Mutations in the gene-encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, are associated with Brody disease.

Brody disease is a rare inherited disorder of skeletal muscle function. Symptoms include exercise-induced impairment of skeletal muscle relaxation, stiffness and cramps. Ca2+ uptake and Ca2+ ATPase activities are reduced in the sarcoplasmic reticulum, leading to the prediction that Brody disease results from defects in the ATP2A1 gene on chromosome 16p12.1-12.2, encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase. A recent search, however, did not reveal any mutations in the ATP2A1 gene in three Brody patients. We have now associated Brody disease with the autosomal recessive inheritance of three ATP2A1 mutations in two families, suggesting that the disease is genetically heterogeneous. One mutation occurs at the splice donor site of intron 3, while the other two mutations lead to premature stop codons, truncating SERCA1, deleting essential functional domains and raising the intriguing question: how have these Brody patients partially compensated for the functional knockout of a gene product believed to be essential for fast-twitch skeletal muscle relaxation?

A mutation in the human ryanodine receptor gene associated with central core disease.

Central core disease (CCD) is a morphologically distinct, autosomal dominant myopathy with variable clinical features. A close association with malignant hyperthermia (MH) has been identified. Since MH and CCD genes have been linked to the skeletal muscle ryanodine receptor (RYR1) gene, cDNA sequence analysis was used to search for a causal RYR1 mutation in a CCD individual. The only amino acid substitution found was an Arg2434His mutation, resulting from the substitution of A for G7301. This mutation was linked to CCD with a lod score of 4.8 at a recombinant fraction of 0.0 in 16 informative meioses in a 130 member family, suggesting a causal relationship to CCD.

Localization of sarcoplasmic reticulum proteins in rat skeletal muscle by immunofluorescence.

Ca++-Mg++-dependent ATPase and calsequestrin, the major intrinsic and extrinsic proteins, respectively, of the sarcoplasmic reticulum, were localized in cryostat sections of adult rat skeletal muscle by immunofluorescent staining and phase-contrast microscopy. Relatively high concentrations of both the ATPase and calsequestrin were found in fast-twitch myofibers while a very low concentration of the ATPase and a moderate concentration of calsequestrin were found in slow-twitch myofibers. These findings are consistent with previous biochemical studies of the isolated sarcoplasmic reticulum of slow-twitch and fast-twitch mammalian muscles. The distribution of the ATPase in muscle fibers is distinctly different from that of calsequestrin. While calsequestrin is present only near the interface between the I- and A-band regions of the sarcomere, the ATPase is found throughout the I-band region as well as in the center of the A-band region. In comparing these results with in situ ultrastructural studies of the distribution of sarcoplasmic reticulum in fast-twitch muscle, it appears that the ATPase is rather uniformly distributed throughout the sarcoplasmic reticulum while calsequestrin is almost exclusively confined to those regions of the membrane system which correspond to terminal cisternae. Fluorescent staining with these antisera was not observed in vascular smooth muscle cells present in the cryostat sections of the mammalian skeletal muscle used in this study.

Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.

The ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat gracilis muscle was determined by indirect immunoferritin labeling of ultrathin frozen sections. Simultaneous visualization of ferritin particles and of adsorption-stained cellular membranes showed that the Ca2+ + Mg2+-ATPase was concentrated in the longitudinal sarcoplasmic reticulum and in the nonjunctional regions of the terminal cisternae membrane but was virtually absent from mitochondria, plasma membranes, transverse tubules, and junctional sarcoplasmic reticulum. Ferritin particles were found preponderantly on the cytoplasmic surface of the membrane, in agreement with published data showing an asymmetry of the Ca2+ + Mg2+-ATPase within the sarcoplasmic reticulum membrane. Comparison of the density of ferritin particles in fast and slow myofibers suggested that the density of the Ca2+ + Mg2+-ATPase in the sarcoplasmic reticulum membrane in a fast myofiber is approximately two times higher than in a slow myofiber.

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle.

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.

Ultrastructural localization of calsequestrin in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.

The ultrastructural localization of calsequestrin in rat skeletal muscle (gracilis) was determined by indirect immunoferritin labeling of ultrathin frozen sections. Calsequestrin was found in the lumen of transversely and longitudinally oriented terminal cisternae but was absent from most of the longitudinal sarcotubules and the fenestrated sarcoplasmic reticulum. Calsequestrin was occasionally observed in vesicular structures found in the central region of the I band. Since calsequestrin is believed to provide the major site of Ca2+ sequestration in the sarcoplasmic reticulum, the present results support the view that Ca2+, transported to the lumen of the sarcoplasmic reticulum, is preferentially sequestered in the terminal cisternae, but they also suggest that additional Ca2+ sequestration may occur near the center of the I band.

Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures.

Immunofluorescent staining techniques were used to study the distribution of the Ca(2) + Mg(2+)-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins could be identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca(2+) medium contained calsequestrin and the ATPase, even though they were unable to fuse. These studies at the cellular level confirm biochemical findings on the biosynthesis of calsequestrin and the ATPase. Immunofluorescent staining of myoblasts showed that calsequestrin first appears in a well-defined region of the cell near one end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. This suggests that calsequestrin first accumulates in the Golgi region and then gradually spreads throughout the cell. In contrast, the ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to one particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident.

Calreticulin is essential for cardiac development.

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.

Malignant hyperthermia.

In humans genetically predisposed to malignant hyperthermia, anesthesia can induce skeletal muscle rigidity, hypermetabolism, and high fever, which, if not immediately reversed, can lead to tissue damage or death. The corresponding condition in swine leads to stress-induced deaths and devalued meat products. Abnormalities in the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been implicated in the cause of both the porcine and human syndromes by physiological and biochemical studies and genetic linkage analysis. In swine, a single founder mutation in the ryanodine receptor gene (RYR1) can account for all cases of malignant hyperthermia in all breeds, but a series of different RYR1 mutations are likely to be uncovered in human families with MH. Moreover, lack of linkage between malignant hyperthermia and RYR1 in some families indicates a heterogeneous genetic basis for the human syndrome.

Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia.

Malignant hyperthermia (MH) causes neurological, liver, and kidney damage and death in humans and major economic losses in the swine industry. A single point mutation in the porcine gene for the skeletal muscle ryanodine receptor (ryr1) was found to be correlated with MH in five major breeds of lean, heavily muscled swine. Haplotyping suggests that the mutation in all five breeds has a common origin. Assuming that this is the causal mutation for MH, the development of a noninvasive diagnostic test will provide the basis for elimination of the MH gene or its controlled inclusion in swine breeding programs.

Solubilization and reconstitution of cholinephosphotransferase from sarcoplasmic reticulum: stabilization of solubilized enzyme by diacylglycerol and glycerol.

Cholinephosphotransferase (CDPcholine: 1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2), which catalyzes the terminal step in phosphatidylcholine synthesis via the CDPcholine pathway, is present in sarcoplasmic reticulum from rabbit skeletal muscle (Cornell, R. and MacLennan, D.H. (1985) Biochim. Biophys. Acta 835, 567-576). The conditions for solubilization and reconstitution of this enzyme were investigated as a preliminary step towards its eventual purification. The activity was not released by treatment of membranes with 1 M KCl, but was solubilized after dissolution of membranes with detergents. Cholinephosphotransferase was inactivated by cholate, deoxycholate, Triton X-100, octylglucoside, Tween-20 or SDS at concentrations which solubilize the membrane. However, the activity could be fully recovered after reconstituting the membrane by adding excess lipid (soybean) and removing detergent by gel filtration, dialysis or by absorption to Bio-Beads. When the membrane was solubilized with octylglucoside or cholate at weight ratios of detergent: membrane protein of at least 10, the activity was irreversibly lost unless stabilizers were added with detergent. The substrate diacylglycerol and glycerol were effective stabilizers.

The capacity of the sarcoplasmic reticulum for phospholipid synthesis: a developmental study.

The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes wee undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was 8-fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.

The genetic basis of malignant hyperthermia.

Anaesthesia can induce skeletal muscle rigidity, hypermetabolism and high fever in humans genetically predisposed to malignant hyperthermia. If not immediately reversed, such episodes can lead to tissue damage and death. In swine with the corresponding condition, stress can induce death or lead to devalued meat products. Since muscle contraction is controlled by sarcoplasmic Ca2+, the abnormality, as reviewed here by David H. MacLennan, could reside in the skeletal muscle Ca(2+)-release channel gene, RYR1. Several observations support the view that a single RYR1 mutation is causal of malignant hyperthermia in all breeds of pigs and in at least some human families: the substitution of Cys for Arg615 as the sole deduced amino acid sequence change in a comparison of malignant hyperthermia and normal porcine RYR1 cDNAs; the linkage of this mutation to malignant hyperthermia in over 450 pigs in six breeds, including 338 meioses; and the appearance of the corresponding mutation, Cys for Arg614, across a species barrier, in a few human families, where it also cosegregates with malignant hyperthermia. Linkage of malignant hyperthermia to RYR1 is, however, not observed in all human families with malignant hyperthermia. Accordingly, other abnormal genes that may cause the condition are being sought.

