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Objective: Increasing evidence points to a molecular disturbance of Ca2+ homeostasis in stunned myocardium. The aim of this study was therefore to investigate the expression of mRNAs for Ca2+ binding proteins related to the sarcoplasmic reticulum in a porcine model of myocardial stunning., Methods: In 22 anaesthetised pigs, stunning was achieved by one or two cycles of 10 min left anterior descending coronary artery occlusion and reperfusion. Hearts were excised at various timepoints of the protocol. Total RNA was extracted from stunned (experimental) as well as normally perfused (control) myocardium., Results: Northern blot analysis using radioactive cDNA probes revealed that the Ca(2+)-ATPase mRNA levels increased 1.6-fold compared to the control value at 90 min of the second reperfusion. The steady state level of phospholamban mRNA rose 2.5-fold at 180 min of reperfusion. A 2.3-fold increase in calsequestrin mRNAs was observed after 90 min of the second reperfusion. The calmodulin and alpha, beta myosin heavy chain mRNA levels were unchanged. A glyceraldehyde-3-phosphate dehydrogenase cDNA probe served as a reference system. Nuclear run-on assays showed increased transcription for Ca(2+)-ATPase and calsequestrin at 90 min of reperfusion, supporting the view that increased mRNA levels seen with northern hybridisation were due to increased transcription of the respective gene., Conclusions: The results suggest specific repair mechanisms of stunned myocardium and point to the involvement of calcium regulatory proteins related to the sarcoplasmic reticulum in the pathogenesis of myocardial stunning.

Intracellular rapidly exchanging Ca2+ stores are identified and defined in terms of intralumenal low-affinity, high-capacity Ca(2+)-binding proteins, of which calsequestrin (CS) is the prototype in striated muscles. In chicken striated muscles, there is a single gene for CS [Choi and Clegg (1990) Dev. Biol. 142, 169-177]. In the chicken brain, the gene for CS was found to be selectively expressed in Purkinje neurons, as judged by Northern blotting, in situ hybridization and immunocytochemistry. The synthetic machinery for CS was found to be restricted to the cell body, i.e. excluded from dendrites and axon.

Fluorescence in situ hybridization (FISH) experiments were performed using genomic and complementary DNA probes in order to determine the location on human chromosomes for five genes expressed in cardiac and skeletal muscle sarcoplasmic reticulum. The chromosome location of each gene was determined in terms of both cytogenetic bands and fractional chromosome length. The ATP2A2 gene, expressing the SERCA2 isoform of the Ca2+ pump, maps to bands 12q23-q24.1, the phospholamban gene (PLN) to 6q22.1, the human skeletal muscle calsequestrin gene (CASQ1) to band 1q21, the cardiac calsequestrin gene (CASQ2) to bands 1p11-p13.3, and the cardiac calcium release channel gene (RYR2) to the interval between band 1q42.1 (distal) and band 1q43 (proximal).

Cytoplasmic free calcium ions (Ca2+) play a central role in excitation-contraction coupling of cardiac muscle. Abnormal Ca2+ handling has been implicated in systolic and diastolic dysfunction in patients with end-stage heart failure. The current study tests the hypothesis that expression of genes encoding proteins regulating myocardial Ca2+ homeostasis is altered in human heart failure. We analyzed RNA isolated from the left ventricular (LV) myocardium of 30 cardiac transplant recipients with end-stage heart failure (HF) and five organ donors (normal control), using cDNA probes specific for the cardiac dihydropyridine (DHP) receptor (the alpha 1 subunit of the DHP-sensitive Ca2+ channel) and cardiac calsequestrin of sarcoplasmic reticulum (SR). In addition, abundance of DHP binding sites was assessed by ligand binding techniques (n = 6 each for the patients and normal controls). There was no difference in the level of cardiac calsequestrin mRNA between the HF patients and normal controls. In contrast, the level of mRNA encoding the DHP receptor was decreased by 47% (P less than 0.001) in the LV myocardium from the patients with HF compared to the normal controls. The number of DHP binding sites was decreased by 35-48%. As reported previously, expression of the SR Ca(2+)-ATPase mRNA was also diminished by 50% (P less than 0.001) in the HF group. These data suggest that expression of the genes encoding the cardiac DHP receptor and SR Ca(2+)-ATPase is reduced in the LV myocardium from patients with HF. Altered expression of these genes may be related to abnormal Ca2+ handling in the failing myocardium, contributing to LV systolic and diastolic dysfunction in patients with end-stage heart failure.

