Alfredo Margreth

Characterization study of the ryanodine receptor and of calsequestrin isoforms of mammalian skeletal muscles in relation to fibre types

SummaryWe have investigated high-affinity ryanodine-binding sites in membrane preparations from representative fast-twitch and slow-twitch muscles of the rabbit and rat, as well as from human mixed muscle. Our results, obtained in high-ionic strength binding buffer, demonstrate extensive similarities in binding affinity for [3H]ryanodine (Kd: about 10 nM) and a two-fold to four-fold difference in membrane density of the ryanodine receptor between fast-twitch and slow-twitch muscle of the rat and rabbit, respectively. The [3H]ryanodine-pCa relationship for the Ca2+-activation curve of ryanodine binding was found to be similar for all mammalian muscles, as tested at 20 nM ryanodine. With 10 mM caffeine or 50 μM doxorubicin the pCa for half-maximal activation of [3H]ryanodine binding invariably shifted from an average pCa value of 6.5 to pCa 7.1–7.3. IC50 values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca2+-release channel blocker, did not differ significantly (range 0.3–1.0 μM). The Ca2+-dependence curve (range 1 nM–10 mM free Ca2+) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cisternae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cisternae from the same species. Cardiac ryanodine receptor is also clearly distinguishable for electrophoretic mobility, Clevelands peptide maps, and, most strikingly, for total lack of cross-reactivity with polyclonal antibody to fast skeletal RyR. By the same properties, the ryanodine receptor of fast- and slow-twitch muscle appear to be the same or a similar protein. On investigating the composition of calsequestrin in rat and human skeletal muscles, both in membrane-bound form and after purification by phenyl-Sepharose chromatography, we have been able to show that, independent of the animal species, the cardiac isoform, as characterized by the identical amino-terminal amino-acid sequence, pattern of immunoreactivity, and lack of Ca2+-dependent shift in mobility on SDS-PAGE, is exclusively expressed in slow-twitch fibres, together with the main fast-skeletal calsequestrin isoform. While our experimental findings strongly argue for the presence of only one population of skeletal-specific Ca2+-release channels in junctional terminal cisternae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of Ca2+-release, depending on its isoform composition.

Calsequestrin, a component of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of chicken cerebellum

The presence and distribution of calsequestrin (CS), Ca2+ pump, and inositol 1,4,5-trisphosphate (IP3) receptor were investigated biochemically and immunologically in microsomal (P3) fractions isolated from chicken cerebrum and cerebellum. Two different batches of polyclonal antibodies specific for chicken skeletal muscle CS identified a Ca2+ binding, CS-like protein that was extremely enriched in cerebellum P3 fractions and absent from all cerebrum fractions. The cerebellum CS-like protein was deemed authentic CS because the N-terminal amino acid domain and peptide mapping were identical to those of skeletal muscle CS in the same species. CS was detected in striated muscles and cerebellum only. Cerebellum P3 fractions were also found to be considerably enriched in Ca2+ pump and IP3 receptor compared with the homologous cerebrum fractions, as judged by measurements of Ca2+ uptake, Ca2(+)-ATPase activity, IP3-induced Ca2+ release, and [3H]IP3 binding, respectively. Cerebellum microsomal fractions therefore appear to contain membrane fragments endowed with Ca2+ pump, IP3 receptor, and CS, i.e., three key components of a Ca2+ storage organelle.

