Skeletal muscle weakness has been associated with different pathological conditions, including sa... more Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mammalian target of rapamycin (mTOR) signalling. We wanted to elucidate the functional role of mTOR in muscle contractility. Most loss‐of‐function studies for mTOR signalling have used the drug rapamycin to inhibit some of the signalling downstream of mTOR. However, given that rapamycin does not inhibit all mTOR signalling completely, we generated a double knockout for mTOR and for the scaffold protein of mTORC1, raptor, in skeletal muscle. We found that double knockout in mice results in a more severe phenotype compared with deletion of raptor or mTOR alone. Indeed, these animals display muscle weakness, increased fibre denervation and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow‐twitch fibres and changes in the expression levels of calcium‐related genes, such as Serca...
Polyglutamine (polyQ)-expanded androgen receptor (AR) causes spinobulbar muscular atrophy. Skelet... more Polyglutamine (polyQ)-expanded androgen receptor (AR) causes spinobulbar muscular atrophy. Skeletal muscle is a primary site of polyQ-expanded AR toxicity, however it remains to be established what the early pathological processes are and how they unfold during disease progression. Using transgenic, knock-in SBMA mice and patient-derived muscle biopsies, we show that polyQ-expanded AR alters the generation of intrinsic muscle force prior to denervation. The pattern of expression of genes encoding key components of the excitation-contraction coupling (ECC) machinery is altered in the muscles of presymptomatic mice and patients. Altered mitochondrial respiration is another early event followed by stimulus-dependent accumulation of calcium into mitochondria during disease onset and structural organization alterations of the muscle triad. Surgical castration and AR silencing prevented early pathological processes. These observations show that androgen-dependent deregulations of ECC and ...
Mitochondrial quality control is essential in highly structured cells such as neurons and muscles... more Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal ...
Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine (polyQ) expansions in the an... more Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. Although clinical and experimental evidence highlight a primary role for skeletal muscle in the onset, progression, and outcome of disease, the pathophysiological and molecular processes underlying SBMA muscle atrophy are poorly understood. Here we show that polyQ-expanded AR alters intrinsic muscle force generation before denervation. Reduced muscle force was associated with a switch in fiber-type composition, disrupted muscle striation, altered calcium (Ca++) dynamics in response to muscle contraction, and aberrant expression of excitation-contraction coupling (ECC) machinery genes in transgenic, knock-in and inducible SBMA mice and patients. Importantly, treatment to suppress polyQ-expanded AR toxicity restored ECC gene expression back to normal. Suppression of AR activation by surgical castration elicited similar ECC gene expression changes in normal mice, ...
Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads t... more Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.
Oxidative stress has been proposed to play a key role in malignant hyperthermia (MH), a syndrome ... more Oxidative stress has been proposed to play a key role in malignant hyperthermia (MH), a syndrome caused by excessive Ca2+release in skeletal muscle. Incidence of mortality in male calsequestrin-1 knockout (CASQ1-null) mice during exposure to halothane and heat (a syndrome closely resembling human MH) is far greater than that in females. To investigate the possible role of sex hormones in this still unexplained gender difference, we treated male and female CASQ1-null mice for 1 month, respectively, with Premarin (conjugated estrogens) and leuprolide (GnRH analog) and discovered that during exposure to halothane and heat Premarin reduced the mortality rate in males (79–27% and 86–20%), while leuprolide increased the incidence of mortality in females (18–73% and 24–82%). We then evaluated the (a) responsiveness of isolated muscles to temperature and caffeine, (b) sarcoplasmic reticulum (SR) Ca2+release in single fibers, and (c) oxidative stress and the expression levels of main enzymes...
In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperther... more In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type-1 (RYR1 Y522S/WT) and calsequestrin-1 knockout (CASQ1-null), 2 proteins that control Ca 2+ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1 Y522S/WT and CASQ1-null mice to an exertional-stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1 Y522S/WT and CASQ1-null mice, respectively, vs. 0% in wild-type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1 Y522S/WT and CASQ1-null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca 2+dependent activation of calpains and NF-kB by lowering myoplasmic Ca 2+ concentration and nitro-oxidative stress, parameters that were elevated in RYR1 Y522S/WT and CASQ1-null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.