We have cloned, sequenced and expressed the cDNA encoding frog skeletal muscle calsequestrin. The processed frog calsequestrin is 398 residues long, with an Mr of 45941 (unglycosylated form), and exhibits 77% sequence similarity with its rabbit counterpart. Consensus sequences for glycosylation and phosphorylation of the protein were conserved. Compared with rabbit calsequestrin, the mature amphibian protein has peculiar structural properties, which include (i) a higher content of negatively charged residues (142 versus 109), and (ii) a striking repeat sequence at the C-terminal region of 44 aspartic acid residues. Furthermore, this is the first report on the expression of calsequestrin cDNA in COS-1 cells; the expressed protein exhibited an Mr and antigenic properties which were indistinguishable from those of the native protein. In addition, it was capable of binding 45Ca in a ligand overlay. Northern blot analysis of frog skeletal muscle, liver, heart and brain RNA showed that the protein is mainly expressed in skeletal muscle. The high density of negative charges at the C-terminus might constitute high-capacity low-affinity Ca(2+)-binding sites, which may account for the higher Ca(2+)-binding capacity of frog calsequestrin compared with other members of the calsequestrin family (56 mol/mol versus 40-44 mol/mol of protein).

The expression of major sarcoplasmic reticulum proteins during cardiac and fast-twitch skeletal muscle development was examined using gene-specific probes. Through the use of S1 nuclease mapping, Northern blot, and RNA slot-blot analysis, sarcoplasmic reticulum proteins were shown to exhibit both narrow tissue specificity and plasticity in their expression during muscle development. In fast-twitch skeletal muscle, the cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin were detected at high levels in fetal stages but were gradually replaced by fast-twitch isoforms in adult muscle. In contrast, cardiac muscle expressed exclusively cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin at all stages. Both fast-twitch and slow-twitch skeletal muscle expressed the same skeletal muscle ryanodine receptor isoform, whereas cardiac muscle expressed a cardiac isoform. Phospholamban expression was restricted to cardiac and slow-twitch skeletal muscle and did not appear in developing fast-twitch skeletal muscle. During in vitro myogenesis of C2C12 cells, the mRNA transcripts encoding sarcoplasmic reticulum proteins were found to be coordinately induced in synchrony with that of contractile protein mRNA. The myogenic factor "myogenin" induced sarcoplasmic reticulum gene transcripts along with contractile protein mRNAs in nonmyogenic cells. These data suggest that the induction of both sarcoplasmic reticulum and contractile protein gene families is under the control of a common myogenic differentiation program.

A cDNA encoding rabbit cardiac calsequestrin was isolated and characterized. The deduced nascent cardiac calsequestrin contains 409 amino acids of which 26% are acidic residues, and had 93% and 67% aa identity with canine cardiac calsequestrin and rabbit fast-twitch skeletal muscle calsequestrin, respectively. RNA blot analyses indicate that this mRNA is expressed in atrium, ventricle and to a lesser amount in slow-twitch skeletal muscle. This mRNA transcript is not expressed in adult fast-twitch skeletal muscle, smooth muscle, or nonmuscle tissues. Analysis of in vitro skeletal muscle myogenesis using a mouse myoblast cell line C2C12, demonstrates that both cardiac and skeletal calsequestrin isoforms are coproduced during muscle differentiation.

Little is known concerning the molecular mechanisms responsible for changes in sarcoplasmic reticulum (SR) function during ontogenic development and aging except that the amount of SR Ca(2+)-ATPase mRNA varies in these conditions. The aim of the present work was to determine whether SR maturation requires expression of specific isoforms and synchronous accumulation of mRNAs encoding proteins located in SR. Thus, we have studied expression of SR Ca(2+)-ATPase and calsequestrin genes in the rat at different developmental stages from 14 fetal days to 24 months of age. Analysis of alternative splicing of the major Ca(2+)-ATPase gene expressed in heart by nuclease S1 mapping led us to conclude that the Ca(2+)-ATPase gene expressed in heart was not differentially spliced during ontogenic development and senescence. A single calsequestrin mRNA isoform was also detected in rat heart whatever the developmental stage. The amount of specific mRNA was then measured by dot blot and normalized to 18S ribosomal RNA or to myosin heavy chain mRNA. The amount of Ca(2+)-ATPase mRNA relative to 18S RNA increases substantially at the end of fetal life and in the early postnatal period (9.5 +/- 0.5% in the 14-15 day fetus versus 99 +/- 7% in the 4-day-old rat). A stable high level is observed during adulthood. In aged rats (24 months), Ca(2+)-ATPase mRNA represents only 44.6% the amount observed in young adults (1-2 months).(ABSTRACT TRUNCATED AT 250 WORDS)

