RESUMO
Sarcolipin (SLN) regulates muscle-based nonshivering thermogenesis and is up-regulated with high-fat feeding (HFF). To investigate whether other muscle-based thermogenic systems compensate for a lack of Sln and to firmly establish SLN as a mediator of diet-induced thermogenesis (DIT), we measured muscle and whole-body energy expenditure in chow- and high-fat-fed Sln(-/-) and wild-type (WT) mice. Following HFF, resting muscle metabolic rate (VO2, µl/g/s) was increased similarly in WT (0.28±0.02 vs. 0.31±0.03) and Sln(-/-) (0.23±0.03 vs. 0.35±0.02) mice due to increased sympathetic nervous system activation in Sln(-/-) mice; however, whole-body metabolic rate (VO2, ml/kg/h) was lower in Sln(-/-) compared with WT mice following HFF but only during periods when the mice were active in their cages (WT, 2894±87 vs. Sln(-/-), 2708±61). Treatment with the ß-adrenergic receptor (ß-AR) antagonist propranolol during HFF completely prevented muscle-based DIT in Sln(-/-) mice; however, it had no effect in WT mice, resulting in greater differences in whole-body metabolic rate and diet-induced weight gain. Our results suggest that ß-AR signaling partially compensates for a lack of SLN to activate muscle-based DIT, but SLN is the primary and more effective mediator.
Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Proteolipídeos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Termogênese/fisiologia , Animais , Cálcio/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Masculino , Camundongos , Camundongos Knockout , Termogênese/genéticaRESUMO
We examined the influence of estrogen receptor-alpha (ERα) activation on estrogen-mediated regulation of heat shock proteins 70 (Hsp70) and 27 (Hsp27) in soleus. Ovariectomized rats received estrogen (EST), an ERα agonist (propyl pyrazole triol, PPT), both (EST+PPT), or a sham, and they served as either unexercised controls or were subjected to exercise by having to run downhill (17 m/min, -13.5° grade) for 90 min. At 72 h postexercise, soleus muscles were removed and either immunohistochemically stained for Hsp70 and myosin heavy chain or homogenized for Western blotting for Hsp70 and Hsp27. Elevated (p < 0.05) basal Hsp70 in both type I and II fibres in the unexercised EST, PPT, and EST+PPT groups relative to unexercised sham animals was noted. Compared with Hsp70 levels in the unexercised animals, that in exercised animals was elevated (p < 0.05) in both sham and PPT groups but not in EST and EST+PPT groups. Western blot determined that Hsp27 levels were not significantly different between groups. Hence, the ability of estrogen to augment resting type I and type II muscle fibre Hsp70 content is primarily mediated via muscle ERα. However, the blunted Hsp70 response following damaging exercise in estrogen-supplemented animals does not appear to be fully accounted for by ERα-mediated effects.
Assuntos
Receptor alfa de Estrogênio/efeitos dos fármacos , Terapia de Reposição de Estrogênios , Estrogênios/farmacologia , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Contração Muscular , Músculo Esquelético/efeitos dos fármacos , Esforço Físico , Pirazóis/farmacologia , Animais , Western Blotting , Receptor alfa de Estrogênio/metabolismo , Feminino , Imunofluorescência , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Ovariectomia , Fenóis , Ratos , Ratos Sprague-Dawley , Corrida , Fatores de TempoRESUMO
BACKGROUND: In the failing human heart, abnormalities of Ca(2+) cycling have been described, but there is scant knowledge about Ca(2+) handling in the skeletal muscle of humans with heart failure (HF). We tested the hypothesis that in humans with HF, Ca(2+) cycling proteins in skeletal muscle are abnormal. METHODS AND RESULTS: Ten advanced HF patients (50.4 ± 3.7 years), and 9 age-matched controls underwent vastus lateralis biopsy. Western blot analysis showed that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a, which is responsible for Ca(2+) sequestration into the sarcoplasmic reticulum(SR), was lower in HF versus controls (4.8 ± 0.5 vs 7.5 ± 0.8 AU, P = .01). Although phospholamban (PLN), which inhibits SERCA2a, was not different in HF versus controls, phosphorylation (SER16 site) of PLN, which relieves this inhibition, was reduced (0.8 ± 0.1 vs 3.9 ± 0.9 AU, P = .004). Dihydropyridine receptors were reduced in HF, (2.1 ± 0.4 vs 3.6 ± 0.5 AU, P = .04). We tested the hypothesis that these abnormalities of Ca(2+) handling protein content and regulation were due to increased oxidative stress, but oxygen radical scavenger proteins were not elevated in the skeletal muscle of HF patients. CONCLUSION: In chronic HF, marked abnormalities of Ca(2+) handling proteins are present in skeletal muscle, which mirror those in failing heart tissue. This suggests a common mechanism, such as chronic augmentation of sympathetic activity and autophosphorylation of Ca(2+)-calmodulin-dependent-protein kinase II.
