk of high-resistance training (8 repetition maximum) of the quadriceps and underwent identical testing procedures pre- and posttraining. EWT significantly increased their SR Ca2+ uptake and Ca2+-ATPase activity in response to training but showed no alterations in speed of relaxation or relative fiber-type areas. In YWT none of the variables was altered after resistance training. These findings suggest that 1) a reduced SR Ca2+ uptake in skeletal muscle of elderly women was partially reversed with resistance training and 2) SR Ca2+ uptake in the vastus lateralis was not the rate-limiting mechanism for the slowing of relaxation measured from electrically evoked quadriceps muscle of elderly women.     

Abnormal features in skeletal muscle from mice lacking mitsugumin29

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.     

A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle

Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.     

Direct measurement of Ca2+ uptake and release by the sarcoplasmic reticulum of saponin permeabilized isolated smooth muscle cells

To make direct measurements of Ca2+ uptake and release by the sarcoplasmic reticulum (SR) of isolated smooth muscle cells, a fluorometric method for monitoring Ca2+ uptake by striated muscle SR vesicles (Kargacin, M.E., C.R. Scheid, and T.W. Honeyman. 1988. American Journal of Physiology. 245:C694-C698) was modified. With the method, it was possible to make continuous measurements of SR function in saponin-skinned smooth muscle cells in suspension. Calcium uptake by the SR was inhibited by thapsigargin and sequestered Ca2+ could be released by Br-A23187 and thapsigargin. From the rate of Ca2+ uptake by the skinned cells and the density of cells in suspension, it was possible to calculate the Ca2+ uptake rate for the SR of a single cell. Our results indicate that the SR Ca2+ pump in smooth muscle cells can remove Ca2+ at a rate that is 45-75% of the rate at which Ca2+ is removed from the cytoplasm of intact cells during transient Ca2+ signals. From estimates of SR volume reported by others and our measurements of the amount of Ca2+ taken up by the skinned cells, we conclude that the SR of a single cell can store greater than 10 times the amount of Ca2+ needed to elicit a single transient contractile response.     

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.     

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Ca2+ entry, efflux and release in smooth muscle

Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30% and 70% respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction.     

High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease

A unique set of high molecular weight proteins was identified in junctional sarcoplasmic reticulum (SR) vesicles isolated from both cardiac muscle and skeletal muscle. These high Mr proteins were not present in free SR vesicles isolated from either tissue, nor were they observed in purified sarcolemmal fractions. The junctional SR high Mr proteins migrated as doublets in sodium dodecyl sulfate-polyacrylamide gels and exhibited apparent Mr values between 290,000 and 350,000. The high Mr proteins bound calmodulin; they were the principal proteins labeled in the cardiac and skeletal muscle SR subfractions by azido-125I-calmodulin. The high Mr proteins were also substrates for an endogenous Ca2+-calmodulin-dependent protein kinase activity, as well as exogenously added catalytic subunit of cAMP-dependent protein kinase. In addition, the junctional SR high Mr proteins were the major SR proteins degraded by a Ca2+-activated protease purified from smooth muscle. Control experiments verified the separation of junctional SR vesicles and free SR vesicles from both muscle types. Junctional SR vesicles were enriched in calsequestrin, and they exhibited Ca2+ uptake which was stimulated up to 10-fold by either ryanodine or ruthenium red. Free SR vesicles were deficient in calsequestrin and were insensitive to these two agents. Localization of the cardiac and skeletal muscle high Mr proteins to the junctional SR, coupled with demonstration of their nearly identical biochemical properties, suggests that the proteins are homologous and are likely to have similar functions in both types of striated muscle.     

Physiology and excitation-contraction coupling in the intestinal muscle of the crayfish Procambarus clarkii

The intestinal muscles of Procambarus clarkii are striated and yet they are specialized to produce slow peristaltic waves of contraction, not unlike those seen in vertebrate visceral smooth muscle. These muscles cannot be tetanized either by repetitive stimulation or by elevated potassium saline. The excitation-contraction (E-C) coupling mechanism was explored and compared with that known in crustacean skeletal muscle. Contraction is dependent on external Ca2+ which triggers the release of intracellular calcium from the sarcoplasmic reticulum (SR) via calcium-induced calcium release (CICR). Whereas contraction force is proportional to [Ca2+]o up to that in normal saline (13.4 mM), higher levels of Ca2+ reduce force. Ryanodine, which blocks calcium release from the SR, abolishes electrically stimulated contractions and CICR. Relaxation is achieved by removal of calcium from the cytosol in at least two ways, first by the re-loading of calcium into the SR by Ca2+-ATPases and second by the movement of calcium out of the cell by extruding it across the sarcolemma via Na+/Ca2+-exchangers. It is hypothesized that the inability of this muscle to show tetanus arises from inactivation of the voltage-gated calcium channels by high calcium. This is supported by the result that caffeine application causes an increase in tonus and size of phasic contractions by circumventing the sarcolemma and dumping SR calcium stores.     

The pore region of the skeletal muscle ryanodine receptor is a primary locus for excitation-contraction uncoupling in central core disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced approximately 90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.     

The C terminus (amino acids 75-94) and the linker region (amino acids 42-54) of the Ca2+-binding protein S100A1 differentially enhance sarcoplasmic Ca2+ release in murine skinned skeletal muscle fibers

S100A1, a Ca2+-binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca2+ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast- and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca2+ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca2+ release and Ca2+-induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca2+-binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca2+ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca2+ release as S100A1 influenced neither SR Ca2+ uptake nor myofilament Ca2+ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast- and slow-twitch skeletal muscle fibers based on enhanced SR Ca2+ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca2+ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Postnatal development of Ca2+-sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice

Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.     

SR Function in malignant hyperthermia

Malignant hyperthermia (MH) is a genetic disease in man and other animal species that predisposes to a catastrophic hypermetabolic syndrome that is triggered by certain anesthetic agents. A working hypothesis is that a defect in regulation of muscle cell calcium is the primary mechanism that initiates the MH syndrome. This paper reviews the evidence for a defect in muscle cell calcium as regulated by the sarcoplasmic reticulum membrane system. Skeletal muscle biopsied from MH man, pigs and dogs has abnormal in vitro contracture response to halothane and caffeine and these responses can be altered by lowering calcium content of the bathing solution and/or the muscle. Measurements of MH muscle cell Ca2+ by Ca2+-specific microelectrodes in vivo and fura-2 in vitro have demonstrated abnormal Ca2+ levels in resting and in caffeine-stimulated states. The SR membrane system is the primary calcium regulating organelle in skeletal muscle and a likely site for the defect in MH muscle. Two Ca2+ regulating functions of the SR have been explored in SR isolated from MH muscle. An abnormality of the 100K Ca2+-ATPase protein that functions to transport Ca2+ from myoplasm to inside the SR does not appear to be responsible for MH. The most probable defective site in the SR appears to be Ca2+ release channels and a Ca2+-induced Ca2+ release pathway has been shown to be abnormal in SR from MH human and pig muscle.