Calsequestrin, a muscle calcium binding protein, has been shown to bind the extracellular matrix protein laminin and evidence has been presented that CAL (initially called aspartactin) is on the cell surface, consistent with a role as a laminin receptor (1). In this report, we present evidence that does not support a laminin receptor function for CAL. We found that CAL immunoreactivity could not be detected on live cultured chick myotubes unless they were permeabilized with detergent. Furthermore, polyclonal anti-CAL antibodies did not perturb myotube adhesion to laminin or the rate of myoblast fusion on laminin. Expression of the CAL cDNA in a melanoma cell line that was poorly adherent to laminin did not increase adhesion to laminin. In these cells, CAL could not be detected on the cell surface, and the majority of CAL was found to be secreted into the media.

Calsequestrin is an acidic Ca2(+)-binding protein of sarcoplasmic reticulum existing as different gene products in cardiac muscle and skeletal muscle. A unique feature of cardiac calsequestrin is a 31-amino acid-long COOH-terminal tail (Scott, B. T., Simmerman, H. K. B., Collins, J. H., Nadal-Ginard, B., and Jones, L. R. (1988) J. Biol. Chem. 263, 8958-8964), which is highly acidic and contains several consensus phosphorylation sites for casein kinase II. In the work described here, we tested whether this cardiac-specific sequence is a substrate for casein kinase II. Both cardiac and skeletal muscle calsequestrins were phosphorylated by casein kinase II, but cardiac calsequestrin was phosphorylated to a higher stoichiometry and at least 50 times more rapidly. The site of rapid phosphorylation of cardiac calsequestrin was localized to the distinct COOH terminus, where a cluster of three closely spaced serine residues are found (S378DEESN-DDSDDDDE-COOH). The slower phosphorylation of skeletal muscle calsequestrin occurred at its truncated COOH terminus, at threonine residue 363 (I351NTEDDDDDE-COOH). The similar sequence in cardiac calsequestrin (I351NTEDDDNEE) was not phosphorylated. Cardiac calsequestrin, as isolated, already contained 1.2 mol of Pi/mol of protein, whereas skeletal muscle calsequestrin contained only trace levels of Pi. The endogenous Pi of cardiac calsequestrin was also localized to the distinct COOH terminus. Our results indicate that the cardiac isoform of calsequestrin is the preferred substrate for casein kinase II both in vivo and in vitro.

Calsequestrin (CAL) is a calcium-binding protein whose primary function is thought to involve sequestration of calcium in the muscle sarcoplasmic reticulum (SR). Little is known about the mechanisms regulating CAL expression, or about the role of this protein in muscle development. In addition, CAL may regulate calcium localization in some nonmuscle cells. We have identified an avian calsequestrin homolog. The predicted amino acid sequence of the avian CAL, first described as a laminin binding protein, and named aspartactin, is 70-80% identical to mammalian CAL sequences. We have used affinity-purified antibodies and cDNA probes to investigate expression in developing and adult chicken tissues. In adult chickens, the avian CAL homolog was expressed in slow and fast twitch skeletal muscle as well as in cardiac muscle. Surprisingly high levels of CAL protein were also detected in cerebellum. During development, CAL mRNA and protein were detected in Embryonic Day 5 (E-5) limb primordia, well before the initiation of myoblast fusion. In leg skeletal muscle, CAL protein and mRNA increase approximately 10-fold from E-8 to E-18 with a time course that just precedes myoblast fusion. This early expression pattern was also observed in cultured chicken pectoral myoblasts, and appears to be regulated at the level of mRNA abundance. The developmental profile of CAL expression is compared to that of other muscle proteins and possible additional functions of CAL are discussed.

We have previously reported the amino terminal sequence of adult chicken calsequestrin, an intraluminal Ca2(+)-binding protein isolated from fast-twitch skeletal muscle. The partial sequence showed homology with mammalian calsequestrins contained in the PIR data bank and complete identity with the amino terminus of a putative laminin-binding protein of the extracellular matrix, aspartactin. Based on these data, oligonucleotide primers were synthesized for PCR amplification and direct DNA sequencing. We report herein the primary sequence of chicken calsequestrin, deduced from cDNA. The sequence has been verified by amino acid sequencing of internal tryptic peptides. Importantly, the data show the primary structure of calsequestrin to be identical to the amino acid sequence reported for aspartactin, with the exception of a single amino acid difference (ileu vs. val) which may be animal strain-related. Based on these data, calsequestrin and aspartactin are the same protein.