The response to resistance training and protein supplementation in the latissimus dorsi muscle (L... more The response to resistance training and protein supplementation in the latissimus dorsi muscle (LDM) has never been investigated. We investigated the effects of resistance training (RT) and protein supplementation on muscle mass, strength, and fiber characteristics of the LDM. Eighteen healthy young subjects were randomly assigned to a progressive eight-week RT program with a normal protein diet (NP) or high protein diet (HP) (NP 0.85 vs. HP 1.8 g of protein·kg(-1)·day(-1)). One repetition maximum tests, magnetic resonance imaging for cross-sectional muscle area (CSA), body composition, and single muscle fibers mechanical and phenotype characteristics were measured. RT induced a significant gain in strength (+17%, p < 0.0001), whole muscle CSA (p = 0.024), and single muscle fibers CSA (p < 0.05) of LDM in all subjects. Fiber isometric force increased in proportion to CSA (+22%, p < 0.005) and thus no change in specific tension occurred. A significant transition from 2X to 2...
Obesity is reaching epidemic proportions in Western countries and is a strong risk factor for car... more Obesity is reaching epidemic proportions in Western countries and is a strong risk factor for cardiovascular disease. Despite the constant recommendations of health care organizations regarding the importance of weight control, this goal often fails. Although there is a common agreement about the concept that exercise and diet are two key factors for the control of body weight, the ideal amount and type of exercise and also the ideal diet for weight control are still under debate. A widely accepted nutritional regime is the Mediterranean diet that has evident health benefits although less attention has been paid to see if the effects are due to other lifestyle factors which may contribute to the health benefits perhaps as much as specific food choices. There are several other options available to the physician that may produce good weight loss results in the short/medium term and also for maintenance of the goal achieved. One of these strategies is the ketogenic diet or VLCKD (very ...
Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum... more Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca 2+-binding protein inside SR, calsequestrin. Mitochondrial free Ca 2+-concentration ([Ca 2+ ] mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca 2+ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca 2+ ] mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca 2+ concentration. The rise in [Ca 2+ ] mito during electrical stimulation occurred in 20230 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s 21. Accordingly, frequency-dependent increase in [Ca 2+ ] mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca 2+ ] mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca 2+ ] mito transients were increased by inhibition of mitochondrial Na + /Ca 2+ exchanger (mNCX). These results provide direct evidence for fast Ca 2+ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca 2+-handling and a dependence of mitochondrial Ca 2+-handling on intracellular (SR) and external Ca 2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca 2+-leakage.
Journal of Muscle Research and Cell Motility, 2014
Calcium storage, release, and reuptake are essential for normal physiological function of muscle.... more Calcium storage, release, and reuptake are essential for normal physiological function of muscle. Several human skeletal muscle disorders can arise from dysfunction in the control and coordination of these three critical processes. The release from the Sarcoplasmic Reticulum stores (SR) is handled by a multiprotein complex called Calcium Release Unit and composed of DiHydroPyridine Receptor or DHPR, Ryanodine Receptor or RYR, Calsequestrin or CASQ, junctin, Triadin, Junctophilin and Mitsugumin 29. Malignant hyperthermia (MH), Central Core Disease (CCD), Exertional/environmental Heat Stroke (EHS) and Multiminicore disease (MmD) are inherited disorders of calcium homeostasis in skeletal muscles directly related to mutations of genes coding for proteins of the CRU, primarily ryanodine receptor (RYR1). To understand the pathophysiology of MH and CCD, four murine lines carrying point mutations of human RYR1 have been developed: Y524S, R163C, I4898T and T4826I. Mice carrying those mutations show a phenotype with the traits of MH and/or CCD. Interestingly, also ablation of skeletal muscle calsequestrin (CASQ1) leads to a phenotype with MH-like lethal episodes in response to halothane and heat stress and development of central cores. In this review, we aim to describe the murine lines with RYR mutations or CASQ ablation, which show a phenotype similar to human MH or CCD, to underline their specific phenotypes and their differences and to discuss their contribution to the understanding of the pathophysiology of the disorders and the development of therapeutic strategies.
Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a count... more Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a counterintuitive positive correlation between MSTN and muscle mass (MM). Our aim was to investigate the influence of 2 months of resistance training (RT) and diets with different protein contents on plasma MSTN, interleukin 1 beta (IL-1b), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), and insulin-like growth factor 1 (IGF-1). Eighteen healthy volunteers were randomly divided in two groups: high protein (HP) and normal protein (NP) groups. Different protein diet contents were 1.8 and 0.85 g of protein$kg bw-1 $day-1 for HP and NP, respectively. Subjects underwent 8 weeks of standardized progressive RT. MSTN, IGF-1, IL-1b, IL-6, and TNF-a were analyzed before and after the first and the last training sessions. Lean body mass, MM, upper-limb muscle area, and strength were measured. Plasma MSTN showed a significant increase (P < .001) after the last training in the HP group compared with NP group and with starting value. IGF-1 plasma concentration showed a positive correlation with MSTN in HP after the last training (r 2 = 0.6456; P = .0295). No significant differences were found between NP and HP for IL-1b, IL-6, TNF-a, and strength and MM or area. These findings suggest a ''paradoxical'' postexercise increase of plasma MSTN after 8 weeks of RT and HP diets. This MSTN elevation correlates positively with IGF-1 plasma level. This double increase of opposite (catabolic/anabolic) mediators could explain the substantial overlapping of MM increases in the two groups.
The aim of our study was to investigate fiber type distribution and contractile characteristics o... more The aim of our study was to investigate fiber type distribution and contractile characteristics of Latissimus Dorsi muscle (LDM). Samples were collected from 18 young healthy subjects (9 males and 9 females) through percutaneous fine needle muscle biopsy. The results showed a predominance of fast myosin heavy chain isoforms (MyHC) with 42% of MyHC 2A and 25% of MyHC 2X, while MyHC 1 represented only 33%. The unbalance toward fast isoforms was even greater in males (71%) than in females (64%). Fiber type distribution partially reflected MyHC isoform distribution with 28% type 1/slow fibers and 5% hybrid 1/2A fibers, while fast fibers were divided into 30% type 2A, 31% type A/X, 4% type X, and 2% type 1/2X. Type 1/slow fibers were not only less abundant but also smaller in cross-sectional area than fast fibers. During maximal isometric contraction, type 1/slow fibers developed force and tension significantly lower than the two major groups of fast fibers. In conclusion, the predominan...
Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular... more Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti‐inflammatory activity. Methods: To address this issue, mdx mice were treated daily with methylprednisolone and non‐steroidal anti‐inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib). Results: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX‐2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin. Conclusions: NSAIDs have a beneficial effect on mdx muscle morpholog...
The influence of nutrition on skin health is a growing research area but the findings of various ... more The influence of nutrition on skin health is a growing research area but the findings of various studies on the effect of diet on the development of acne have often been contradictory. The general opinion among researchers has oscillated between two different, opposing positions: that diet either is or is not a key factor for acne development. This review examines the evidence supporting an influence of various dietary components on the development of acne particularly focusing on the role played by carbohydrates. The physiological and biochemical effects of the ketogenic diet are examined from this perspective and mechanisms will be proposed via which this type of diet could have a role in the treatment of acne.
Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cel... more Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6‐C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6‐C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin‐positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA‐2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin‐dominant negative protein, suggesting the involvement in this process of the Ca...
This study aimed to analyse the sarcolemma of Col6a1−/−fibers in comparison with wild type and md... more This study aimed to analyse the sarcolemma of Col6a1−/−fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1−/−and increased in mdx fibers. In the membrane of Col6a1−/−fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane ...
