Biomedical subjects

Carlo Reggiani

Publications and source records attributed to Carlo Reggiani.

At least 19 recordsLinked to original sources

PubMed2006

The sarcomeric myosin heavy chain gene family in the dog: analysis of isoform diversity and comparison with other mammalian species.

Sarcomeric myosin heavy chains (MyHC) are the major contractile proteins of cardiac and skeletal muscles and belong to class II MyHC. In this study the sequences of nine sarcomeric MyHC isoforms were obtained by combining assembled contigs of the dog genome draft available in the NCBI database. With this information available the dog becomes the second species, after human, for which the sequences of all members of the sarcomeric MyHC gene family are identified. The newly determined sequences of canine MyHC isoforms were aligned with their orthologs in mammals, forming a set of 38 isoforms, to search for the molecular features that determine the structural and functional specificity of each type of isoform. In this way the structural motifs that allow identification of each isoform and are likely determinants of functional properties were identified in six specific regions (surface loop 1, loop 2, loop 3, converter, MLC binding region, and S2 proximal segment).

Amino Acid Sequence↗

PubMed2006

RyR isoforms and fibre type-specific expression of proteins controlling intracellular calcium concentration in skeletal muscles.

Muscle fibres which shorten with high maximum shortening velocity also exhibit fast kinetics of contraction, i.e. short values of time to peak tension and time to half relaxation. This short review aims to discuss the molecular basis of such correlation, to reach, based on the available literature, an answer to the question whether there is a correlation in expression of proteins determining shortening velocity, myosin isoforms in the first place, and proteins controlling cytosolic calcium concentration and its variations at rest or during contraction. Although the isoforms of RyR, the sarcoplasmic calcium release channels, do not show a tightly coordinated expression with myosin isoforms, other proteins involved in controlling intracellular calcium do. This is likely sufficient to guarantee the correlation between maximum shortening velocity and speed of isometric contraction.

Animals↗

PubMed2006

Effect of swimming on myostatin expression in white and red gastrocnemius muscle and in cardiac muscle of rats.

The aim of this study was to test the hypothesis that swimming training might impact differentially myostatin expression in skeletal muscles, depending on fibre type composition, and in cardiac muscle of rats. Myostatin expression was analysed by real time reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry of the red deep portion (mainly composed of slow and type II A fibres) and in the superficial, white portion (composed of fast type II X and II B fibres) of the gastrocnemius muscle in adult male Wistar rats: (i) subjected to two consecutive swimming bouts for 3 h; (ii) subjected to intensive swimming training for 4 weeks; and (iii) sedentary control rats. Myostatin mRNA content was in all cases higher in white than in red muscles. Two bouts of swimming did not alter myostatin expression, whereas swimming training for 4 weeks resulted in a significant reduction of myostatin mRNA contents, significant both in white and red muscles but more pronounced in white muscles. Western blot did not detect any change in the amount of myostatin protein. Immunohistochemistry showed that, in control rats, myostatin was localized in presumptive satellite cells of a few muscle fibres. After training, the number of myostatin-positive spots decreased significantly. Myostatin mRNA content in cardiac muscle was lower than in skeletal muscle and was significantly increased by swimming training. In conclusion, the results obtained showed that intense training caused a decreased expression of myostatin mRNA in white and red skeletal muscles but an increase in cardiac muscle.

Animals↗

PubMed2006

NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle.

Calcineurin-NFAT signaling has been shown to control activity-dependent muscle gene regulation and induce a program of gene expression typical of slow oxidative muscle fibers. Following Ca2+-calmodulin stimulation, calcineurin dephosphorylates NFAT proteins and induces their translocation into the nucleus. However, NFAT nuclear translocation has never been investigated in skeletal muscle in vivo. To determine whether NFATc1 nucleocytoplasmic shuttling depends on muscle activity, we transfected fast and slow mouse muscles with plasmids coding for an NFATc1-GFP fusion protein. We found that NFATc1-GFP has a predominantly cytoplasmic localization in the fast tibialis anterior muscle but a predominantly nuclear localization in the slow soleus muscle, with a characteristic focal intranuclear distribution. Two hours of complete inactivity, induced by denervation or anaesthesia, cause NFATc1 export out of the nucleus in soleus muscle fibers, whereas electrostimulation of tibialis anterior with a low-frequency tonic impulse pattern, mimicking the firing pattern of slow motor neurons, causes NFATc1 nuclear translocation. The activity-dependent nuclear import and export of NFATc1 is a rapid event, as visualized directly in vivo by two-photon microscopy. The calcineurin inhibitor cain/cabin1 causes nuclear export of NFATc1 both in normal soleus and stimulated tibialis anterior muscle. These findings support the notion that in skeletal muscle NFATc1 is a calcineurin-dependent nerve activity sensor.

