Role of nitric oxide and nitric oxide synthases in experimental models of denervation and reinnervation

Nitric oxide (NO) is a short-living free molecule synthesized by three different isoforms of nitric oxide synthases (NOS)—neuronal NOS, endothelial NOS, and inducible NOS—associated with neuromuscular transmission, muscle contractility, mitochondrial respiration, and carbohydrate metabolism in skeletal muscle. Neuronal NOS is constitutively expressed at the muscle fiber sarcolemma linked to the dystrophin-glycoprotein complex and concentrated at the neuromuscular endplate. There is increasing evidence that altered expression of neuronal NOS plays a role in muscle fiber damage in neuromuscular diseases such as dystrophinopathies and denervating disorders. Although there have been some previous conflicting results on the neuronal NOS expression pattern in denervated muscle fibers, it is now well established that denervation is associated with a down-regulation and disappearance of sarcolemmal neuronal NOS at synaptic/extrasynaptic or both sites. As NO has been shown to induce collapse and growth arrest on neuronal growth cones, down-regulation of sarcolemmal neuronal NOS may contribute to axonal regeneration and attraction to muscle fibers aiming at the formation of new motor endplates providing reinnervation and reconstitution of NOS expression. As NO serves as a retrograde messenger, it may trigger structural downstream events responsible for neuromuscular synaptogenesis and preventing polyneural innervation. Nevertheless, decreased NO production in denervation reduces the cytoprotective scavenger function of NO for superoxide anions promoting oxidative stress that is likely to be involved in muscle fiber damage and death. However, the multifaced role of NOS and NO under physiological and pathological conditions remains poorly understood on the basis of the current knowledge. Microsc. Res. Tech. 55:181–186, 2001. © 2001 Wiley-Liss, Inc.     

Role of nitric oxide in the pathogenesis of muscular dystrophies: a "two hit" hypothesis of the cause of muscle necrosis.

Although the genetic and biochemical bases of many of the muscular dystrophies have been elucidated, the pathophysiological mechanisms leading to muscle cell death and degeneration remain elusive. Among the most well studied of the dystrophies are those due to defects in proteins that make up the dystrophin-glycoprotein complex (DGC). There has been much interest in the role of nitric oxide (NO(*)) in the pathogenesis of these diseases because the enzyme that synthesizes NO(*), nitric oxide synthase (NOS), is associated with the DGC. Recent studies of dystrophies related to DGC defects suggest that one mechanism of cellular injury is functional ischemia related to alterations in cellular NOS and disruption of a normal protective action of NO(*). This protective action is the prevention of local ischemia during contraction-induced increases in sympathetic vasoconstriction. However, the loss of this protection, alone, does not explain the subsequent muscle cell death and degeneration since mice lacking neuronal NOS (the predominant isoform expressed in muscle) do not develop a muscular dystrophy. Thus, there must be additional biochemical changes conferred upon the cells by these DGC defects, and these changes are discussed in terms of a proposed "two hit" hypothesis of the pathogenetic mechanisms that underlie the muscular dystrophies. According to this hypothesis, pathogenic defects in the DGC have at least two biochemical consequences: a reduction in NO(*)-mediated protection against ischemia, and an increase in cellular susceptibility to metabolic stress. Either one alone may be insufficient to lead to muscle cell death. However, in combination, the biochemical consequences are sufficient to cause muscle degeneration. The role of oxidative stress as a final common pathophysiologic pathway is discussed in terms of data showing that oxidative injury precedes pathologic changes and that muscle cells with defects in the DGC have an increased susceptibility to oxidant challenges. Accordingly, this "two hit" hypothesis may explain many of the complex spatial and temporal variations in disease expression that characterize the muscular dystrophies, such as grouped necrosis, a pre-necrotic phase of the disease, and selective muscle involvement.     