Skeletal muscle weakness has been associated with different pathological conditions, including sa... more Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mammalian target of rapamycin (mTOR) signalling. We wanted to elucidate the functional role of mTOR in muscle contractility. Most loss‐of‐function studies for mTOR signalling have used the drug rapamycin to inhibit some of the signalling downstream of mTOR. However, given that rapamycin does not inhibit all mTOR signalling completely, we generated a double knockout for mTOR and for the scaffold protein of mTORC1, raptor, in skeletal muscle. We found that double knockout in mice results in a more severe phenotype compared with deletion of raptor or mTOR alone. Indeed, these animals display muscle weakness, increased fibre denervation and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow‐twitch fibres and changes in the expression levels of calcium‐related genes, such as Serca...
Polyglutamine (polyQ)-expanded androgen receptor (AR) causes spinobulbar muscular atrophy. Skelet... more Polyglutamine (polyQ)-expanded androgen receptor (AR) causes spinobulbar muscular atrophy. Skeletal muscle is a primary site of polyQ-expanded AR toxicity, however it remains to be established what the early pathological processes are and how they unfold during disease progression. Using transgenic, knock-in SBMA mice and patient-derived muscle biopsies, we show that polyQ-expanded AR alters the generation of intrinsic muscle force prior to denervation. The pattern of expression of genes encoding key components of the excitation-contraction coupling (ECC) machinery is altered in the muscles of presymptomatic mice and patients. Altered mitochondrial respiration is another early event followed by stimulus-dependent accumulation of calcium into mitochondria during disease onset and structural organization alterations of the muscle triad. Surgical castration and AR silencing prevented early pathological processes. These observations show that androgen-dependent deregulations of ECC and ...
Mitochondrial quality control is essential in highly structured cells such as neurons and muscles... more Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal ...
Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine (polyQ) expansions in the an... more Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. Although clinical and experimental evidence highlight a primary role for skeletal muscle in the onset, progression, and outcome of disease, the pathophysiological and molecular processes underlying SBMA muscle atrophy are poorly understood. Here we show that polyQ-expanded AR alters intrinsic muscle force generation before denervation. Reduced muscle force was associated with a switch in fiber-type composition, disrupted muscle striation, altered calcium (Ca++) dynamics in response to muscle contraction, and aberrant expression of excitation-contraction coupling (ECC) machinery genes in transgenic, knock-in and inducible SBMA mice and patients. Importantly, treatment to suppress polyQ-expanded AR toxicity restored ECC gene expression back to normal. Suppression of AR activation by surgical castration elicited similar ECC gene expression changes in normal mice, ...
Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads t... more Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.
Oxidative stress has been proposed to play a key role in malignant hyperthermia (MH), a syndrome ... more Oxidative stress has been proposed to play a key role in malignant hyperthermia (MH), a syndrome caused by excessive Ca2+release in skeletal muscle. Incidence of mortality in male calsequestrin-1 knockout (CASQ1-null) mice during exposure to halothane and heat (a syndrome closely resembling human MH) is far greater than that in females. To investigate the possible role of sex hormones in this still unexplained gender difference, we treated male and female CASQ1-null mice for 1 month, respectively, with Premarin (conjugated estrogens) and leuprolide (GnRH analog) and discovered that during exposure to halothane and heat Premarin reduced the mortality rate in males (79–27% and 86–20%), while leuprolide increased the incidence of mortality in females (18–73% and 24–82%). We then evaluated the (a) responsiveness of isolated muscles to temperature and caffeine, (b) sarcoplasmic reticulum (SR) Ca2+release in single fibers, and (c) oxidative stress and the expression levels of main enzymes...
In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperther... more In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type-1 (RYR1 Y522S/WT) and calsequestrin-1 knockout (CASQ1-null), 2 proteins that control Ca 2+ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1 Y522S/WT and CASQ1-null mice to an exertional-stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1 Y522S/WT and CASQ1-null mice, respectively, vs. 0% in wild-type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1 Y522S/WT and CASQ1-null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca 2+dependent activation of calpains and NF-kB by lowering myoplasmic Ca 2+ concentration and nitro-oxidative stress, parameters that were elevated in RYR1 Y522S/WT and CASQ1-null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.