Coexpression of two isoforms of calsequestrin in rabbit slow-twitch muscle

SummaryThe cardiac and fast-twitch skeletal muscle forms of the Ca2+ -binding protein calsequestrin (CS) are the products of two different genes, both of which are transcribed in slow-twitch skeletal muscle, though at much different rates (Scottet al., 1988., Fliegelet al., 1989). We have investigated this problem more closely at the protein level, on isolated terminal cisternae (TC) of the sarcoplasmic reticulum (SR) of rabbit slow-twitch muscle, and following purification of two distinct forms of CS from whole tissue by DEAE-Cellulose chromatography and Ca2+ -dependent elution from phenyl-Sepharose.Two electrophoretically (apparent molecular mass of 64 kDa and 54 kDa, respectively), and antigenically distinct forms of CS, here shown to be related to the fast-twitch skeletal muscle and to cardiac-type isoform of CS, respectively, colocalize to junctional TC of slow-twitch muscle. The cardiac-type isoform that is expressed in slow-twitch muscle accounts for about 25% of total CS present in isolated TC, it binds Ca2+ as effectively as the major CS form, using a45Ca-overlay technique, and it shares extensive similarities with dog cardiac CS, not only in size and antigenically, but also in pl, as well as in the DEAE-elution characteristics. No difference in behaviour with phenyl-Sepharose resin were observed between the two CS isoforms from slow-twitch muscle. Purified CS from fast-twitch muscle and the major CS isoform of slow-twitch muscle were found to be identical proteins, when compared between each other for peptide composition, after partial digestion withS. aureus V8 protease and for amino-terminal amino acid sequences, as well as concerning Ca2+ -induced structural changes, as determined by intrinsic fluorescense spectroscopy. On the other hand, by all these properties the minor CS form from slow-twitch muscle appears to be closely related or identical to cardiac CS.

Low-affinity Ca2+-binding sites versus Zn2+-binding sites in histidine-rich Ca2+-binding protein of skeletal muscle sarcoplasmic reticulum

Abstract Histidine-rich Ca 2+ -binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [ 125 I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca 2+ -binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45 Ca at low affinity, as well as being able to bind 65 Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca 2+ -binding protein calsequestrin not having detectable 65 Zn-binding sites, HRC protein bound selectively to immobilized Zn 2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.

QUANTITATION OF RYANODINE RECEPTOR OF RABBIT SKELETAL MUSCLE, HEART AND BRAIN

The total number of high-affinity ryanodine receptor (RyR) binding sites present in skeletal and cardiac muscle and in brain tissue of the rabbit was determined by [3H]ryanodine binding to subfractions obtained by differential centrifugation of homogenates prepared in a low-ionic strength medium, containing 0.5% Chaps. In all three tissues at least 80% of [3H]ryanodine binding was recovered in the total membrane (TM) fraction obtained by centrifuging between 650 g for 10 min and 120,000 x g for 90 min. Skeletal muscle displayed higher contents of high-affinity RyR sites (about 49 pmol/g wet wt) than heart and brain (about 12 pmol and 3.5 pmol/g wet wt, respectively). The affinity for ryanodine, as well as the affinity for Ca2+, in the absence or presence of Ca2(+)-releasing drugs (caffeine and doxorubicin) of TM from skeletal muscle, were found to be identical to those of purified terminal cisternae. As low as 1 g of tissue was sufficient to perform several experiments.

Postnatal development of rabbit fast-twitch skeletal muscle: accumulation, isoform transition and fibre distribution of calsequestrin

SummaryThe time-course of disappearance of slow-cardiac calsequestrin (CS) and that of appearance of the skeletal CS isoform were investigated in developing fast-twitch skeletal muscle of the rabbit between postnatal days 1 and 60, along with changes in density of the ryanodine receptor (RyR)/Ca2+ release channel. Western blot data on skeletal muscle membranes, purification of two CS isoforms by phenyl-Sepharose chromatography, and their immunolocalization in muscle fibres, all show that both CS isoforms are coexpressed in neonatal muscle. Our results, at the protein level, indicate that the turning off of synthesis of cardiac CS and its total disappearance from fast-twitch fibres take place at critical periods between two and four weeks postnatally, i.e. past changes in the respective mRNA. In contrast, the accumulation in muscle membranes of both the RyR and the skeletal CS isoform proceeds steadily up to one month, to reach adult values at about two months of age. These findings seem to argue that myogenic factors, in addition to the morphogenetic influence on the sarcoplasmic reticulum from the neural input to the muscle, may be involved in the developmental transition of CS isoforms in mammalian fast-twitch muscle fibres.

