Roberta Sacchetto

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: An animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G>A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.

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.

Crystal structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle.

The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9Å resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.

A missense mutation in the skeletal muscle chloride channel 1 (CLCN1) as candidate causal mutation for congenital myotonia in a New Forest pony

A 7-month-old New Forest foal presented for episodes of recumbency and stiffness with myotonic discharges on electromyography. The observed phenotype resembled congenital myotonia caused by CLCN1 mutations in goats and humans. Mutation of the CLCN1 gene was considered as possible cause and mutation analysis was performed. The affected foal was homozygous for a missense mutation (c.1775A>C, p.D592A) located in a well conserved domain of the CLCN1 gene. The mutation showed a recessive mode of inheritance within the reported pony family. Therefore, this CLCN1 polymorphism is considered to be a possible cause of congenital myotonia.

Pseudomyotonia, a muscle function disorder associated with an inherited ATP2A1 (SERCA1) defect in a Dutch Improved Red and White cross-breed calf

A Dutch Improved Red and White cross-breed heifer calf was evaluated for a muscular disorder resulting in exercise induced muscle stiffness. Clinical findings included generalized exercise-induced muscle spasms with normal response to muscle percussion. Electromyography showed no myotonic discharges, thus ruling out myotonia. Whereas histological examination of muscle tissue was unremarkable, Ca(2+)-ATPase activity of sarcoplasmatic reticulum membranes (SERCA1) was markedly decreased compared to control animals. Mutation analysis revealed the presence of a missense mutation in the ATP2A1 gene encoding the SERCA1 protein (p.Arg559Cys). The present case presents similarities to human Brodys disease, but also to pseudomyotonia and congenital muscular dystonia previously described in different cattle breeds.

Clues to calcineurin function in mammalian fast-twitch muscle

It is believed that brief, high amplitude Ca2+ transients, as found in fast-twitch muscles, are not sufficient to activate the calcineurin (Cn)-dependent signaling pathway involved in regulation of slow myosin and slow sarcoplasmic reticulum Ca2+-ATPase genes (Olson and Williams, Cell101: 689–692, 2000). The results reported here try to fill the gap between this molecular knowledge, and the still fragmentary pieces of information on a possible different role of calcineurin in the same type of muscles. In the present work calcineurin was determined immunocytochemically by labeling fast- and slow-twitch fibers of representative rabbit muscles with anti-CnB antibodies, and was assessed by western blotting of isolated subcellular fractions. Calcineurin was found to be largely soluble and to be constitutively overexpressed in fast muscle as CnAα and CnAβ isoforms, the latter appearing to be predominant. Particulate calcineurin was not only associated with myofibrils but also with membranes of various origins. Fluorescence microscopy showed that calcineurin was distributed in the same pattern with respect to sarcomeres in both types of fibers, and formed punctate dots spanning the I-Z-I region, rather than being exclusively located at the Z-line, a disposition described for cardiomyocytes (Frey et al., Proc Natl Acad Sci USA97: 14,632–14,637, 2000). From knowledge that, in mammalian skeletal muscle fibers, junctional triads are located at the A-I band boundary, we explored the distribution of calcineurin between triadic components, after having verified that it was present in very low amounts in dystrophin-enriched sarcolemmal membranes. Our results demonstrate that a small but significant proportion of calcineurin coenriched with transverse tubules (TT), and copurified with the DHPR and with DHPR-associated PKA-AKAP15/18, thus suggesting that it is assembled as a multiprotein complex in the junctional membrane domain of TT. The membrane specificity of this association is further corroborated by the negative evidence for the presence of calcineurin in SR terminal cisternae. Calcineurin was separated from the DHPR and isolated as a AKAP15/18 subcomplex, including β2 adrenergic receptor, in addition to PKA and calcineurin, following equilibrium centrifugation of detergent extracts on a linear sucrose gradient. We show that the α1 subunit skeletal isoform of the DHPR, is a substrate for calcineurin dephosphorylation, after previous phosphorylation by endogenous PKA.