Active Transport, Cell Nucleus↗

PubMed2005

Denervation in murine fast-twitch muscle: short-term physiological changes and temporal expression profiling.

Denervation deeply affects muscle structure and function, the alterations being different in slow and fast muscles. Because the effects of denervation on fast muscles are still controversial, and high-throughput studies on gene expression in denervated muscles are lacking, we studied gene expression during atrophy progression following denervation in mouse tibialis anterior (TA). The sciatic nerve was cut close to trochanter in adult CD1 mice. One, three, seven, and fourteen days after denervation, animals were killed and TA muscles were dissected out and utilized for physiological experiments and gene expression studies. Target cDNAs from TA muscles were hybridized on a dedicated cDNA microarray of muscle genes. Seventy-one genes were found differentially expressed. Microarray results were validated, and the expression of relevant genes not probed on our array was monitored by real-time quantitative PCR (RQ-PCR). Nuclear- and mitochondrial-encoded genes implicated in energy metabolism were consistently downregulated. Among genes implicated in muscle contraction (myofibrillar and sarcoplasmic reticulum), genes typical of fast fibers were downregulated, whereas those typical of slow fibers were upregulated. Electrophoresis and Western blot showed less pronounced changes in myofibrillar protein expression, partially confirming changes in gene expression. Isometric tension of skinned fibers was little affected by denervation, whereas calcium sensitivity decreased. Functional studies in mouse extensor digitorum longus muscle showed prolongation in twitch time parameters and shift to the left in force-frequency curves after denervation. We conclude that, if studied at the mRNA level, fast muscles appear not less responsive than slow muscles to the interruption of neural stimulation.

Animals↗

PubMed2005

Selective expression of the type 3 isoform of ryanodine receptor Ca2+ release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits.

The expression pattern of the RyR3 isoform of Ca2+ release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform.

Animals↗

PubMed2005

Post-transcriptional silencing and functional characterization of the Drosophila melanogaster homolog of human Surf1.

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C-GAL4 or elav-GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C-GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav-GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C-GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav-GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.

Actins↗

PubMed2005

Drosophila CAKI/CMG protein, a homolog of human CASK, is essential for regulation of neurotransmitter vesicle release.

Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, we employed a caki null mutant in the model organism Drosophila melanogaster. By means of electrophysiological methods, we analyzed, in adult flies, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system. We found that in caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ was increased, the response of the giant fiber pathway to continuous stimulation was impaired, and electroretinographic responses to single and continuous repetitive stimuli were altered and optomotor behavior was abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function.

Animals↗

PubMed2005

Imaging and elasticity measurements of the sarcolemma of fully differentiated skeletal muscle fibres.

This study aimed to describe the three-dimensional structure and the elastic properties of the sarcolemma of adult, fully differentiated, skeletal muscle fibres combining Atomic Force Microscopy (AFM) and optical microscopy. Single fibres were enzymatically dissociated from Flexor Digitorum Brevis of adult mice and were maintained in culture up to 3 weeks. On the sixth day after dissociation, the upper surface of intact fibres, either alive in solution or fixed and kept in solution or fixed and exposed in air, was analysed with AFM. The most prominent features in AFM images were periodic transversal foldings with an interval that corresponded to the sarcomere length. More detailed analysis of the topography profile showed that the depth in the folding decreased with increasing sarcomere length and that the crests of the foldings corresponded to the Z-lines. Minor periodic structures could be detected in the valleys between the major foldings. AFM images also showed deep depressions on the sarcolemma likely corresponding to openings of T tubules and caveolae. Two-dimensional elasticity maps were obtained using AFM as an indenter and showed that the crests of the transversal foldings correspond to higher stiffness regions. This study provides the first complete three-dimensional topography and mechanical characterization of intact, living skeletal muscle fibres and might form the basis for further investigations aimed to compare healthy and dystrophic muscles.

Animals↗

PubMed2005

Nerve influence on myosin light chain phosphorylation in slow and fast skeletal muscles.