Distribution of nitric oxide synthase immunoreactivity in the mouse brain

Nitric oxide (NO) is a gaseous intercellular messenger with a wide range of neural functions. NO is synthesized by activation of different isoforms of nitric oxide synthases (NOS). At present NOS immunoreactivity has been described in mouse brain in restricted and definite areas and no detailed mapping studies have yet been reported for NOS immunoreactivity. We have studied the distribution of neuronal NOS-containing neurons in the brain of three months male mice, using a specific commercial polyclonal antibody against the neuronal isoform of nitric oxide synthase (nNOS). Neuronal cell bodies exhibiting nNOS immunoreactivity were found in several distinct nuclei throughout the brain. The neurons that were positively stained exhibited different intensities of reaction. In some brain areas (i.e., cortex, striatum, tegmental nuclei) neurons were intensely stained in a Golgi-like fashion. In other regions, immunoreactive cells are moderately stained (i.e., magnocellular nucleus of the posterior commissure, amygdaloid nucleus, interpeduncular nucleus, lateral periaqueductal gray) or weakly stained (i.e., vascular organ of the lamina terminalis, hippocampus, inferior colliculus, reticular nucleus). In the mouse, the NO-producing system appears well developed and widely diffused. In particular, nNOS immunoreactive neurons seem chiefly present in several sensory pathways like all the nuclei of the olfactory system, as well as in many regions of the lymbic system. These data suggest a widespread role for the NO system in the mouse nervous system.     

Nitric oxide: relation to integrity, injury, and healing of the gastric mucosa

Nitric oxide (NO) plays a multifaceted role in mucosal integrity. The numerous functions of NO and the double-edged role played by NO in most of them provide a great complexity to the NO action. The three enzymatic sources of NO, neuronal NO-synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), have been characterised in the gastrointestinal tract. The protective properties of the NO derived from constitutive NO-synthases (eNOS and nNOS) have already been well established. Less clear is the role assigned to iNOS. The simplistic initial view of low levels of NO synthesised by constitutive NOS being protective while exaggerated NO levels after iNOS induction leading irremediably to cytotoxicity is being questioned by new evidence. As initially reported for constitutive NOS, iNOS activity may be associated to reduced leukocyte-endothelium interaction and platelet aggregation as well as protection of mucosal microcirculation. Moreover, iNOS activity may be important to resolve inflammation by increasing apoptosis in inflammatory cells. It is entirely possible that a low level of expression of iNOS will reflect a positive host-defense response to challenge, but that exaggerated or uncontrolled expression of iNOS itself becomes detrimental. There is no doubt about the protective role of NO in physiological conditions. However, when the mucosa is threatened, the role of NO becomes multiple and the final effect will probably depend on the nature of the insult, the environment involved, and the interaction with other mediators.     

Expression of Leu-19 (CD56, N-CAM) and nitric oxide synthase (NOS) I in denervated and reinnervated human skeletal muscle.

Neural cell adhesion molecule (N-CAM, Leu-19, CD 56) expression appears during muscle fiber regeneration and after denervation. Sarcolemma-associated nitric oxide synthase (NOS) I, however, disappears from denervated myofibers. The dynamics of expression of both proteins were studied in 5 cases of acute/subacute denervation, 28 cases of chronic denervation with and without collateral reinnervation, 5 cases of the intermediate type spinal muscular atrophy (SMA 2), and in 2 normal biopsies. NOS I and its NADPH diaphorase (NADPHd) activity disappeared from the sarcolemma region shortly after denervation, and before the appearance of denervation atrophy. N-CAM was found diffusely distributed in the sarcoplasm at the most severe phase of denervation atrophy in the majority of highly atrophic fibers. During reinnervation, NOS I expression remained absent and in part of the cases the target/targetoid phenomenon appeared. In parallel with the increase in volume of the reinnervated muscle fibers, the intensity of N-CAM immunoreactivity decreased progressively. After full restitution of muscle fiber caliber, the target/targetoid phenomenon and N-CAM immunostaining disappeared completely, and, finally, NOS I reappeared in the sarcolemma region. The sarcolemmal expression of dystrophin and dystrophin-associated proteins was unchanged during denervation. NOS I was completely absent in children with SMA 2, since the protein does not appear before 5 years of age in skeletal muscle, while N-CAM was very intensely expressed in the sarcoplasm of highly atrophic denervated muscle fibers. In conclusion, this study suggests that innervation is an important factor for selective gene expression and positioning of NOS I and N-CAM in skeletal muscle and gives practical information for the assessment of the phase and developmental stage of the denervation and reinnervation process.     