Isolation of the calmodulin-dependent protein kinase system from rabbit skeletal muscle sarcoplasmic reticulum.

A calmodulin-dependent protein kinase system from the sarcoplasmic reticulum was dissolved in Nonidet P40, adsorbed to a CaM affinity column in the presence of Ca2+ and eluted in the presence of EGTA. The purified fraction contained major proteins of 60 and 20 kDa and minor components of 89 and 34 kDa, all of which were phosphorylated with dependencies on Ca2+, CaM, ATP and pH similar to those observed in the sarcoplasmic reticulum. Differences in the phosphopeptides produced by partial proteolysis of the individual phosphoproteins indicated that they are distinct entities. 125I-CaM labeled only the 60 kDa protein, suggesting that it is a kinase.

Rabbit cardiac and slow-twitch muscle express the same phospholamban gene.

The nucleotide sequences of cDNAs encoding phospholamban were found to be virtually identical when the cDNA clones were isolated from rabbit slow-twitch (soleus) and rabbit cardiac muscle libraries. These findings demonstrate that both types of muscle express the same phospholamban gene. The deduced amino acid sequences of rabbit and dog phospholamban were identical except for a change from Asp (dog) to Glu (rabbit) at position 2. The nucleotide sequences of the 5'- and the very long 3'-untranslated regions of rabbit and dog phospholamban cDNAs also exhibited a high percentage of identity.

Co-expression of slow-twitch/cardiac muscle Ca2(+)-ATPase (SERCA2) and phospholamban.

Full length cDNAs encoding both slow-twitch/cardiac (SERCA2) and fast-twitch skeletal muscle (SERCA1) Ca2(+)-ATPases were expressed by transient transfection of COS-1 cells. Studies of the Ca2(+)-dependency of Ca2(+)-transport in microsomes isolated from these cells showed that both isoforms had an affinity for Ca2+ of about 0.2 microM. The Ca2(+)-affinity of SERCA2 was lowered when phospholamban was co-expressed with it, demonstrating that the two proteins interact in this expression system. These studies support the view that phospholamban inhibition accounts for the low Ca2(+)-affinity and low activity of SERCA2 in cardiac muscle sarcoplasmic reticulum.

Phospholamban domain I/cytochrome b5 transmembrane sequence chimeras do not inhibit SERCA2a.

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b5 were coexpressed with the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four-base circuit.

The fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twitch soleus muscle.

The major form of calsequestrin in rabbit slow-twitch soleus muscle is shown to be identical to that isolated and cloned from rabbit fast-twitch muscle on the following bases: identity of cDNAs cloned from mRNAs from the two muscle sources; equivalent hybridization of a fast-twitch calsequestrin cDNA probe to mRNAs isolated from fast-twitch and slow-twitch muscles; identity of the 23 amino-terminal amino acids; strong binding of 45Ca2+ in a gel overlay of slow muscle sarcoplasmic reticulum protein to a band at the level of the fast-twitch calsequestrin isoform and only weak binding at the level of the cardiac isoform. No evidence was obtained for developmentally regulated alternative splicing of the calsequestrin transcript in mature slow or fast-twitch muscle.

Deletion of NH2- and COOH-terminal sequences destroys function of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum.

Deletion mutants of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS-1 cells. The mutants were expressed at levels 7- to 15-fold lower than the wild-type and were inactive. In vitro transcription-translation-insertion experiments showed that deletion of transmembrane sequences M1 and M2, but not of M8, M9, M10 or the NH2-terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1 and M2.

The role of the calcium release channel of skeletal muscle sarcoplasmic reticulum in malignant hyperthermia.

In this short review, we have described studies that have identified Arg415 in the Ca2+ release channel as a residue that influences channel sensitivity to Ca2+ induced Ca2+ release, rate of Ca2+ release, and channel closing. We have also described studies that confirm Dr. Numa's predictions that residues 4246-4267, 4382-4417, and 4478-4512 contain Ca2+ binding sites. The site between residues 4483 and 4494 (the PE repeat sequence) may be a key binding site for Ca2+ activation of the channel. Other residues in the sequence 4478-4512 may also contribute to activation of the channel. Thus our studies have contributed to basic knowledge of regulation of Ca2+ release function. They have also provided practical benefits in defining a disease gene, in development of a diagnostic test for porcine MH that is of economic benefit, and in laying the foundation for human MH diagnostic tests that may prevent anesthesia-induced morbidity and mortality.