Neural stimulation controls the contractile properties of skeletal muscle fibres through transcriptional regulation of a number of proteins, including myosin isoforms. To study whether neural stimulation is also involved in the control of post-translational modifications of myosin, we analysed the phosphorylation of alkali myosin light chains (MLC1) and regulatory myosin light chains (MLC2) in rat slow (soleus) and fast (extensor digitorum longus EDL) muscles using 2D-gel electrophoresis and mass spectrometry. In control rats, soleus and EDL muscles differed in the proportion of the fast and slow isoforms of MLC1 and MLC2 that they contained, and also in the distribution of the variants with distinct isoelectric points identified on 2D gels. Denervation induced a slow-to-fast transition in myosin isoforms and increased MLC2 phosphorylation in soleus, whereas the opposite changes in myosin isoform expression and MLC2 phosphorylation were observed in EDL. Chronic low-frequency stimulation of EDL, with a pattern mimicking that of soleus, induced a fast-to-slow transition in myosin isoforms, accompanied by a decreased MLC2 phosphorylation. Chronic administration (10 mg x kg(-1) x d(-1) intraperitoneally) of cyclosporin A, a known inhibitor of calcineurin, did not change significantly the distribution of fast and slow MLC2 isoforms or the phosphorylation of MLC2. All changes in MLC2 phosphorylation were paralleled by changes in MLC kinase expression without any variation of the phosphatase subunit, PP1. No variation in MLC1 phosphorylation was detectable after denervation or cyclosporin A administration. These results suggest that the low-frequency neural discharge, typical of soleus, determines low levels of MLC2 phosphorylation together with expression of slow myosin, and that MLC2 phosphorylation is regulated by controlling MLC kinase expression through calcineurin-independent pathways.

Animals↗

PubMed2004

Muscle plasticity and high throughput gene expression studies.

Changes in gene expression are known to contribute to muscle plasticity. Until recently most studies have described differences of one or few genes at a time, in the last few years, however, the development of new technology of high throughput mRNA expression analysis has allowed the study of a large part if not all transcripts in the same experiment. Knowledge on any muscle adaptive response has already gained from the application of this novel approach, but the most important new findings have come from studies on muscle atrophy. A new and unexpected groups of genes, which increase their expression during atrophy and are, therefore, designated as atrogins, have been discovered. In spite of the impressive power of the new technology many problems are still to be resolved to optimize the experimental design and to extract all information which are provided by the outcome of the global mRNA assessment.

Animals↗

PubMed2004

Clenbuterol antagonizes glucocorticoid-induced atrophy and fibre type transformation in mice.

Beta-agonists and glucocorticoids are frequently coprescribed for chronic asthma treatment. In this study the effects of 4 week treatment with beta-agonist clenbuterol (CL) and glucocorticoid dexamethasone (DEX) on respiratory (diaphragm and parasternal) and limb (soleus and tibialis) muscles of the mouse were studied. Myosin heavy chain (MHC) distribution, fibres cross sectional area (CSA), glycolytic (phosphofructokinase, PFK; lactate dehydrogenase, LDH) and oxidative enzyme (citrate synthase, CS; cytochrome oxidase, COX) activities were determined. Muscle samples were obtained from four groups of adult C57/B16 mice: (1) Control (2) Mice receiving CL (CL, 1.5 mg kg(-1) day(-1) in drinking water) (3) Mice receiving DEX (DEX, 5.7 mg kg(-1) day(-1) s.c.) (4) Mice receiving both treatments (DEX + CL). As a general rule, CL and DEX showed opposite effects on CSA, MHC distribution, glycolytic and mitochondrial enzyme activities: CL alone stimulated a slow-to-fast transition of MHCs, an increase of PFK and LDH and an increase of muscle weight and fibre CSA; DEX produced an opposite (fast-to-slow transition) change of MHC distribution, a decrease of muscle weight and fibre CSA and in some case an increase of CS. The response varied from muscle to muscle with mixed muscles, as soleus and diaphragm, being more responsive than fast muscles, as tibialis and parasternal. In combined treatments (DEX + CL), the changes induced by DEX or CL alone were generally minimized: in soleus, however, the effects of CL predominated over those of DEX, whereas in diaphragm DEX prevailed over CL. Taken together the results suggest that CL might counteract the unwanted effects on skeletal muscles of chronic treatment with glucocorticoids.

Adrenergic beta-Agonists↗

PubMed2004

Fast fibres in a large animal: fibre types, contractile properties and myosin expression in pig skeletal muscles.