The response to resistance training and protein supplementation in the latissimus dorsi muscle (L... more The response to resistance training and protein supplementation in the latissimus dorsi muscle (LDM) has never been investigated. We investigated the effects of resistance training (RT) and protein supplementation on muscle mass, strength, and fiber characteristics of the LDM. Eighteen healthy young subjects were randomly assigned to a progressive eight-week RT program with a normal protein diet (NP) or high protein diet (HP) (NP 0.85 vs. HP 1.8 g of protein·kg(-1)·day(-1)). One repetition maximum tests, magnetic resonance imaging for cross-sectional muscle area (CSA), body composition, and single muscle fibers mechanical and phenotype characteristics were measured. RT induced a significant gain in strength (+17%, p < 0.0001), whole muscle CSA (p = 0.024), and single muscle fibers CSA (p < 0.05) of LDM in all subjects. Fiber isometric force increased in proportion to CSA (+22%, p < 0.005) and thus no change in specific tension occurred. A significant transition from 2X to 2...
Obesity is reaching epidemic proportions in Western countries and is a strong risk factor for car... more Obesity is reaching epidemic proportions in Western countries and is a strong risk factor for cardiovascular disease. Despite the constant recommendations of health care organizations regarding the importance of weight control, this goal often fails. Although there is a common agreement about the concept that exercise and diet are two key factors for the control of body weight, the ideal amount and type of exercise and also the ideal diet for weight control are still under debate. A widely accepted nutritional regime is the Mediterranean diet that has evident health benefits although less attention has been paid to see if the effects are due to other lifestyle factors which may contribute to the health benefits perhaps as much as specific food choices. There are several other options available to the physician that may produce good weight loss results in the short/medium term and also for maintenance of the goal achieved. One of these strategies is the ketogenic diet or VLCKD (very ...
Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum... more Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca 2+-binding protein inside SR, calsequestrin. Mitochondrial free Ca 2+-concentration ([Ca 2+ ] mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca 2+ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca 2+ ] mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca 2+ concentration. The rise in [Ca 2+ ] mito during electrical stimulation occurred in 20230 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s 21. Accordingly, frequency-dependent increase in [Ca 2+ ] mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca 2+ ] mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca 2+ ] mito transients were increased by inhibition of mitochondrial Na + /Ca 2+ exchanger (mNCX). These results provide direct evidence for fast Ca 2+ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca 2+-handling and a dependence of mitochondrial Ca 2+-handling on intracellular (SR) and external Ca 2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca 2+-leakage.
Journal of Muscle Research and Cell Motility, 2014
Calcium storage, release, and reuptake are essential for normal physiological function of muscle.... more Calcium storage, release, and reuptake are essential for normal physiological function of muscle. Several human skeletal muscle disorders can arise from dysfunction in the control and coordination of these three critical processes. The release from the Sarcoplasmic Reticulum stores (SR) is handled by a multiprotein complex called Calcium Release Unit and composed of DiHydroPyridine Receptor or DHPR, Ryanodine Receptor or RYR, Calsequestrin or CASQ, junctin, Triadin, Junctophilin and Mitsugumin 29. Malignant hyperthermia (MH), Central Core Disease (CCD), Exertional/environmental Heat Stroke (EHS) and Multiminicore disease (MmD) are inherited disorders of calcium homeostasis in skeletal muscles directly related to mutations of genes coding for proteins of the CRU, primarily ryanodine receptor (RYR1). To understand the pathophysiology of MH and CCD, four murine lines carrying point mutations of human RYR1 have been developed: Y524S, R163C, I4898T and T4826I. Mice carrying those mutations show a phenotype with the traits of MH and/or CCD. Interestingly, also ablation of skeletal muscle calsequestrin (CASQ1) leads to a phenotype with MH-like lethal episodes in response to halothane and heat stress and development of central cores. In this review, we aim to describe the murine lines with RYR mutations or CASQ ablation, which show a phenotype similar to human MH or CCD, to underline their specific phenotypes and their differences and to discuss their contribution to the understanding of the pathophysiology of the disorders and the development of therapeutic strategies.
Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a count... more Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a counterintuitive positive correlation between MSTN and muscle mass (MM). Our aim was to investigate the influence of 2 months of resistance training (RT) and diets with different protein contents on plasma MSTN, interleukin 1 beta (IL-1b), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), and insulin-like growth factor 1 (IGF-1). Eighteen healthy volunteers were randomly divided in two groups: high protein (HP) and normal protein (NP) groups. Different protein diet contents were 1.8 and 0.85 g of protein$kg bw-1 $day-1 for HP and NP, respectively. Subjects underwent 8 weeks of standardized progressive RT. MSTN, IGF-1, IL-1b, IL-6, and TNF-a were analyzed before and after the first and the last training sessions. Lean body mass, MM, upper-limb muscle area, and strength were measured. Plasma MSTN showed a significant increase (P < .001) after the last training in the HP group compared with NP group and with starting value. IGF-1 plasma concentration showed a positive correlation with MSTN in HP after the last training (r 2 = 0.6456; P = .0295). No significant differences were found between NP and HP for IL-1b, IL-6, TNF-a, and strength and MM or area. These findings suggest a ''paradoxical'' postexercise increase of plasma MSTN after 8 weeks of RT and HP diets. This MSTN elevation correlates positively with IGF-1 plasma level. This double increase of opposite (catabolic/anabolic) mediators could explain the substantial overlapping of MM increases in the two groups.
The aim of our study was to investigate fiber type distribution and contractile characteristics o... more The aim of our study was to investigate fiber type distribution and contractile characteristics of Latissimus Dorsi muscle (LDM). Samples were collected from 18 young healthy subjects (9 males and 9 females) through percutaneous fine needle muscle biopsy. The results showed a predominance of fast myosin heavy chain isoforms (MyHC) with 42% of MyHC 2A and 25% of MyHC 2X, while MyHC 1 represented only 33%. The unbalance toward fast isoforms was even greater in males (71%) than in females (64%). Fiber type distribution partially reflected MyHC isoform distribution with 28% type 1/slow fibers and 5% hybrid 1/2A fibers, while fast fibers were divided into 30% type 2A, 31% type A/X, 4% type X, and 2% type 1/2X. Type 1/slow fibers were not only less abundant but also smaller in cross-sectional area than fast fibers. During maximal isometric contraction, type 1/slow fibers developed force and tension significantly lower than the two major groups of fast fibers. In conclusion, the predominan...
Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular... more Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti‐inflammatory activity. Methods: To address this issue, mdx mice were treated daily with methylprednisolone and non‐steroidal anti‐inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib). Results: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX‐2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin. Conclusions: NSAIDs have a beneficial effect on mdx muscle morpholog...
The influence of nutrition on skin health is a growing research area but the findings of various ... more The influence of nutrition on skin health is a growing research area but the findings of various studies on the effect of diet on the development of acne have often been contradictory. The general opinion among researchers has oscillated between two different, opposing positions: that diet either is or is not a key factor for acne development. This review examines the evidence supporting an influence of various dietary components on the development of acne particularly focusing on the role played by carbohydrates. The physiological and biochemical effects of the ketogenic diet are examined from this perspective and mechanisms will be proposed via which this type of diet could have a role in the treatment of acne.
Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cel... more Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6‐C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6‐C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin‐positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA‐2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin‐dominant negative protein, suggesting the involvement in this process of the Ca...
This study aimed to analyse the sarcolemma of Col6a1−/−fibers in comparison with wild type and md... more This study aimed to analyse the sarcolemma of Col6a1−/−fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1−/−and increased in mdx fibers. In the membrane of Col6a1−/−fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane ...
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Papers by Marta Canato