Elements Of Sarcoplasmic Reticulum Research Articles

Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation

Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation.

Mitsugumin 56 is an MBOAT Family Member and Contributes to Postnatal Muscle Maturation

The sarcoplasmic reticulum (SR) functions as a highly specialized intracellular Ca2+ store controlling muscle contraction cycle. Although major SR proteins involved in Ca2+ handling have been extensively studied, there are still SR components with unknown functions, which would potentially open new study fields in muscle biology. Here we report mitsugumin 56 (MG56), a new SR membrane protein predominantly expressed in striated muscle. MG56 belongs to the MBOAT (membrane-bound O-acyltransferase) family, and is specifically localized to the junctional SR composing the triad in skeletal muscle. Mg56-knockout mice grew normally for a week after birth, however, they gradually developed suckling failure and died within two weeks under starvation conditions. In the skeletal muscle of Mg56-knockout mice, the SR elements began to swell near the Z-line prior to physical debilitation, and further developed enormous vacuoles spreading over the sarcomeres. However, in tension measurements, regular contractile features were largely preserved in Mg56-knockout muscle that contained swelling SR elements. Meanwhile, biochemical analysis demonstrated that unfolded protein response was highly activated in Mg56-knockout muscle, suggesting that the suckling failure was caused by disrupted muscle maturation under ER stress conditions. Therefore, MG56 exerts anti-ER stress activity in the developing SR and is essential for postnatal muscle maturation.

Mechanisms of the sarcoplasmic reticulum Ca2+ release induced by P2X receptor activation in mesenteric artery myocytes

BackgroundATP is one of the principal sympathetic neurotransmitters which contracts vascular smooth muscle cells (SMCs) via activation of ionotropic P2X receptors (P2XRs). We have recently demonstrated that contraction of the guinea pig small mesenteric arteries evoked by stimulation of P2XRs is sensitive to inhibitors of IP3 receptors (IP3Rs). Here we analyzed contribution of IP3Rs and ryanodine receptors (RyRs) to [Ca2+]i transients induced by P2XR agonist αβ-meATP (10μM) in single SMCs from these vessels. MethodsThe effects of inhibition of L-type Ca2+ channels (VGCCs), RyRs and IP3Rs (5μM nicardipine, 100μM tetracaine and 30μM 2-APB, respectively) on αβ-meATP-induced [Ca2+]i transients were analyzed using fast x–y confocal Ca2+ imaging. ResultsThe effect of IP3R inhibition on the [Ca2+]i transient was significantly stronger (67±7%) than that of RyR inhibition (40±5%) and was attenuated by block of VGCCs. The latter indicates that activation of VGCCs is linked to IP3R-mediated Ca2+ release. Immunostaining of RyRs and IP3Rs revealed that RyRs are located mainly in deeper sarcoplasmic reticulum (SR) while sub-plasma membrane (PM) SR elements are enriched with type 1 IP3Rs. This structural peculiarity makes IP3Rs more accessible to Ca2+ entering the cell via VGCCs. Thus, IP3Rs may serve as an “intermediate amplifier” between voltage-gated Ca2+ entry and RyR-mediated Ca2+ release. ConclusionsP2X receptor activation in mesenteric artery SMCs recruits IP3Rs-mediated Ca2+ release from sub-PM SR, which is facilitated by activation of VGCCs. Sensitivity of IP3R-mediated release to VGCC antagonists in vascular SMCs makes this mechanism of special therapeutic significance.

The Mboat Family Protein Mitsugumin 56 Contributes to Postnatal Maturation in the Muscle Sarcoplasmic Reticulum

The sarcoplasmic reticulum (SR) is a highly-specialized form of the smooth endoplasmic reticulum (ER), and functions intensively as an intracellular Ca2+ store in muscle cells. Although major proteins contributing to Ca2+ handling have been extensively studied, there are still as-yet-unknown SR components, which would potentially open new fields in muscle physiology. We currently identified mitsugumin 56 (MG56), that is a novel SR membrane protein expressed in striated muscle. MG56 belongs to the MBOAT (membrane-bound O-acyltransferase) family, and is specifically localized to the junctional SR composing the triad in skeletal muscle. Although Mg56-knockout mice normally grew until postnatal day 7 (P7), they exhibited suckling failure and died within two weeks under starvation conditions. In skeletal muscle from Mg56-knockout mice, SR elements began to swell near the Z-line on P5, and further developed enormous vacuoles spreading over sarcomeres afterwards. In tension measurements, Mg56-knockout extensor digitorum longus extensor digitorum longus muscle retained apparently normal contractile features on P7, but attenuated its vital contractility on P9. On the other hand, spontaneous Ca2+ sparks/waves under resting conditions were observed in isolated flexor digitarium breris fibers of Mg56-knockout mice on P7-9. Therefore, MG56 essentially contributes to postnatal SR maturation in skeletal muscle, although its contribution to SR Ca2+ handling remains to be defined at the molecular level.