Little is known about the influence of Myosin Heavy Chain (MHC) isoforms on the contractile properties of single muscle fibres in large animals. We have studied MHC isoform composition and contractile properties of single muscle fibres from the pig. Masseter, diaphragm, longissimus, semitendinosus, rectractor bulbi and rectus lateralis were sampled in female pigs (aged 6 months, mass 160 kg). RT-PCR, histochemistry, immunohistochemistry and gel electrophoresis were combined to identify and separate four MHC isoforms: MHC-slow and three fast MHC (2A, 2X, 2B). Maximum shortening velocity (V(o)) and isometric tension (P(o)) were measured in single muscle fibres with known MHC isoform composition. Six groups of fibres (pure: slow, 2A, 2X and 2B, and hybrid: 2A-2X and 2X-2B) with large differences in V(o) and P(o) were identified. Slow fibres had mean V(o)=0.17+/-0.01 length s(-1) and P(o)=25.1+/-3.3 mN mm(-2). For fast fibres 2A, 2X and 2B, mean V(o) values were 1.86+/-0.18, 2.55+/-0.19 and 4.06+/-0.33 length s(-1) and mean P(o) values 74.93+/-8.36, 66.85+/-7.58 and 32.96+/-7.47 mN mm(-2), respectively. An in vitro motility assay confirmed that V(o) strictly reflected the functional properties of the myosin isoforms. We conclude that pig muscles express high proportions of fast MHC isoforms, including MHC-2B, and that V(o) values are higher than expected on the basis of the scaling relationship between contractile parameters and body size.

Analysis of Variance↗

PubMed2003

Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency.

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

Animals↗

PubMed2003

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms.

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca(2+) activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0-1 degrees C to the test temperature (approximately 12 degrees C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40-70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin-myosin interactions under stretch become independent of the MHC isoform.

Adult↗

PubMed2003

Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus.

In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 +/- 3.9% vs. 8.5 +/- 3.9%) and was separated into two spots, the less acidic spot being 73.5 +/- 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 +/- 5.6%, HS: 62.4 +/- 6.8%, CB-HS: 56.4 +/- 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9-5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 +/- 3.1%, HS: 23.9 +/- 3.3%, CB-HS: 25.3 +/- 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 +/- 6.7%, HS: 28.7 +/- 6.5%, CB-HS: 21.8 +/- 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops.

Adrenergic beta-Agonists↗

PubMed2002

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells.

Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) variation is a key event in myoblast differentiation, but the mechanism by which it occurs is still debated. Here we show that increases of extracellular Ca(2+) concentration ([Ca(2+)](o)) produced membrane hyperpolarization and a concentration-dependent increase of [Ca(2+)](i) due to Ca(2+) influx across the plasma membrane. Responses were not related to inositol phosphate turnover and Ca(2+)-sensing receptor. [Ca(2+)](o)-induced [Ca(2+)](i) increase was inhibited by Ca(2+) channel inhibitors and appeared to be modulated by several kinase activities. [Ca(2+)](i) increase was potentiated by depletion of intracellular Ca(2+) stores and depressed by inactivation of the Na(+)/Ca(2+) exchanger. The response to arginine vasopressin (AVP), which induces inositol 1,4,5-trisphosphate-dependent [Ca(2+)](i) increase in L6-C5 cells, was not modified by high [Ca(2+)](o). On the contrary, AVP potentiated the [Ca(2+)](i) increase in the presence of elevated [Ca(2+)](o). Other clones of the L6 line as well as the rhabdomyosarcoma RD cell line and the satellite cell-derived C2-C12 line expressed similar responses to high [Ca(2+)](o), and the amplitude of the responses was correlated with the myogenic potential of the cells.

Animals↗

PubMed2002

Bupivacaine myotoxicity is mediated by mitochondria.

We have investigated the effects of the myotoxic local anesthetic bupivacaine on rat skeletal muscle mitochondria and isolated myofibers from flexor digitorum brevis, extensor digitorum longus, soleus, and from the proximal, striated portion of the esophagus. In isolated mitochondria, bupivacaine caused a concentration-dependent mitochondrial depolarization and pyridine nucleotide oxidation, which were matched by an increased oxygen consumption at bupivacaine concentrations of 1.5 mm or less at pH 7.4, whereas respiration was inhibited at higher concentrations. As a consequence of depolarization, bupivacaine caused the opening of the permeability transition pore (PTP), a cyclosporin A-sensitive inner membrane channel that plays a key role in many forms of cell death. In intact flexor digitorum brevis fibers bupivacaine caused mitochondrial depolarization and pyridine nucleotides oxidation that were matched by increased concentrations of cytosolic free Ca(2+), release of cytochrome c, and eventually, hypercontracture. Both mitochondrial depolarization and cytochrome c release were inhibited by cyclosporin A, indicating that PTP opening rather than bupivacaine as such was responsible for these events. Similar responses to bupivacaine were observed in the soleus, which is highly oxidative. In contrast, fibers from the esophagus (which we show to be more fatigable than flexor digitorum brevis fibers) and from the highly glycolytic extensor digitorum longus didn't undergo pyridine nucleotide oxidation upon the addition of bupivacaine and were resistant to bupivacaine toxicity. These results suggest that active oxidative metabolism is a key determinant in bupivacaine toxicity, that bupivacaine myotoxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca(2+) dysregulation, and that it represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role.

Anesthetics, Local↗