Coexistence of two calsequestrin isoforms in rabbit slow-twitch skeletal muscle fibers

The cardiac and skeletal muscle isoforms of calsequestrin (CS), the low affinity, high capacity Ca2+ binding protein localized in the lumen of sarcoplasmic reticulum, are the products of two different genes (Fliegel, L., Leberer, E., Green, N.M. and MacLennan, D.H. (1982) FEBS Lett. 242, 297–300), and can be both purified from slow‐twitch skeletal muscle of the rabbit (Damiani, E., Volpe, P. and Margreth, A. (1990) J. Muscle Res. Cell Motil. 11, 522–530). Here we show that both CS isoforms coexist in slow‐twitch muscle fibers as indicated by indirect immunofluorescent staining of cryosections with affinity‐purified antibodies specific for each CS isoform.

Characteristics of skeletal muscle calsequestrin: comparison of mammalian, amphibian and avian muscles.

SummaryCalsequestrin was identified in the isolated sarcoplasmic reticulum from skeletal muscle of three mammalian species (man, rat and rabbit) and from frog and chicken muscle, using electrophoretic and immunoblot techniques. It was further characterized in sarcoplasmic reticulum protein mixtures and at several stages of purification, following extraction with EDTA.We found extensive similarities in apparent molecular weight values, Stains All staining properties and in Clevelands peptide maps, between mammalian calsequestrins, and no detectable difference within a species between fast and slow muscle. Human calsequestrin, with an apparent molecular weight of 60 000 when measured at alkaline pH and of 41 000 when measured at neutral pH, appears to be the smallest in size. Frog calsequestrin, although weakly cross-reactive with rabbit calsequestrin and having a relatively higher apparent molecular weight at alkaline pH (72 000), shares several significant properties with mammalian calsequestrins. It bound calcium with a high capacity (1300 nmol per mg protein), it contained about 32% acidic amino acid residues and focused at closely similar pI values. We observed the formation of a complex with Stains All absorbing maximally at 535 nm, rather than at 600 nm, and an even more marked shift in apparent molecular weight at neutral pH.We found distinct differences in the case of chicken calsequestrin, in addition to those previously reported. It is a highly acidic, calcium-precipitable protein, but its amino acid composition is contradistinguished by a higher ratio of glutamate to aspartate and its rate of electrophoretic mobility is minimally affected by changes in pH. It stained deep bluish with Stains All after gel electrophoresis and yielded a protein-dye complex in aqueous solution, absorbing maximally at 560 nm, and finally, it bound fluorescent Concanavalin A.

Specific protein-protein interactions of calsequestrin with junctional sarcoplasmic reticulum of skeletal muscle

Minor protein components of triads and of sarcoplasmic reticulum (SR) terminal cisternae (TC), i.e. 47 and 37 kDa peptides and 31-30 kDa and 26-25 kDa peptide doublets, were identified from their ability to bind 125I calsequestrin (CS) in the presence of EGTA. The CS-binding peptides are specifically associated with the junctional membrane of TC, since they could not be detected in junctional transverse tubules and in longitudinal SR fragments. The 31-30 kDa peptide doublet, exclusively, did not bind CS in the presence of Ca2+. Thus, different types of protein-protein interactions appear to be involved in selective binding of CS to junctional TC.

Fast to slow change of myosin in nemaline myopathy: Electrophoretic and immunologic evidence

Muscle biopsies from two familial and one sporadic case with congenital nemaline myopathy and seven healthy family members were examined for myosin composition. Myosin was characterized with respect to light chain (LC) composition by one-dimensional and two-dimensional electrophoresis, and by immunologic methods (enzyme-linked immunosorbent assay [ELISA]), using specific antibody for rabbit fast myosin LC1 (LC1F). Type I fiber predominance was associated with the substitution of a hybrid, predominantly “slow” to a virtually pure “slow” myosin LC pattern for the “mixed” pattern found with myosin of normal muscle. Muscle myosin from the relatives had apparently normal light chain composition.