String mitochondria in mouse soleus muscle

Red myofibers in mouse soleus muscle have two spatially distinct populations of mitochondria: one where these organelles are disposed in large clusters just inside the sarcolemma and the other situated between the myofibrils. In most cases, the interfibrillar mitochondria (IFM), which are much smaller than the subsarcolemmal ones (SSM), are arranged as pairs, with each member on opposite sides of the Z-line. In some myofibers, the IFM have fused end-to-end to form greatly elongated organelles, which we call "string mitochondria." Although narrow, these can be many sarcomeres in length. The SSM do not form string mitochondria. Most of the string mitochondria exhibit many instances of "pinching," a process involved in mitochondrial division. Elements of sarcoplasmic reticulum are intimately involved with each mitochondrial membrane invagination. It appears as if the fusion:fission balance of IFM in the soleus muscle is slightly out of kilter, with end-to-end fusion predominating over fission.

Mitochondrial Division in Rat Cardiomyocytes: An Electron Microscope Study

In cardiomyocytes of rats, two distinct mitochondrial division processes are in operation. The predominant process involves extension of a single crista until it spans the full width of a mitochondrion. Ingrowth of the outer membrane ultimately results in scission. The second division process involves "pinching," in which narrowing of the organelle at specific surface locations leads to its attenuation. When limiting membranes from opposite sides meet, mitochondrial fission ensues. When pinching is the operative mode, elements of sarcoplasmic reticulum always are associated with the membrane constrictions. The nuclear control mechanisms that determine which modality of mitochondrial division will prevail are unknown.

Mitochondrial division in rat cardiomyocytes: An electron microscope study

In cardiomyocytes of rats, two distinct mitochondrial division processes are in operation. The predominant process involves extension of a single crista until it spans the full width of a mitochondrion. Ingrowth of the outer membrane ultimately results in scission. The second division process involves “pinching”, in which narrowing of the organelle at specific surface locations leads to its attenuation. When limiting membranes from opposite sides meet, mitochondrial fission ensues. When pinching is the operative mode, elements of sarcoplasmic reticulum always are associated with the membrane constrictions. The nuclear control mechanisms that determine which modality of mitochondrial division will prevail are unknown.

A Single Luminally Continuous Sarcoplasmic Reticulum with Apparently Separate Ca2+ Stores in Smooth Muscle

Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca2+ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca2+ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca2+ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP3R, by the [Ca2+] within the SR, may create several apparent SR elements with various receptor arrangements. IP3R and RyR may appear to exist entirely on a single store, and there may seem to be additional SR elements that express either only RyR or only IP3R. The various SR receptor arrangements and apparently separate Ca2+ storage elements exist in a single luminally continuous SR entity.

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca2+ channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca2+ entry through VGCCs triggers RyR-mediated Ca2+ release via a Ca2+-induced Ca2+ release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca2+ release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP3Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca2+]i. The initial abrupt sub-PM [Ca2+]i upstroke was all but abolished by block of VGCCs (by 5μM nicardipine), depletion of intracellular Ca2+ stores (with 10μM cyclopiazonic acid) or inhibition of IP3Rs (by 2μM xestospongin C or 30μM 2-APB), but was not affected by block of RyRs (by 50–100μM tetracaine or 100μM ryanodine). Inhibition of either IP3Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation–contraction coupling in this phasic visceral smooth muscle occurs by Ca2+ entry through VGCCs which evokes an initial IP3R-mediated Ca2+ release activated via a CICR mechanism.

Phosphorylation-dependent Translocation of Glycogen Synthase to a Novel Structure during Glycogen Resynthesis

Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively. Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed that glycogen was present in dynamic cellular organelles that were termed glycosomas but no such cellular entities have ever been demonstrated. The aim of this study was to characterize muscle GS and GPh intracellular distribution and to identify possible translocation processes of both enzymes. Using in situ stimulation of rabbit tibialis anterior muscle, we show GS and GPh intracellular redistribution at the beginning of glycogen resynthesis after contraction-induced glycogen depletion. We identify a new "player," a new intracellular compartment involved in skeletal muscle glycogen metabolism. They are spherical structures that were not present in basal muscle, and we present evidence that indicate that they are products of actin cytoskeleton remodeling. Furthermore, for the first time, we show a phosphorylation-dependent intracellular distribution of GS. Here, we present evidence of a new regulatory mechanism of skeletal muscle glycogen metabolism based on glycogen enzyme intracellular compartmentalization.

Ultrastructural organization of the transverse tubules and the sarcoplasmic reticulum in a fish sound-producing muscle.

The ultrastructural basis for the extremely rapid contraction-relaxation cycle (up to 300 s(-1)) in the swim-bladder muscle (SBM) of a scorpionfish (Sebastiscus marmoratus), producing characteristic sounds for communication, was investigated by electron microscopy. The SBM fibres contained well-developed sarcoplasmic reticulum (SR) showing triadic contacts with well-organized transverse tubules (T tubules). It was newly found that different types of triadic contacts were present within the single SBM fibre. In the middle region of the fibre (approximately 54% of the fibre length), the triadic contacts were located around the level of boundary between the A- and I-bands (AI-type triad). However in the two end regions of the fibre (approximately 21% and approximately 12% of the fibre length), the triadic contacts were seen around the level of the Z-band (Z-type triad). Between the middle and end regions of the fibre, T tubule-SR contacts exhibited the form of pentads composed of a pair of T tubules and three SR elements, and newly found heptads composed of three T tubules and four SR elements. The fractional volume of SR relative to the fibre volume was estimated to be approximately 26% in the middle region of the fibre with the AI-type triads and approximately 15% in the fibre ends with the Z-type triads. These results are discussed in connection with the mechanism, by which the mechanical activity of the SBM muscle is neurally controlled.

Supramolecular calsequestrin complex.

As recently demonstrated by overlay assays using calsequestrin-peroxidase conjugates, the major 63 kDa Ca(2+)-binding protein of the sarcoplasmic reticulum forms complexes with itself, and with junctin (26 kDa), triadin (94 kDa) and the ryanodine receptor (560 kDa) [Glover, L., Culligan, K., Cala, S., Mulvey, C. & Ohlendieck, K. (2001) Biochim. Biophys. Acta1515, 120-132]. Here, we show that variations in the relative abundance of these four central elements of excitation-contraction coupling in different fiber types, and during chronic electrostimulation-induced fiber type transitions, are reflected by distinct alterations in the calsequestrin overlay binding patterns. Comparative immunoblotting with antibodies to markers of the junctional sarcoplasmic reticulum, in combination with the calsequestrin overlay binding patterns, confirmed a lower ryanodine receptor expression in slow soleus muscle compared to fast fibers, and revealed a drastic reduction of the RyR1 isoform in chronic low-frequency stimulated tibialis anterior muscle. The fast-to-slow transition process included a distinct reduction in fast calsequestrin and triadin and a concomitant reduction in calsequestrin binding to these sarcoplasmic reticulum elements. The calsequestrin-binding protein junctin was not affected by the muscle transformation process. The increase in calsequestrin and decrease in junctin expression during postnatal development resulted in similar changes in the intensity of binding of the calsequestrin conjugate to these sarcoplasmic reticulum components. Aged skeletal muscle fibers tended towards reduced protein interactions within the calsequestrin complex. This agrees with the physiological concept that the key regulators of Ca(2+) homeostasis exist in a supramolecular membrane assembly and that protein-protein interactions are affected by isoform shifting underlying the finely tuned adaptation of muscle fibers to changed functional demands.

Intracellular Ca2+ store in embryonic cardiac myocytes.

In mature cardiac myocytes, Ca2+ influx through the L-type Ca2+ channel activates the ryanodine receptor and triggers Ca2+ release from the sarcoplasmic reticulum (SR). This Ca2+ signal amplification, termed Ca2+-induced Ca2+ release (CICR), occurs within the junctional membrane complex between the plasma membrane and the SR, and is essential for cardiac excitation-contraction (E-C) coupling. On the other hand, Ca2+ available during E-C coupling is predominantly derived from Ca2+ influx in embryonic cardiac myocytes. To examine the role of the intracellular Ca2+ store in immature cardiac myocytes, we have generated knockout mice lacking the cardiac type of the ryanodine receptor (RyR-2), or junctophilin (JP-2) contributing to formation of the junctional membrane complex. Both RyR-2- and JP-2-knockout mice show lethality at early embryonic stages immediately after beginning of heart beating. The loss of RyR-2 produced abnormal SR elements exhibiting vacuolated structures and Ca2+-overloading in embryonic cardiac myocytes. In JP-2-deficient cardiac myocytes, formation of junctional membrane complexes, called pheripheral couplings, was disturbed, and abnormal Ca2+ transients without spatial and temporal synchronization were observed. Therefore, the knockout mice have demonstrated that RyR-2-mediated Ca2+ release at the junctional membrane complex is essential for cellular Ca2+ homeostasis in immature cardiac myocytes.