Assuntos
Cálcio/metabolismo , Tolerância ao Exercício , Insuficiência Cardíaca/metabolismo , Coração , Músculo Esquelético/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Estatística como Assunto , Sistema Nervoso Simpático , Adulto JovemRESUMO
OBJECTIVES: To determine the state of the literature on assessing heat-stress using physiological parameters. To provide recommendations to the nuclear industry regarding worker heat-stress management practices. METHODS: A scoping review identified relevant articles. A search strategy was developed based on a research question concepts. Identified records were screened with inclusion-exclusion criteria. Included articles underwent data extraction using a qualitative data charting method. A thematic analysis and frequency counts were performed. RESULTS: 1687 articles were identified through four databases. The final inclusion consisted of 34 studies. Articles were classified by determinants of heat exposure risks: core body temperature (direct and indirect), scoring scale including core body temperature, scoring scale including human perception, and others. Heart rate and rectal temperature were the two most utilized physiological measurements. CONCLUSION: A significant amount of literature examined estimation of core temperature using non-invasive methods, sometimes integrated into wearables. Heat-stress management practices could include perceptual measures to better evaluate heat-strain.
Assuntos
Transtornos de Estresse por Calor , Temperatura Alta , Frequência Cardíaca , Resposta ao Choque Térmico , HumanosRESUMO
Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.
Assuntos
Transporte Biológico/fisiologia , Cálcio/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Relaxamento Muscular/fisiologia , Proteolipídeos/genética , Proteolipídeos/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Mutação , Miocárdio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
In this study, we aimed to directly quantify the relative contribution of Ca(2+) cycling to resting metabolic rate in mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) skeletal muscle. Resting oxygen consumption of isolated muscles (Vo(2), microl.g wet wt(-1).s(-1)) measured polarographically at 30 degrees C was approximately 25% higher in soleus (0.61 +/- .03) than in EDL (0.46 +/- .03). To quantify the specific contribution of Ca(2+) cycling to resting metabolic rate, cyclopiazonic acid (CPA), a highly specific inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPases (SERCAs), was added to the bath at different concentrations (1, 5, 10, and 15 microM). There was a concentration-dependent effect of CPA on Vo(2), with increasing CPA concentrations up to 10 microM resulting in progressively greater reductions in muscle Vo(2). There were no differences between 10 and 15 microM CPA, indicating that 10 microM CPA induces maximal inhibition of SERCAs in isolated muscle preparations. Relative reduction in muscle Vo(2) in response to CPA was nearly identical in EDL (1 microM, 10.6 +/- 3.0%; 5 microM, 33.2 +/- 3.4%; 10 microM, 49.2 +/- 2.9%; 15 microM, 50.9 +/- 2.1%) and soleus (1 microM, 11.2 +/- 1.5%; 5 microM, 37.7 +/- 2.4%; 10 microM, 50.0 +/- 1.3%; 15 microM, 49.9 +/- 1.6%). The results indicate that ATP consumption by SERCAs is responsible for approximately 50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30 degrees C. Thus SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.