Multiple/heterogeneous Ca2+ stores in cerebellum purkinje neurons

1. The rapid and transient redistribution of Ca2+ from intracellular membrane-bound compartments (stores) is a key event of cell activation. 2. The cytological nature and molecular composition of such Ca2+ stores have been the object of intense investigation in recent years. 3. Here we review: (a) the current knowledge on intracellular Ca2+ stores of Purkinje neurons at the functional, biochemical, molecular, morphological and ultrastructural level; and discuss: (b) the relationship between Ca2+ stores and the endoplasmic reticulum, and (c) the occurrence of multiple/heterogeneous Ca2+ stores.

Coordinate expression of Ca2+‐ATPase slow‐twitch isoform and of β calmodulin‐dependent protein kinase in phospholamban‐deficient sarcoplasmic reticulum of rabbit masseter muscle

Modulation of sarcoplasmic reticulum (SR) Ca2+ transport by endogenous calmodulin‐dependent protein kinase II (CaM K II) involves covalent changes of regulatory protein phospholamban (PLB), as a common, but not the only mechanism, in limb slow‐twitch muscles of certain mammalian species, such as the rabbit. Here, using immunofluorescent techniques in situ, and biochemical and immunological methods on the isolated SR, we have demonstrated that rabbit masseter, a muscle with a distinct embryological origin, lacks PLB. Accommodating embryological heterogeneity in the paradigm of neural‐dependent expression of specific isogenes in skeletal muscle fibers, our results provide novel evidence for the differential expression in the SR of 72 kDa β components of CaM K II, together with the expression of a slow‐twitch sarcoendoplasmic reticulum Ca2+‐ATPase isoform, both in limb muscle and in the masseter.

Two splice variants of CaMKII-anchoring protein are present in the sarcoplasmic reticulum of rabbit fast-twitch muscle.

Anchoring protein alphaKAP targets calmodulin kinase II (CaMKII) to the sarcoplasmic reticulum (SR), and in the rabbit is a substrate of CaMKII itself in fast-twitch, but not in slow-twitch muscle. This work was aimed at elucidating the molecular basis for differential phosphorylation of alphaKAP. Here we show that two, immunologically related, size forms (23 and 21 kDa) of alphaKAP are present in fast-twitch muscle SR in a 3:1 stoichiometry. Phosphorylation experiments identified the shorter form as the CaMKII specific substrate. Both forms are shown to be stably integrated into the holoenzyme. Two splice variants of alphaKAP were found in rabbit fast-twitch muscle and only one in slow-twitch muscle, using RT-PCR. Mobilities on SDS-PAGE are those expected. The shorter splice variants lacks the 33-nucleotide sequence inserted by alternative splicing present in full-length alphaKAP, akin to differences between variants A and B of brain alphaCaMKII. The absence of the 11-amino acid sequence creates a novel CaMKII phosphorylation site. Taken together our results show that alternative splicing regulates alphaKAP phosphorylation in a fiber-type specific manner.

Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum

Skeletal muscle triadin is a sarcoplasmic reticulum (SR) membrane protein that had been shown to interact structurally and functionally at the cytoplasmic domain (amino acid residues 1–47) with the ryanodine receptor (RyR1), and to undergo phosphorylation by endogenous calmodulin protein kinase (CaM K II) in isolated terminal cisternae from rabbit fast-twitch muscle. Here we show that triadin cytoplasmic domain expressed as glutathione-S-transferase fusion protein, is a substrate of the protein kinase. This finding is corroborated by identification of a specific consensus sequence in the deduced amino sequence between residue 34 and 37 of triadin. Confirming the regulatory features of CaM K II, we show the phosphorylation of triadin cytoplasmic segment by the kinase, when converted to the autonomous form. We propose that triadin modulates RyR1 in a phosphorylation-dependent manner.