Nitric oxide synthase expression and function in embryonic and adult cardiomyocytes.

Nitric oxide (NO) is an important signalling molecule that plays a relevant role in different cell systems, among them the adult heart. The effects of NO are primarily mediated through modulation of Ca(2+) homeostasis, myofibrillar contractility, and metabolic regulation in cardiomyocytes. Recent evidence also suggests an important role of NO for cardiomyogenesis by modulating proliferation and differentiation and regulating cardiac function. In the embryonic, but also the healthy and diseased, adult mammalian heart, the inducible (iNOS) and the endothelial (eNOS) nitric oxide synthases (NOS) are detected. However, the expression pattern of NO and its function differ during development. Furthermore, under pathophysiological conditions NOS expression can also change and cause impairment of cardiac performance and cytotoxic effects. The present review focuses on the role and function of NO during cardiomyogenesis, the mechanisms responsible for eNOS availability, and the paracrine effects of NO generated by cardiomyocytes.     

NADPH-diaphorase activity in the superficial layers of the superior colliculus of rats during aging

Neurons in the superficial layers of the superior colliculus are key elements in the visual system of rodents since they receive extensive afferent projections from retinal ganglion cells. The NADPH-diaphorase histochemical technique was used to detect differences in neuronal nitric oxide synthase (nNOS) in the superficial layers of the superior colliculus (sSC) of young adult (3 months) and aged (24 and 26 months) rats. The orientation of the dendritic processes of NADPH-diaphorase-positive neurons, cross-sectional area, and number of neurons per mm2 were analyzed. NADPH-d histochemistry revealed a high number of NADPH-d-positive cells in the stratum zonale and stratum griseum superficiale in adult and aged animals. NADPH-d-positive neurons were classified into the following morphological types: marginal, horizontal, pyriform, narrow-field vertical, wide-field vertical, and stellate. During aging, narrow field vertical and wide field vertical neurons present somatic atrophy and an increase in dendritic processes with dorsoventral orientation, whereas wide field vertical neurons show a decrease in those with lateromedial orientation. Marginal neurons undergo somatic hypertrophy at 26 months when compared with those at 3 months. The remaining types of neurons do not undergo size changes. Finally, the number of NADPH-d-positive neurons per mm2 in the various types of morphology does not significantly change with age. It is suggested to be likely that the aging process in the nitrergic neurons of the sSC does not lead to significant changes in the synthesis of NO from the constitutive NOS isoforms.     

Nitric oxide metabolism in heart mitochondria

Normal cardiac function is accomplished through a continuous energy supply provided by mitochondria. Heart mitochondria are the major source of reactive oxygen and nitrogen species: superoxide anion (O₂-) and nitric oxide (NO). NO production by mitochondrial NOS (mtNOS) is modified by metabolic state and shows an exponential dependence on Δψ. The interaction between mtNOS and complexes I and IV might be a mechanism involved in the regulation of mitochondrial NO production. NO exerts a high affinity, reversible and physiological inhibition of cytochrome c oxidase activity. A second effect of NO on the respiratory chain is accomplished through its interaction with ubiquinol-cytochrome c oxidoreductase. The ability of mtNOS to regulate mitochondrial O₂ uptake and O₂- and H₂O₂ productions through the interaction of NO with the respiratory chain is named mtNOS functional activity. Together, heart mtNOS allows NO to optimize the balance between cardiac energy production and utilization, and to regulate the steady-state concentrations of other oxygen and nitrogen species.     