Assuntos
Trifosfato de Adenosina/metabolismo , Metabolismo Basal , Cálcio/metabolismo , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Lenta/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Metabolismo Basal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Indóis/farmacologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/metabolismo , Consumo de Oxigênio , Polarografia , Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Sulfonamidas/farmacologia , Tolueno/análogos & derivados , Tolueno/farmacologiaRESUMO
Aging skeletal muscle shows an increased time to peak force and relaxation and a decreased specific force, all of which could relate to changes in muscle Ca(2+) handling. The purpose of this study was to determine if Ca(2+)-handling protein content and function are decreased in senescent gastrocnemius muscle and if initiating a training program in late middle age (LMA, 29 mo old) could improve function in senescent (34- to 36-mo-old) muscle. LMA male Fischer 344 x Brown-Norway rats underwent 5-7 mo of treadmill training. Aging resulted in a decrease in maximal sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity and a decrease in Ca(2+) release rate but no change in Ca(2+) uptake rate. Efficiency of the Ca(2+) pump was increased with age, as was the content of SERCA2a. Training caused a further increase in SERCA2a content. Aging also caused an increase in protein carbonyl and reactive nitrogen species damage accumulation, and both further increased with training. Consistent with the increase in oxidative damage, heat shock protein 70 content was increased with age and further increased with training. Together, these results suggest that while initiating exercise training in LMA augments the age-related increase in expression of heat shock protein 70 and the more efficient SERCA2a isoform, it did not prevent the decrease in SERCA activity and exacerbated oxidative damage in senescent gastrocnemius muscle.
Assuntos
Adaptação Fisiológica/fisiologia , Envelhecimento/fisiologia , Cálcio/metabolismo , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Fatores Etários , Animais , Western Blotting , Teste de Esforço , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Resistência Física/fisiologia , Carbonilação Proteica/fisiologia , Ratos , Ratos Endogâmicos F344 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
This study examined the effects of 10 days of buthionine sulfoximine (BSO) treatment on in vitro contractility and sarcoplasmic reticulum calcium pump (SERCA) expression and function in adult (AD; 6-8 months old) and middle aged (MA; 14-17 months old) rat diaphragm in both the basal state and following fatiguing stimulation. BSO treatment reduced the cellular concentrations of free glutathione (GSH) by >95% and oxidized glutathione (GSSG) by >80% in both age cohorts. GSH content in AD Control diaphragm was 32% higher (P < 0.01) than in MA Control, with no differences in GSSG. The ratio of GSH:GSSG, an indicator of cellular oxidative state, was 34.6 ± 7.4 in MA Control, 52.5 ± 10.1 in AD Control, 10.6 ± 1.7 in MA BSO, and 9.5 ± 1.1 in AD BSO (BSO vs. Control, P < 0.05). Several findings suggest that the effects of BSO treatment are age dependent. AD BSO diaphragm had 26% higher twitch and 28% higher tetanic force (both P < 0.05) than AD Controls, whereas no significant difference existed between the two MA groups. In contrast to our previous work on BSO-treated AD rats, BSO treatment did not influence maximal SERCA ATPase activity in MA rat diaphragm, nor did SERCA2a expression increase in BSO-treated MA diaphragm. Biotinylated iodoacetamide binding to SERCA1a, a specific marker of free cysteine residues, was reduced by 35% (P < 0.05) in AD Control diaphragm following fatiguing stimulation, but was not reduced in any other group. Collectively, these results suggest an important role for redox regulation in both contractility and SERCA function which is influenced by aging.
RESUMO
Centronuclear myopathy (CNM) is a congenital myopathy that is histopathologically characterized by centrally located nuclei, central aggregation of oxidative activity, and type I fiber predominance and hypotrophy. Here, we obtained commercially available mice overexpressing phospholamban (Pln(OE)), a well-known inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), in their slow-twitch type I skeletal muscle fibers to determine the effects on SERCA function. As expected with a 6- to 7-fold overexpression of phospholamban, SERCA dysfunction was evident in Pln(OE) muscles, with marked reductions in rates of Ca(2+) uptake, maximal ATPase activity and the apparent affinity of SERCA for Ca(2+). However, our most significant discovery was that the soleus and gluteus minimus muscles from the Pln(OE) mice displayed overt signs of myopathy: they histopathologically resembled human CNM, with centrally located nuclei, central aggregation of oxidative activity, type I fiber predominance and hypotrophy, progressive fibrosis and muscle weakness. This phenotype is associated with significant upregulation of