A genomic clone encoding human fast-twitch skeletal muscle calsequestrin was isolated, and the amino acid sequence of the protein and the exon-intron boundaries of the gene were deduced from its sequence. A comparison with the rabbit gene showed that the sequence Glu-Asp-Asp-Asp-Asp near the COOH terminus of the rabbit sequence is lacking in the human gene. The calsequestrin gene was assigned to human chromosome 1 through the use of a human-mouse somatic cell hybrid mapping panel.

The interaction of calsequestrin (CSQ) with the channel-containing region of the sarcoplasmic reticulum (junctional face membrane, jfm) is involved in Ca2+ release, and it seemed of interest to identify the jfm-binding region of the CSQ molecule. For this purpose, CSQ was digested with trypsin, and peptides were screened for jfm binding. Partial amino acid sequencing of selected peptides allowed us to localize a critical site for jfm binding in the stretch encompassing residues 86-191.

cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites.

We have isolated two overlapping 17-kilobase (kb) genomic clones which, together, encode the rabbit fast-twitch skeletal muscle calsequestrin. The calsequestrin gene is 8.6 kb in length and consists of 11 exons. S1 nuclease protection and primer extension analyses position the transcription initiation site 191 nucleotides upstream of the translation initiation codon. The 5'-flanking sequence contains a CCAAT sequence and two CG-rich sequences but does not have an identifiable TATA box sequence in the anticipated position just upstream of the transcription initiation site. The sequence of the calsequestrin gene confirmed the sequence deduced from cDNA clones derived from neonatal muscle mRNA (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171). We could not detect any potential alternative splicing of the transcript in an analysis of this region of the gene that would account for the different sequence found at the carboxyl terminus of adult rabbit fast-twitch skeletal muscle calsequestrin.

Partial amino acid sequence analysis of rabbit fast-twitch skeletal muscle calsequestrin permitted the construction of synthetic oligonucleotides that were used as both primers and probes for the synthesis and isolation of cDNAs encoding calsequestrin from neonatal rabbit skeletal muscle libraries. The cDNA sequence encodes a processed protein of 367 residues with a Mr of 42,435 and a 28-residue amino-terminal signal sequence. The deduced amino acid sequence agreed closely with the portions of the mature protein that were sequenced using standard protein sequencing. The neonatal protein, however, contains an acidic carboxyl-terminal extension not present in the adult protein, suggesting that the cDNA sequence may have arisen from an alternatively spliced neonatal transcript. A single transcript of 1.9-2.0 kilobases was seen in neonatal skeletal muscle mRNA. A glycosylation site and two potential phosphorylation sites were detected. Although the protein contains about two acidic residues for each Ca2+ bound, there is no repeating distribution of acidic residues and no evidence of EF hand structures. Hydropathy plots show no transmembrane sequences, and structural analyses suggest that less than half of the protein is likely to be highly structured. This sequence defines the characteristics of a class of high-capacity, moderate-affinity, Ca2+ binding proteins.

Tissue contents of the sarcoplasmic-reticulum Ca2+-ATPase (Ca2+ +Mg2+-dependent ATPase), of calsequestrin and of parvalbumin were immunochemically quantified in homogenates of fast- and slow-twitch muscles of embryonic, maturing and adult rabbits. Unlike parvalbumin, Ca2+-ATPase and calsequestrin were expressed in embryonic muscles. Presumptive fast-twitch muscles displayed higher contents of these two proteins than did presumptive slow-twitch muscles. Calsequestrin steeply increased before birth and reached adult values in the two muscle types 4 days after birth. The main increase in Ca2+-ATPase occurred during the first 2 weeks after birth. Denervation of postnatal fast- and slow-twitch muscles decreased calsequestrin to amounts typical of embryonic muscle and suppressed further increases of Ca2+-ATPase. Denervation caused slight decreases in Ca2+-ATPase in adult fast-twitch, but not in slow-twitch, muscles, whereas calsequestrin was greatly decreased in both. Chronic low-frequency stimulation induced a rapid decrease in parvalbumin in fast-twitch muscle, which was preceded by a drastic decrease in the amount of its polyadenylated RNA translatable in vitro. Tissue amounts of Ca2+-ATPase and calsequestrin were essentially unaltered up to periods of 52 days stimulation. These results indicate that in fast- and slow-twitch muscles different basal amounts of Ca2+-ATPase and calsequestrin are expressed independent of innervation, but that neuromuscular activity has a modulatory effect. Conversely, the expression of parvalbumin is greatly enhanced by phasic, and drastically decreased by tonic, motor-neuron activity.

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.