Nitric oxide averts hypoxia-induced damage during reoxygenation in rat heart

Nitric oxide (NO), synthesized by the hemoproteins NO synthases (NOS), is known to play important roles in physiological and pathological conditions in the heart, including hypoxia/reoxygenation (H/R). This work investigates the role that endogenous NO plays in the cardiac H/R-induced injury. A follow-up study was conducted in Wistar rats subjected to 30 min of hypoxia, with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analysing parameters of cell, and tissue damage (lipid peroxidation, apoptosis, and protein nitration), as well as in situ NOS activity and NO production (NOx). The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated in all the experimental groups, and consequently, NOx levels fell. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. These results reveal that NOS inhibition exacerbates the peroxidative and apoptotic damage observed before the treatment with L-NAME in the hypoxic heart, pointing to a cardioprotective role of NOS-derived NO against H/R-induced injury. These findings could open the possibility of future studies to design new therapies for H/R-dysfunctions based on NO-pharmacology.     

Melatonin behavior in restoring chemical damaged C2C12 myoblasts

It is known that, besides a wide range of functions, melatonin provides protection against oxidative stress, thanks to its ability to act, directly, as a free radical scavenger and, indirectly, by stimulating antioxidant enzymes production and mitochondrial electron transport chain efficiency. Oxidative stress is one of the major players in initiating apoptotic cell death in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in several myopathies, in sarcopenia, as well as in denervation and disuse. Melatonin activity was investigated in undifferentiated C2C12 skeletal muscle cells, after exposure to various apoptotic chemical triggers, chosen for their different mechanisms of action. Cells were pretreated with melatonin and then exposed to hydrogen peroxide_, etoposide and staurosporine. Morphofunctional and molecular analyses show that in myoblasts melatonin prevents oxidative stress and apoptosis induced by chemicals following, at least in part, the mitochondria pathway. These results confirm melatonin ability to act as an antioxidant and antiapoptotic molecule in skeletal muscle cells, thus suggesting a possible therapeutic strategy for myopathies involving apoptosis misregulation. Microsc. Res. Tech. 79:532–540, 2016. © 2016 Wiley Periodicals, Inc.     

Association of neuronal nitric oxide synthase (nNOS) with alpha1-syntrophin at the sarcolemma.

alpha1-syntrophin is a PDZ-containing dystrophin-associated protein, expressed predominantly in striated muscle and brain. alpha1-syntrophin null mice generated by gene targeting technique showed no overt muscular dystrophic phenotype. Though other dystrophin-associated proteins were localized at the sarcolemma, neuronal nitric oxide synthase (nNOS) was selectively lost from the membrane fraction but remained in the cytoplasm. Thus, the alpha1-syntrophin null mice are useful in the elucidation of the functional importance of nNOS targeting at the sarcolemma. In addition, the mice would facilitate identification of other signaling molecules, which are targeted to dystrophin complex via interaction with alpha1-syntrophin.     

Nitric oxide regulates mitochondrial re‐modelling in interscapular brown adipose tissue: ultrastructural and morphometric‐stereologic studies

As a complex, cell‐specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of l ‐arginine‐nitric oxide (NO)‐producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving l ‐arginine, a substrate of NO synthases (NOSs), or N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub‐groups – acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. l ‐Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold‐induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in l ‐arginine‐treated rats. Ultrastructural examinations of l ‐arginine‐treated cold‐acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well‐ordered cristae in their profiles), which however were inactive in l ‐arginine‐receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, l ‐NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.     