muscle sarcolipin and dynamin 2, increased Ca(2+)-activated proteolysis, oxidative stress and protein nitrosylation. Moreover, in our assessment of muscle biopsies from three human CNM patients, we found a significant 53% reduction in SERCA activity and increases in both total and monomeric PLN content compared with five healthy subjects, thereby justifying future studies with more CNM patients. Altogether, our results suggest that the commercially available Pln(OE) mouse phenotypically resembles human CNM and could be used as a model to test potential mechanisms and therapeutic strategies. To date, there is no cure for CNM and our results suggest that targeting SERCA function, which has already been shown to be an effective therapeutic target for murine muscular dystrophy and human cardiomyopathy, might represent a novel therapeutic strategy to combat CNM.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Adolescente , Adulto , Animais , Cálcio/farmacologia , Dinamina II/metabolismo , Fibrose , Humanos , Masculino , Camundongos , Músculos/enzimologia , Músculos/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Distrofia Muscular Animal/patologia , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Proteólise/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Calcium/calmodulin-dependent protein kinase (CaMK) activation induces mitochondrial biogenesis in response to increasing cytosolic calcium concentrations. Calcium leak from the ryanodine receptor (RyR) is regulated by reactive oxygen species (ROS), which is increased with high-fat feeding. We examined whether ROS-induced CaMKII-mediated signaling induced skeletal muscle mitochondrial biogenesis in selected models of lipid oversupply. In obese Zucker rats and high-fat-fed rodents, in which muscle mitochondrial content was upregulated, CaMKII phosphorylation was increased independent of changes in calcium uptake because sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) protein expression or activity was not altered, implicating altered sarcoplasmic reticulum (SR) calcium leak in the activation of CaMKII. In support of this, we found that high-fat feeding increased mitochondrial ROS emission and S-nitrosylation of the RyR, whereas hydrogen peroxide induced SR calcium leak from the RyR and activation of CaMKII. Moreover, administration of a mitochondrial-specific antioxidant, SkQ, prevented high-fat diet-induced phosphorylation of CaMKII and the induction of mitochondrial biogenesis. Altogether, these data suggest that increased mitochondrial ROS emission is required for the induction of SR calcium leak, activation of CaMKII, and induction of mitochondrial biogenesis in response to excess lipid availability.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dieta Hiperlipídica , Mitocôndrias/metabolismo , Células Musculares/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Retículo Sarcoplasmático/fisiologia , Animais , Glicemia/metabolismo , Western Blotting , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio , Ativação Enzimática , Peróxido de Hidrogênio/metabolismo , Masculino , Fosforilação , Ratos , Ratos Zucker , Trocador de Sódio e Cálcio/metabolismo , Regulação para CimaRESUMO
The main purpose of this study was to directly quantify the relative contribution of Ca²âº cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO2, µL/g wet weight/s) measured polarographically at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca²âº cycling to resting metabolic rate, the concentration of MgCl2 in the bath was increased to 10 mM to block Ca²âº release through the ryanodine receptor, thus eliminating a major source of Ca²âº leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca²âº-ATPases (SERCAs). The relative (%) reduction in muscle VO2 in response to 10 mM MgCl2 was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca²âº cycling in resting isolated muscles. The results of the MgCl2 experiments indicate that ATP consumption by SERCAs is responsible for 40-50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C, or 12-15% of whole body resting VO2. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.
Assuntos
Trifosfato de Adenosina/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Metabolismo Basal , Cálcio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Oxigênio/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/análiseRESUMO
The purpose of this study was to examine the effects of sarcolipin (SLN) on sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA pump) energetics in vivo and resting skeletal muscle metabolic rate. Using SLN knockout (Sln(-/-)) mice we show that SLN ablation increases the transport stoichiometry of SERCA pumps (Ca(2+) uptake/Ca(2+)-ATPase activity) and decreases the relative contribution of SERCA pumps to resting oxygen consumption (VO2) in soleus without affecting soleus or whole body VO2. These data suggest that the lower energy requirements for Ca(2+) cycling in resting skeletal muscle of Sln(-/-) mice do not impact significantly either skeletal muscle or whole body metabolic rate.