Ultrastructural distribution of the S100A1 Ca2+-binding protein in the human heart

Impaired calcium homeostasis and altered expression of Ca2+-binding proteins are associated with cardiomyopathies, myocardial hypertrophy, infarction or ischemia. S100A1 protein with its modulatory effect on different target proteins has been proposed as one of potential candidates which could participate in these pathological processes. The exact localization of S100A1 in human heart cells on the ultrastructural level accompanied with biochemical determination of its target proteins may help clarify the role of S100A1 in heart muscle. In the present study the distribution of the S100A1 protein using postembedding (Lowicryl K4M) immunocytochemical method in human heart muscle has been determined quantitatively, relating number of antigen sites to the unit area of a respective structural component. S100A1 antigen sites have been detected in elements of sarcoplasmic reticulum (SR), in myofibrils at all levels of sarcomere and in mitochondria, the density of immunolabeling at Z-lines being about 3 times and at SR more than 5 times higher than immunolabeling of remaining structural components. The presence of the S100A1 in SR and myofibrils may be related to the known target proteins for S100A1 at these sites.

Ca2+ Spark as a Regulator of Ion Channel Activity

Ca2+ spark is a local and transient Ca2+ release from sarcoplasmic reticulum (SR) through the ryanodine receptor Ca2+-releasing channel (RyR). In cardiac myocytes, Ca2+ spark is an elementary unit of Ca2+-induced Ca2+ release (CICR) by opening of RyR(s) in junctional SR (jSR), which is triggered by Ca2+-influx through L-type Ca2+ channels to the narrow space between a transverse tubule and jSR. Ca2+ spark has, therefore, been described as the evidence for “the local control of excitation-contraction coupling”. In contrast, Ca2+ sparks in smooth muscle have been reported in relation to Ca2+-dependent K+ (KCa) channel activation and muscle relaxation. A spontaneous Ca2+ spark in a superficial area activates 10–100 KCa channels nearby and induces membrane hyperpolarization, which reduces Ca2+ channel activity. In several types of smooth muscle cells, which have relatively high membrane excitability, an action potential (AP) elicits 5–20 Ca2+ hot spots (evoked sparks with long life) in the early stage via CICR in discrete superficial SR elements and activates KCa-channel current highly responsible for AP repolarization and afterhyperpolarization. CICR available for contraction may occur more slowly by the propagation of CICR from superficial SR to deeper ones. The regulatory mechanism of ion channel activity on plasma membrane by superficial SR via Ca2+ spark generation in smooth muscle cells may be analogously common in several types of cells including neurons.

Localization of ryanodine receptors in smooth muscle.

The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide-stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca2+-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca2+-release channels and origins of Ca2+ sparks, Ca2+ oscillations, and Ca2+ waves.

Giant sarcoplasmic reticulum vesicles: a study of membrane morphogenesis.

Rabbit sarcoplasmic reticulum vesicles were fused into giant proteoliposomes in a medium of 0.1 M KCl, 10 mM Tris-maleate, pH 7.0, 10 micrograms ml-1 antipain, 10 micrograms ml-1 leupeptin, 25 IU per ml Trasylol, 3 mM NaN3, 3.75% PEG 1500 and 3% DMSO by brief exposure to 37 degrees C, followed by incubation for 4 h at 25 degrees C. Approximately 5-10% of the sarcoplasmic reticulum elements underwent fusion, forming single-walled spherical vesicles of 1-25 microns diameter, in which the polarity of the native membrane was preserved. The Ca(2+)-stimulated ATPase activity remained essentially unchanged after fusion. On exposure to decavanadate in a Ca(2+)-free medium the spherical vesicles assumed a corrugated appearance with the formation of long ridges separated by deep furrows that eventually pinched off longitudinally and separated into numerous long crystalline tubules of uniform (approximately 0.1 microns) diameter. The vanadate-induced transformation of giant vesicles into tubules implies that the geometry of the sarcoplasmic reticulum membrane is determined by the conformation of the Ca(2+)-ATPase.

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/μm2 filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 1∶6. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.

Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate.

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.

Functional heterogeneity of the sarcoplasmic reticulum within sarcomeres of skinned muscle fibers.

Precipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations. Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 microM Ca++ and saturated above about 1.0 microM. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations greater than or equal to mM and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25 mM. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.