Detection of reactive oxygen and reactive nitrogen species in skeletal muscle.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are usually identified with pathological states and mediators of cellular injury. However, over the last decade ROS and RNS have been identified in skeletal muscle under physiological conditions. Detection of ROS and RNS production by skeletal muscle cells is fundamental to the problem of differentiating between physiological and pathological levels. The goal of this paper is to review the techniques that have been used to detect ROS and RNS in skeletal muscle. Electron spin resonance, fluorescent assays, cyotchrome c reduction, chemiluminescence, hydroxylation of salicylate, and nitration of phenylalanine are some of the assay systems that have been used thus far. A large body of evidence now indicates that ROS and RNS are continually produced by many different skeletal muscle types studied in vivo, in situ, and in vitro. Under resting conditions, ROS and RNS are detectable in both intracellular and extracellular compartments. Production increases during both non-fatiguing and fatiguing muscle contractions. In the absence of disease, the individual molecular species detected in skeletal muscle include parent radicals for the ROS and RNS cascades: superoxide anions and nitric oxide. Both are generated at rates estimated to range from pmol-to-nmol/mg muscle/minute. Evidence indicates that hydrogen peroxide, hydroxyl radicals, and peroxynitrite are also present under physiological conditions. However, the molecular species that mediate specific biological effects remains largely undetermined, as do the sources of ROS and RNS within muscle fibers. Eventual delineation of the mechanisms whereby ROS and RNS regulate cellular function will hinge on our understanding of the production and distribution of ROS and RNS within skeletal muscle.     

Heart mitochondrial dysfunction in diabetic rats

Diabetic cardiomyopathy, i.e. the ventricular dysfunction in the absence of hypertension or coronary arterial disease, is a common complication of diabetes mellitus that leads to a heightened risk of heart failure and death among diabetic patients. This contractile dysfunction could be associated to mitochondrial dysfunction, in which mitochondrial biogenesis could emerge as a compensatory mechanism triggered in response to hyperglycemia. It has been proposed that nitric oxide synthase activities with enhanced NO production are involved in this process. Alterations in the contractile response and lusitropic reserve were observed in streptozotocin diabetic rats after β-adrenergic stimuli. Additionally, tissue O₂ consumption was declined. A condition of mitochondrial dysfunction with decreased mitochondrial state 3 O₂ consumption, respiratory control ratio, mitochondrial respiratory complexes activities and ATP production were present in hearts of diabetic animals. We observed an increase in NO production by heart mitochondria and in cytochrome oxidase activity in heart homogenates. The latter suggests an increase of newly formed mitochondria. Thus, the impairment of mitochondrial function with increased mitochondrial biogenesis may precede the onset of diabetic cardiomyopathy. However, mitochondrial biogenesis does not necessarily imply that the resultant mitochondria are functional, which might explain the changes in cardiac energy metabolism occurring in hearts of diabetic rats.     

Localization of sarcoglycan, neuronal nitric oxide synthase, beta-dystroglycan, and dystrophin molecules in normal skeletal myofiber: triple immunogold labeling electron microscopy.

In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), beta-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, beta-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of alpha-, beta-, gamma- and delta-sarcoglycans is located intracellularly or extracellularly near the muscle plasma membrane mostly in accordance with the sarcoglycan antigenic sites against which the antibodies were generated. The association of different two and/or three sarcoglycan molecules out of alpha-, beta-, gamma- and delta-sarcoglycan molecules was frequently observed. Each molecule of nNOS, beta-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with beta-dystroglycan and dystrophin, and the association of nNOS with dystrophin were also confirmed ultrastructurally. Thus, this study demonstrated that the constituting molecules of the sarcoglycan complex, nNOS, beta-dystroglycan, and dystrophin existed in the form of a cluster at the normal muscle plasma membrane. The association of nNOS with dystrophin and its associated glycoproteins may form a macromolecular signaling complex at the muscle plasma membrane.     

