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1.  Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice 
Skeletal Muscle  2015;5:10.
Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans.
We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.
About 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40 ± 1 vs. 86 ± 1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46 ± 3 to 59 ± 2 mN/gr) in CASQ1-null mice.
Our findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-015-0035-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4464246  PMID: 26075051
Excitation-contraction (EC) coupling; Mitochondria; Ryanodine receptor (RYR)
2.  Persistent muscle fiber regeneration in long term denervation. Past, present, future 
Despite the ravages of long term denervation there is structural and ultrastructural evidence for survival of muscle fibers in mammals, with some fibers surviving at least ten months in rodents and 3–6 years in humans. Further, in rodents there is evidence that muscle fibers may regenerate even after repeated damage in the absence of the nerve, and that this potential is maintained for several months after denervation. While in animal models permanently denervated muscle sooner or later loses the ability to contract, the muscles may maintain their size and ability to function if electrically stimulated soon after denervation. Whether in mammals, humans included, this is a result of persistent de novo formation of muscle fibers is an open issue we would like to explore in this review. During the past decade, we have studied muscle biopsies from the quadriceps muscle of Spinal Cord Injury (SCI) patients suffering with Conus and Cauda Equina syndrome, a condition that fully and irreversibly disconnects skeletal muscle fibers from their damaged innervating motor neurons. We have demonstrated that human denervated muscle fibers survive years of denervation and can be rescued from severe atrophy by home-based Functional Electrical Stimulation (h-bFES). Using immunohistochemistry with both non-stimulated and the h-bFES stimulated human muscle biopsies, we have observed the persistent presence of muscle fibers which are positive to labeling by an antibody which specifically recognizes the embryonic myosin heavy chain (MHCemb). Relative to the total number of fibers present, only a small percentage of these MHCemb positive fibers are detected, suggesting that they are regenerating muscle fibers and not pre-existing myofibers re-expressing embryonic isoforms. Although embryonic isoforms of acetylcholine receptors are known to be re-expressed and to spread from the end-plate to the sarcolemma of muscle fibers in early phases of muscle denervation, we suggest that the MHCemb positive muscle fibers we observe result from the activation, proliferation and fusion of satellite cells, the myogenic precursors present under the basal lamina of the muscle fibers. Using morphological features and molecular biomarkers, we show that severely atrophic muscle fibers, with a peculiar cluster reorganization of myonuclei, are present in rodent muscle seven-months after neurectomy and in human muscles 30-months after complete Conus-Cauda Equina Syndrome and that these are structurally distinct from early myotubes. Beyond reviewing evidence from rodent and human studies, we add some ultrastructural evidence of muscle fiber regeneration in long-term denervated human muscles and discuss the options to substantially increase the regenerative potential of severely denervated human muscles not having been treated with h-bFES. Some of the mandatory procedures, are ready to be translated from animal experiments to clinical studies to meet the needs of persons with long-term irreversible muscle denervation. An European Project, the trial Rise4EU (Rise for You, a personalized treatment for recovery of function of denervated muscle in long-term stable SCI) will hopefully follow
PMCID: PMC4383182  PMID: 25844146
Human muscle; Conus-Cauda Equina syndrome; Spinal cord injury; Permanent muscle denervation; Severe atrophy and nuclear clumpings; Muscle fiber regeneration; Home-based functional electrical stimulation (h-b FES); Recovery of tetanic contractility; Myogenic stem cells
3.  Risk Assessment of BRONJ in Oncologic Patients Treated with Bisphosphonates: Follow-Up to 18 Months 
Objectives. Bisphosphonates related osteonecrosis of the jaw (BRONJ) is a pathological condition characterized by bone exposure or latent infection in patients treated with the drug. The aim of the study is to monitor the BRONJ level of risk health in patients with cancer, according to a preventive clinical protocol, which is firstly aimed at reducing risk factors such as the periodontal infections. Materials and Methods. 10 patients participated in the protocol and were evaluated at baseline and after 3 and 18 months of treatment with bisphosphonates, through full mouth plaque and bleeding scores (FMPS and FMBS), clinical attachment level (CAL) measurement, and the occurrence of osteonecrosis. Results. The mean plaque and bleeding were reduced and the CAL has not shown significant changes and in no cases was there manifestation of BRONJ. Conclusion. The protocol proved crucial for the maintenance of good oral health conditions by eliminating the risk of BRONJ during the observation period.
PMCID: PMC4167445  PMID: 25258628
4.  Muscle Research and Gene Ontology: New standards for improved data integration 
The Gene Ontology Project provides structured controlled vocabularies for molecular biology that can be used for the functional annotation of genes and gene products. In a collaboration between the Gene Ontology (GO) Consortium and the muscle biology community, we have made large-scale additions to the GO biological process and cellular component ontologies. The main focus of this ontology development work concerns skeletal muscle, with specific consideration given to the processes of muscle contraction, plasticity, development, and regeneration, and to the sarcomere and membrane-delimited compartments. Our aims were to update the existing structure to reflect current knowledge, and to resolve, in an accommodating manner, the ambiguity in the language used by the community.
The updated muscle terminologies have been incorporated into the GO. There are now 159 new terms covering critical research areas, and 57 existing terms have been improved and reorganized to follow their usage in muscle literature.
The revised GO structure should improve the interpretation of data from high-throughput (e.g. microarray and proteomic) experiments in the area of muscle science and muscle disease. We actively encourage community feedback on, and gene product annotation with these new terms. Please visit the Muscle Community Annotation Wiki .
PMCID: PMC2657163  PMID: 19178689
5.  Luminal Ca2+ Regulation of Single Cardiac Ryanodine Receptors: Insights Provided by Calsequestrin and its Mutants 
The Journal of General Physiology  2008;131(4):325-334.
The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)–linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 μM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below ∼0.5 mM.
PMCID: PMC2279168  PMID: 18347081

Results 1-5 (5)