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1.
Food Chem ; 340: 127914, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889207

RESUMO

The objective of this study was to investigate the influence of oxidation on heat shock protein 27 (HSP27) and cytochrome c translocation, myofibrils degradation and endogenous enzymes activities, perfecting tenderization mechanism after slaughter. Bovine muscle (longissimus thoracis) was obtained at 30 min postmortem. Bovine muscle was cut and exposed to saline solution with or without H2O2 at 4 °C for 0.25, 1, 3 and 5 days, followed by detection of proteins degradation, location and enzymes activities. Results showed that oxidation promoted the translocation of HSP27 and cytochrome c from the cytoplasm to the cell membrane, which reduced µ-calpain activity, but increased caspase-3 activity through mediating the interaction with the two enzymes. Oxidation retarded troponin-T degradation, but accelerated desmin degradation, which is probably because oxidative modification of myofibrils induced different susceptibility to proteolysis. Therefore, oxidation leads to different regulatory mechanism on µ-calpain and caspase-3, as well as the degree of degradation of myofibrillar proteins, possibly through mediating HSP27 and cytochrome c.


Assuntos
Calpaína/metabolismo , Caspase 3/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Animais , Bovinos , Citocromos c/metabolismo , Peróxido de Hidrogênio/química , Masculino , Músculo Esquelético/química , Oxirredução , Mudanças Depois da Morte , Proteólise , Carne Vermelha , Troponina T/metabolismo
2.
Nat Metab ; 2(12): 1459-1471, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33288952

RESUMO

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using 15N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.


Assuntos
Amônia/toxicidade , Hibernação/fisiologia , Músculo Esquelético/fisiologia , Nitrogênio/metabolismo , Sciuridae/fisiologia , Aminoácidos/metabolismo , Animais , Regiões Árticas , Nível de Alerta , Rim/metabolismo , Miofibrilas/metabolismo , Torpor/fisiologia , Ureia/metabolismo , gama-Glutamil Hidrolase/metabolismo
3.
Sci Rep ; 10(1): 16372, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009449

RESUMO

Muscles perform a wide range of motile functions in animals. Among various types are skeletal and cardiac muscles, which exhibit a steady auto-oscillation of force and length when they are activated at an intermediate level of contraction. This phenomenon, termed spontaneous oscillatory contraction or SPOC, occurs devoid of cell membranes and at fixed concentrations of chemical substances, and is thus the property of the contractile system per se. We have previously developed a theoretical model of SPOC and proposed that the oscillation emerges from a dynamic force balance along both the longitudinal and lateral axes of sarcomeres, the contractile units of the striated muscle. Here, we experimentally tested this hypothesis by developing an imaging-based analysis that facilitates detection of the structural changes of single sarcomeres at unprecedented spatial resolution. We found that the sarcomere width oscillates anti-phase with the sarcomere length in SPOC. We also found that the oscillatory dynamics can be altered by osmotic compression of the myofilament lattice structure of sarcomeres, but they are unchanged by a proteolytic digestion of titin/connectin-the spring-like protein that provides passive elasticity to sarcomeres. Our data thus reveal the three-dimensional mechanical dynamics of oscillating sarcomeres and suggest a structural requirement of steady auto-oscillation.


Assuntos
Contração Muscular/fisiologia , Músculo Estriado/metabolismo , Músculo Estriado/fisiologia , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Animais , Conectina/metabolismo , Elasticidade/fisiologia , Masculino , Modelos Biológicos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Miocárdio/metabolismo , Miofibrilas/metabolismo , Coelhos
4.
PLoS One ; 15(9): e0238441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881965

RESUMO

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.


Assuntos
Disferlina/genética , Galectina 1/farmacologia , Distrofia Muscular do Cíngulo dos Membros/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Disferlina/metabolismo , Galectina 1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Miofibrilas/metabolismo
5.
Life Sci ; 261: 118342, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32853655

RESUMO

AIMS: The increased incidence of heart failure with reduced ejection fraction in men compared with women suggests that male sex hormones significantly impact myocardial contractile activation. This study aims to examine associations among molecular alterations, cellular modulations and in vivo cardiac contractile function upon deprivation of testicular hormones. MAIN METHODS: Myocardial structure and functions were compared among sham-operated control and twelve-week orchidectomized (ORX) male rats with and without testosterone supplementation. KEY FINDINGS: Echocardiography and pressure-volume relationships demonstrated a decreased left ventricular ejection fraction compared with sham-operated controls. The percentage of contractility reduction was generally similar to the decrease in tension development detected in both right ventricular trabeculae and skinned isolated left ventricular cardiomyocytes of ORX rats. Reductions in tension cost and the rate constant of tension redevelopment (ktr) in ORX samples suggested a decrease in the rate of cross-bridge formation, reflecting a reduced number of cross-bridges. Slow cross-bridge detachment in ORX rat hearts could result from a shift of myosin heavy chain isoforms towards a slower ATPase activity ß-isoform and reductions in the phosphorylation levels of cardiac troponin I and myosin binding protein-C. All the changes in the ORX rat heart, including ejection fractions and myofilament protein expression and phosphorylation, were completed attenuated by a physiological dose of testosterone. SIGNIFICANCE: Testosterone plays a critical role in regulating the mechanical and contractile dynamics of the heart. Deprivation of male sex hormones cause the loss of normal preserved cardiac contractile function leading to a high risk of severe cardiomyopathy progression.


Assuntos
Cardiomiopatias/fisiopatologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Testosterona/metabolismo , Animais , Progressão da Doença , Coração/fisiologia , Masculino , Cadeias Pesadas de Miosina/metabolismo , Orquiectomia , Ratos , Ratos Sprague-Dawley , Volume Sistólico/fisiologia , Testosterona/administração & dosagem , Testosterona/farmacologia , Função Ventricular Esquerda/fisiologia
6.
Nat Commun ; 11(1): 3711, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709891

RESUMO

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.


Assuntos
Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Sarcolema/fisiologia , Sarcolema/ultraestrutura , Animais , Canais de Cálcio/metabolismo , Canais de Cálcio/ultraestrutura , Canais de Cálcio Tipo L/metabolismo , Proteínas de Transporte/metabolismo , Biologia do Desenvolvimento , Complexo de Golgi/metabolismo , Masculino , Microscopia Eletrônica , Proteínas Musculares/química , Músculo Esquelético/química , Miofibrilas/metabolismo , Sarcolema/química , Retículo Sarcoplasmático/metabolismo , Peixe-Zebra
7.
PLoS One ; 15(7): e0232137, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614896

RESUMO

In sarcomeres, α-actinin crosslinks thin filaments and anchors them at the Z-disc. Drosophila melanogaster Zasp52 also localizes at Z-discs and interacts with α-actinin via its extended PDZ domain, thereby contributing to myofibril assembly and maintenance, yet the detailed mechanism of Zasp52 function is unknown. Here we show a strong genetic interaction between actin and Zasp52 during indirect flight muscle assembly, indicating that this interaction plays a critical role during myofibril assembly. Our results suggest that Zasp52 contains an actin-binding site, which includes the extended PDZ domain and the ZM region. Zasp52 binds with micromolar affinity to monomeric actin. A co-sedimentation assay indicates that Zasp52 can also bind to F-actin. Finally, we use in vivo rescue assays of myofibril assembly to show that the α-actinin-binding domain of Zasp52 is not sufficient for a full rescue of Zasp52 mutants suggesting additional contributions of Zasp52 actin-binding to myofibril assembly.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Miofibrilas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Domínios PDZ , Ligação Proteica
8.
Biochim Biophys Acta Mol Cell Res ; 1867(10): 118788, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32603758

RESUMO

Muscle atrophy is an inevitable sequel of fasting, denervation, aging, exposure to microgravity, and many human diseases including, cancer, type-2 diabetes, and renal failure. During atrophy the destruction of the muscle's fundamental contractile machinery, the myofibrils, is accelerated leading to a reduction in muscle mass, weakness, frailty, and physical disability. Recent findings indicate that atrophy can be a major cause of death in affected individuals, and inhibition of muscle wasting is likely to prolong survival. Major advances in our understanding of the mechanisms for myofibril breakdown in atrophy include the discovery of biological pathways and key components that play prominent roles. On fasting or denervation, degradation of myofibrillar proteins requires an initial dissociation of the desmin cytoskeleton, whose integrity is critical for myofibril stability. This loss of desmin filaments involves phosphorylation, ubiquitination, and subsequent depolymerization by calpain-1, and appears to reduce myofibrils integrity and facilitate their destruction. Consequently, depolymerization of desmin filament in atrophy seems to be an early key event for overall proteolysis. A focus of this review is to discuss these new insights and the specific role of calpain-1 in promoting desmin filaments loss, and to highlight important key questions that merit further study.


Assuntos
Calpaína/metabolismo , Desmina/metabolismo , Atrofia Muscular/metabolismo , Polimerização , Animais , Humanos , Miofibrilas/metabolismo , Ubiquitinação
9.
Nat Commun ; 11(1): 2699, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483185

RESUMO

Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.


Assuntos
Proteínas Musculares/genética , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Heterozigoto , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miofibrilas/patologia , Miofibrilas/ultraestrutura , Miopatias da Nemalina/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologia , Sarcômeros/ultraestrutura , Tropomiosina/química , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismo , Difração de Raios X
10.
PLoS One ; 15(5): e0227720, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407314

RESUMO

Numerous mutational studies have demonstrated that circadian clock proteins regulate behavior and metabolism. Nr1d1(Rev-erbα) is a key regulator of circadian gene expression and a pleiotropic regulator of skeletal muscle homeostasis and lipid metabolism. Loss of Rev-erbα expression induces muscular atrophy, high adiposity, and metabolic syndrome in mice. Here we show that, unlike knockout mice, Nr1d1 heterozygous mice are not susceptible to muscular atrophy and in fact paradoxically possess larger myofiber diameters and improved neuromuscular function, compared to wildtype mice. Heterozygous mice lacked dyslipidemia, a characteristic of Nr1d1 knockout mice and displayed increased whole-body fatty-acid oxidation during periods of inactivity (light cycle). Heterozygous mice also exhibited higher rates of glucose uptake when fasted, and had elevated basal rates of gluconeogenesis compared to wildtype and knockout littermates. Rev-erbα ablation suppressed glycolysis and fatty acid-oxidation in white-adipose tissue (WAT), whereas partial Rev-erbα loss, curiously stimulated these processes. Our investigations revealed that Rev-erbα dose-dependently regulates glucose metabolism and fatty acid oxidation in WAT and muscle.


Assuntos
Dislipidemias/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Tecido Adiposo Branco/metabolismo , Adiposidade/genética , Animais , Comportamento Animal/fisiologia , Relógios Circadianos/genética , Dislipidemias/metabolismo , Dislipidemias/patologia , Ácidos Graxos/metabolismo , Gluconeogênese/genética , Glucose/metabolismo , Heterozigoto , Humanos , Metabolismo dos Lipídeos/genética , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Camundongos , Camundongos Knockout , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Miofibrilas/genética , Miofibrilas/metabolismo , Miofibrilas/patologia , Fotoperíodo
11.
Am J Physiol Heart Circ Physiol ; 319(1): H235-H241, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32469635

RESUMO

To maximize data obtainment from valuable cardiac tissue, we hypothesized that myocardium fixed in optimal cutting temperature (OCT) medium for histology could also be used to investigate the function of myofilament proteins in situ. We compared tissue prepared via conventional liquid nitrogen (LN) snap freezing with tissue fixed in OCT and then sectioned in fiber-parallel orientation. We found that actin-myosin Ca2+ sensitivity, activation rate by Ca2+, cooperativity along the thin filament, as well as cross-bridge cycling rate were unaffected by OCT storage and could reliably be interpreted after sectioning. Absolute values in maximum force generation per cross-sectional area, as well as passive strain, are difficult to investigate after sectioning, as myofibrillar continuity along the preparation cannot be guaranteed. We have shown that myocardial tissue stored in OCT and sectioned before analysis is available for functional analysis, a valuable means of maximizing usage of precious cardiac biopsies.NEW & NOTEWORTHY Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.


Assuntos
Criopreservação/métodos , Secções Congeladas/métodos , Miocárdio/citologia , Miofibrilas/metabolismo , Inclusão em Parafina/métodos , Animais , Criopreservação/normas , Secções Congeladas/normas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miofibrilas/ultraestrutura , Inclusão em Parafina/normas
12.
FASEB J ; 34(6): 8204-8216, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32294300

RESUMO

Chronic excessive ethanol consumption has distinct toxic and adverse effects on a variety of tissues. In skeletal muscle, ethanol causes alcoholic myopathy, which is characterized by myofiber atrophy and the loss of muscle strength. Alcoholic myopathy is more prevalent than all inherited muscle diseases combined. Current evidence indicates that ethanol directly impairs muscle organization and function. However, the underlying mechanism by which ethanol causes toxicity in muscle is poorly understood. Here, we show that the nematode Caenorhabditis elegans exhibits the key features of alcoholic myopathy when exposed to ethanol. As in mammals, ethanol exposure impairs muscle strength and induces the expression of protective genes, including oxidative stress response genes. In addition, ethanol exposure causes the fragmentation of mitochondrial networks aligned with myofibril lattices. This ethanol-induced mitochondrial fragmentation is dependent on the mitochondrial fission factor DRP-1 (dynamin-related protein 1) and its receptor proteins on the outer mitochondrial membrane. Our data indicate that this fragmentation contributes to the activation of the mitochondrial unfolded protein response (UPR). We also found that robust, perpetual mitochondrial UPR activation effectively reduces muscle weakness caused by ethanol exposure. Our results strongly suggest that the modulation of mitochondrial stress responses may provide a method to ameliorate alcohol toxicity and damage to muscle.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Etanol/farmacologia , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Caenorhabditis elegans/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Debilidade Muscular/induzido quimicamente , Debilidade Muscular/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/induzido quimicamente , Doenças Musculares/metabolismo , Miofibrilas/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
13.
Nutrients ; 12(3)2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245197

RESUMO

BACKGROUND: The aim of this study was to investigate the effect of whey protein supplementation on myofibrillar protein synthesis (myoPS) and muscle recovery over a 7-d period of intensified resistance training (RT). METHODS: In a double-blind randomised parallel group design, 16 resistance-trained men aged 18 to 35 years completed a 7-d RT protocol, consisting of three lower-body RT sessions on non-consecutive days. Participants consumed a controlled diet (146 kJ·kg-1·d-1, 1.7 g·kg-1·d-1 protein) with either a whey protein supplement or an isonitrogenous control (0.33 g·kg-1·d-1 protein). To measure myoPS, 400 ml of deuterium oxide (D2O) (70 atom %) was ingested the day prior to starting the study and m. vastus lateralis biopsies were taken before and after RT-intervention. Myofibrillar fractional synthetic rate (myoFSR) was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Pyr-IRMS). Muscle recovery parameters (i.e., countermovement jump height, isometric-squat force, muscle soreness and serum creatine kinase) were assessed daily. RESULTS: MyoFSR PRE was 1.6 (0.2) %∙d-1 (mean (SD)). Whey protein supplementation had no effect on myoFSR (p = 0.771) or any recovery parameter (p = 0.390-0.989). CONCLUSIONS: Over an intense 7-d RT protocol, 0.33 g·kg-1·d-1 of supplemental whey protein does not enhance day-to-day measures of myoPS or postexercise recovery in resistance-trained men.


Assuntos
Suplementos Nutricionais , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Biossíntese de Proteínas , Treinamento de Resistência , Proteínas do Soro do Leite/administração & dosagem , Adolescente , Adulto , Biomarcadores , Expressão Gênica , Humanos , Masculino , Força Muscular , Adulto Jovem
14.
Int J Mol Sci ; 21(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244448

RESUMO

Although the presence of cardiac dysfunction and cardiomyopathy in chronic diabetes has been recognized, the pathophysiology of diabetes-induced metabolic and subcellular changes as well as the therapeutic approaches for the prevention of diabetic cardiomyopathy are not fully understood. Cardiac dysfunction in chronic diabetes has been shown to be associated with Ca2+-handling abnormalities, increase in the availability of intracellular free Ca2+ and impaired sensitivity of myofibrils to Ca2+. Metabolic derangements, including depressed high-energy phosphate stores due to insulin deficiency or insulin resistance, as well as hormone imbalance and ultrastructural alterations, are also known to occur in the diabetic heart. It is pointed out that the activation of the sympathetic nervous system and renin-angiotensin system generates oxidative stress, which produces defects in subcellular organelles including sarcolemma, sarcoplasmic reticulum and myofibrils. Such subcellular remodeling plays a critical role in the pathogenesis of diabetic cardiomyopathy. In fact, blockade of the effects of neurohormonal systems has been observed to attenuate oxidative stress and occurrence of subcellular remodeling as well as metabolic abnormalities in the diabetic heart. This review is intended to describe some of the subcellular and metabolic changes that result in cardiac dysfunction in chronic diabetes. In addition, the therapeutic values of some pharmacological, metabolic and antioxidant interventions will be discussed. It is proposed that a combination therapy employing some metabolic agents or antioxidants with insulin may constitute an efficacious approach for the prevention of diabetic cardiomyopathy.


Assuntos
Complicações do Diabetes , Diabetes Mellitus/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/metabolismo , Cálcio/metabolismo , Cardiomiopatias Diabéticas/etiologia , Coração , Insuficiência Cardíaca/metabolismo , Humanos , Insulina/deficiência , Resistência à Insulina , Miofibrilas/metabolismo , Sistema Renina-Angiotensina/fisiologia , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Sistema Nervoso Simpático
15.
Biochim Biophys Acta Mol Basis Dis ; 1866(8): 165800, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305450

RESUMO

Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.


Assuntos
Conexina 43/genética , Conexinas/genética , Disferlina/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/prevenção & controle , Miofibrilas/genética , Animais , Cálcio/metabolismo , Conexina 43/deficiência , Conexinas/deficiência , Creatina Quinase/sangue , Creatina Quinase/genética , Modelos Animais de Doenças , Disferlina/deficiência , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação , Miofibrilas/metabolismo , Miofibrilas/patologia , Permeabilidade , Condicionamento Físico Animal , Teste de Desempenho do Rota-Rod , Sarcolema/metabolismo
16.
Nutrients ; 12(4)2020 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-32290521

RESUMO

BACKGROUND: Leucine-enriched essential amino acids (LEAAs) acutely enhance post-exercise myofibrillar protein synthesis (MyoPS), which has been suggested to be important for muscle repair and recovery. However, the ability of LEAAs to concurrently enhance MyoPS and muscle damage recovery in free-living humans has not been studied. METHODS: In a randomized, double-blind, placebo-controlled, parallel-group design, twenty recreationally active males consuming a controlled diet (1.2 g/kg/d of protein) were supplemented thrice daily with 4 g of LEAAs (containing 1.6 g leucine) or isocaloric placebo for four days following an acute bout of lower-body resistance exercise (RE). MyoPS at rest and integrated over 96 h of recovery was measured by D2O. Isometric and isokinetic torque, muscle soreness, Z-band streaming, muscle heat shock protein (HSP) 25 and 72, plasma creatine kinase (CK), and plasma interleukin-6 (IL-6) were measured over 96 h post-RE to assess various direct and indirect markers of muscle damage. RESULTS: Integrated MyoPS increased ~72% over 96 h after RE (p < 0.05), with no differences between groups (p = 0.98). Isometric, isokinetic, and total peak torque decreased ~21% by 48 h after RE (p < 0.05), whereas total peak torque was ~10% greater overall during recovery in LEAAs compared to placebo (p < 0.05). There were moderate to large effects for peak torque in favour of LEAAs. Muscle soreness increased during recovery with no statistical differences between groups but small to moderate effects in favour of LEAAs that correlated with changes in peak torque. Plasma CK, plasma IL-6, and muscle HSP25 increased after RE (p < 0.05) but were not significantly different between groups (p ≥ 0.13). Consistent with a trend toward attenuated Z-band streaming in LEAAs (p = 0.07), muscle HSP72 expression was lower (p < 0.05) during recovery in LEAAs compared with placebo. There were no correlations between MyoPS and any measures of muscle damage (p ≥ 0.37). CONCLUSION: Collectively, our data suggest that LEAAs moderately attenuated muscle damage without concomitant increases in integrated MyoPS in the days following an acute bout of resistance exercise in free-living recreationally active men.


Assuntos
Aminoácidos Essenciais/farmacologia , Suplementos Nutricionais , Exercício Físico/fisiologia , Leucina/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Miofibrilas/metabolismo , Biossíntese de Proteínas , Fenômenos Fisiológicos da Nutrição Esportiva/fisiologia , Adulto , Aminoácidos Essenciais/administração & dosagem , Método Duplo-Cego , Expressão Gênica , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Leucina/administração & dosagem , Masculino , Adulto Jovem
17.
Food Chem ; 319: 126571, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32169769

RESUMO

This study aims to investigate the changes in mitochondrial apoptotic factors and proteolysis of two porcine muscles (psoas major - PM and longissimus dorsi - LD) during aging. Results found that during 2-168 h postmortem mitochondrial membrane permeability, mitochondrial lipid peroxidation, Ca2+ levels were increased, while the reduction level and abundance of cytochrome c were decreased (P < 0.05) in both muscle types. Furthermore, the activation of caspase-3 along with increases in troponin-T and desmin degradation, and µ-calpain autolysis were found (P < 0.05), regardless of muscle type. PM maintained higher mitochondrial apoptotic factors, but had more intact desmin, less troponin-T degradation and less extent of autolyzed products of µ-calpain compared to LD (P < 0.05). These results indicate that the rapid onset of mitochondrial apoptosis of PM would not lead to a subsequent impact on myofibrillar protein degradation, suggesting that the mitochondrial apoptosis mediated tenderization process could be muscle-specific.


Assuntos
Apoptose , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Carne Vermelha , Animais , Autólise/metabolismo , Calpaína/metabolismo , Desmina/metabolismo , Miofibrilas/metabolismo , Proteólise , Suínos , Fatores de Tempo , Troponina T/metabolismo
18.
Food Chem ; 315: 126226, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32018081

RESUMO

The aim of this study was to investigate the effects of oxidation on the structure of pork myofibrillar proteins (MPs) and the water retention mechanism of MPs gel. In a Fenton reaction system, protein oxidation increases (P < 0.05) with hydrogen peroxide (H2O2) concentration (0, 0.5, 1, 3, 5, 10, and 20 mmol/L). The Brunauer-Emmett-Teller surface area of the proteins gel gradually increased (P < 0.05) from 6.17 m2/g to 14.73 m2/g. Low field nuclear magnetic resonance results showed that immobilized water in the gel gradually decreased but free water content gradually increased (P < 0.05). Gel strength and water holding capacity (WHC) increased and then decreased. The results reveal that moderate oxidation contributes to the compact and uniform pore structure, higher WHC of proteins gel as well. However, excessive oxidation leads to increase pores and changes in water states of gel, leading to lower WHC.


Assuntos
Proteínas Musculares/metabolismo , Músculos/metabolismo , Miofibrilas/metabolismo , Carne Vermelha/análise , Água/química , Animais , Géis/química , Peróxido de Hidrogênio/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas Musculares/química , Músculos/química , Miofibrilas/química , Oxirredução , Suínos
19.
J Biol Chem ; 295(12): 3794-3807, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32024695

RESUMO

The troponin complex regulates the Ca2+ activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid-rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+ buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.


Assuntos
Proteínas de Drosophila/metabolismo , Ácido Glutâmico/metabolismo , Músculo Esquelético/metabolismo , Troponina T/metabolismo , Processamento Alternativo , Animais , Ligante CD27/química , Ligante CD27/metabolismo , Cálcio/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/classificação , Proteínas de Drosophila/genética , Feminino , Voo Animal , Masculino , Contração Muscular , Mutagênese , Miofibrilas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Tropomiosina/química , Tropomiosina/metabolismo , Troponina T/química , Troponina T/classificação , Troponina T/genética , Cromossomo X
20.
J Biol Chem ; 295(14): 4398-4410, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32086378

RESUMO

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.


Assuntos
Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Troponina I/metabolismo , Animais , Cálcio/metabolismo , Humanos , Masculino , Miofibrilas/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Peptídeos/análise , Peptídeos/química , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais , Troponina I/química , Troponina I/genética
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