Assuntos
Cálcio/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteolipídeos/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Metabolismo Energético , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/deficiência , Consumo de Oxigênio , Proteolipídeos/deficiência , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologiaRESUMO
Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) by reducing their apparent affinity for Ca(2+). A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca(2+) affinity of SERCA (K Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (µmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Proteolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adolescente , Adulto , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Ativação Enzimática , Expressão Gênica , Humanos , Isoenzimas , Masculino , Camundongos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/genética , Cadeias Pesadas de Miosina/metabolismo , Ligação Proteica , Proteolipídeos/genética , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Adulto JovemRESUMO
The purpose of this investigation was to determine the effects of acute physiological GSH administration on endothelium-mediated reduction in coronary vascular resistance (CVR) using isolated perfused Sprague-Dawley rat hearts. A dose-response curve to GSH was conducted to determine a threshold concentration of GSH. We demonstrate that 30 µM GSH was sufficient to reduce CVR, and maximal dilation was achieved with 1 mM. In subsequent experiments, GSH was administered at concentrations of 0 [control (CON)], 1 µM, or 10 µM (GSH(10)), and dose-response curves to the endothelial agonist bradykinin (BK) were constructed. These GSH concentrations were chosen because of the physiological relevance and because the effects of GSH on BK action could be assessed independent of baseline differences in CVR. Sensitivity to BK (EC(50)) was enhanced in GSH(10) vs. CON (P < 0.05). This enhancement remained in the presence of nitric oxide (NO) synthase inhibition l-(ω)nitro-l-arginine (lNAME) and/or soluble guanylate cyclase (sGC) inhibition. Treatment with 4-hydroxy (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPOL) enhanced the sensitivity to BK in CON, similar to the effects of GSH(10) and GSH(10) + TEMPOL. However, the GSH(10)-dependent enhancement of EC(50) observed in the presence of lNAME did not occur in the presence of lNAME + TEMPOL or in the presence of lNAME + sGC inhibition and NO scavenging. Collectively, these results suggest that GSH enhances BK-mediated dilation and reduction in CVR through an antioxidant-dependent mechanism that involves a NO intermediate but is unrelated to acute production of NO and GC-dependent effects of NO. These results suggest a mechanism whereby physiologically relevant levels of GSH modulate the endogenous reactive oxygen species and NO control of endothelium-dependent coronary vascular function.
Assuntos
Vasos Coronários/fisiologia , Endotélio Vascular/fisiologia , Glutationa/farmacologia , Óxido Nítrico/química , Espécies Reativas de Oxigênio/metabolismo , Resistência Vascular/fisiologia , Animais , Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resistência Vascular/efeitos dos fármacosRESUMO
The senescent heart has decreased systolic and diastolic functions, both of which could be related to alterations in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) handling. The purpose of this study was to determine if SR protein content and rates of Ca(2+) release and uptake and ATPase activity are lower in the senescent (34-36 mo) Fisher 344×Brown-Norway F1 hybrid rat heart and if a long-term exercise training program could maintain SR function. Late middle aged (29 mo) male rats underwent 5-7 mo of treadmill training. Aging resulted in a decrease in SERCA activity and modest decrease in the rate of Ca(2+) uptake but no change in Ca(2+) release rate. SERCA2a content was not decreased with age but nitrotyrosine accumulation was increased and Ser16 phosphorylated phospholamban (PLN) was decreased. Ryanodine receptor content was not decreased with age but dihydropyridine receptor content was decreased in the senescent heart. Treadmill training had no significant effect on any of the SR properties or protein contents in the senescent rat heart. These results suggest that decreases in Ca(2+) uptake and SERCA activity in the senescent F344BN rat heart are due to increased SERCA2a damage from nitrotyrosine accumulation and inhibition by PLN and that exercise training initiated at late middle age is unable to prevent these age-related changes in cardiac SR function.
Assuntos
Adaptação Fisiológica , Envelhecimento/metabolismo , Cálcio/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Condicionamento Físico Animal , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Envelhecimento/fisiologia , Animais , Western Blotting , Masculino , Estresse Oxidativo , Fosforilação , Resistência Física , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344RESUMO
The overexpression of heat shock protein 70 (Hsp70) provides cytoprotection to cells, making them resistant to otherwise lethal levels of stress. In this review, the role Hsp70 plays in protecting both cardiac and skeletal muscle against the pathophysiological effects of oxidative stress are examined, with a focus on the molecular basis for the cytoprotective effects of Hsp70. The ability of Hsp70 to maintain cell survival undoubtedly involves the regulation of multiple steps within apoptotic pathways, but could also involve the regulation of key upstream mediators of apoptosis (i.e., oxidative stress, Ca2+ overload). Hsp70 can stabilize the structure and function of both the skeletal muscle and cardiac Ca2+ pump under heat stress conditions. Given that Ca2+ overload has long been implicated in cell death, Hsp70 might protect muscle cells by maintaining cellular Ca2+ homeostasis, thereby preventing the initiation of apoptosis. The functional interaction between Hsp70 and Ca2+ pumps might also promote improvements in muscle contractility after exposure to oxidative stress.