Origin and Co-localization of nitric oxide synthase, CGRP, PACAP, and VIP in the cerebral circulation of the rat

The origin of perivascular nerve fibres storing nitric oxide synthase (NOS) and co-localisation with perivascular neuropeptides were examined in the rat middle cerebral artery (MCA) by retrograde tracing with True Blue (TB) in combination with immunocytochemistry. Application of TB to the proximal part of the middle cerebral artery labelled nerve cell bodies ipsilaterally in the trigeminal, sphenopalatine, otic, and superior cervical ganglia. A few labelled cell bodies were seen contralaterally, suggesting bilateral innervation. In the parasympathetic sphenopalatine and otic ganglia, numerous TB-labelled cell bodies contained neuronal NOS (C- and N-terminal), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase activating peptide (PACAP). In the trigeminal ganglion, almost all TB-labelled cell bodies contained calcitonin gene-related peptide (CGRP) but only a few cells contained NOS. In the superior cervical ganglion, the majority of the TB-labelled nerve cells contained neuropeptide Y (NPY) but none of them contained NOS. Removal of the ipsilateral sphenopalatine ganglion caused a slight reduction in the number of perivascular VIP-, PACAP-, and NOS-containing fibres after 3 days in the MCA while there was no difference at 2 and 4 weeks after the denervation as compared to control. This indicates that the parasympathetic VIP-, PACAP-, and NOS-immunoreactive nerve fibres in the rat MCA originate from several sources.     

Distribution of NADPH-diaphorase in rat mesencephalon: A light and electron microscopical study

NADPH-diaphorase is a useful technique to reveal NO producing neurons at light microscopic level (LM). A modification of the technique using the tetrazolium salt BSPT as susbtrate, is useful to study the ultrastructure of NO neurons. The aim of this work was to perform a detailed analysis of NADPH diaphorase reactive neurons in rat mesencephalon both at light and electron microscopic levels.
NADPH-diaphorase reactive neurons were observed in superior colliculus, in central gray matter, in dorsal and medial raphe and in the pedunculopontine tegmental nucleus using two histochemical techniques at LM. Electron microscopy showed deposits on membranes of the endoplasmic reticulum, Golgi apparatus and nuclear envelope of dorsal raphe neurons. Presynaptic and postsynaptic terminals showed deposits on membranous elements but postsynaptic terminals also showed deposits on the inner surface of their membranes.
Further physiological studies are needed to clarify the meaning of the ultrastructural findings such as the putative interaction of NOS with postsynaptic proteins, receptors or membranous channels.     

Coordinated expression of myosin heavy chains, metabolic enzymes, and morphological features of porcine skeletal muscle fiber types

Combined methodologies of electrophoresis, immunoblots, immunohistochemistry, histochemistry, and photometric image analysis were applied to characterize porcine skeletal muscle fibers according to their myosin heavy chain (MyHC) composition, and to determine on a fiber-to-fiber basis the correlation between contractile [MyHC (s), myofibrillar ATPase (mATPase), and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [succinate dehydrogenase (SDH), and glycerol-3-phosphate dehydrogenase (GPDH) activities, glycogen, and phospholamban (PLB) contents], and morphological [cross-sectional area (CSA), capillary, and nuclear densities] features of individual myofibers. An accurate delineation of MyHC-based fiber types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibers with overwhelming dominance of one MyHC isoform. The phenotypic differences in contractile, metabolic, and morphological properties seen between fiber types were related with MyHC content. Slow fibers had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPDH activity (glycolytic metabolism), and glycogen content, the smallest CSA, the greatest capillary, and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IIB fibers, whereas type IIA and IIX fibers had intermediate properties. Hybrid fibers had mean values intermediate in-between their respective pure phenotypes. Discrimination of myofibers according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafiber interrelationships suggest that myofibers of control pigs exhibit a high degree of co-ordination in their physiological, biochemical, and anatomical features. This study may well be a useful baseline for future work on the pig meat industry and also offers new prospects for muscle fiber typing in porcine experimental studies.     

Melatonin prevents mitochondrial dysfunctions and death in differentiated skeletal muscle cells

Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho‐functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders.