RESUMO
The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca2+ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca2+, and the calculated rate of Ca2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca2+ release, and thus could be involved in voltage sensor rearrangements or Ca2+ release activation.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo L/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Acoplamento Excitação-Contração , Ativação do Canal Iônico , Camundongos , Coelhos , Retículo Sarcoplasmático/metabolismoRESUMO
Insulin resistance (IR) is one of the hallmarks of heart failure (HF). Abnormalities in skeletal muscle (SM) metabolism have been identified in patients with HF. However, the underlying mechanisms of IR development in SM in HF are poorly understood. Herein, we hypothesize that HF upregulates miR-133b in SM and in turn alters glucose metabolism and the propensity toward IR. Mitochondria isolated from SM of mice with HF induced by transverse aortic constriction (TAC) showed lower respiration and downregulation of muscle-specific components of the tricarboxylic acid (TCA) cycle, AMP deaminase 1 (AMPD1), and fumarate compared with those from control animals. RNA-Seq and subsequent qPCR validation confirmed upregulation of SM-specific microRNA (miRNA), miR-133b, in TAC versus sham animals. miR-133b overexpression alone resulted in significantly lower mitochondrial respiration, cellular glucose uptake, and glycolysis along with lower ATP production and cellular energy reserve compared with the scramble (Scr) in C2C12 cells. miR-133b binds to the 3'-untranslated region (UTR) of KLF15, the transcription factor for the insulin-sensitive glucose transporter, GLUT4. Overexpression of miR-133b lowers GLUT4 and lowers pAkt in presence of insulin in C2C12 cells. Finally, lowering miR-133b in primary skeletal myocytes isolated from TAC mice using antagomir-133b reversed the changes in KLF15, GLUT4, and AMPD1 compared with the scramble-transfected myocytes. Taken together, these data demonstrate a role for SM miR-133b in altered glucose metabolism in HF and suggest the therapeutic potential in HF to improve glucose uptake and glycolysis by restoring GLUT4 abundance. The data uncover a novel mechanism for IR and ultimately SM metabolic abnormalities in patients with HF.NEW & NOTEWORTHY Heart failure is associated with systemic insulin resistance and abnormalities in glucose metabolism but the underlying mechanisms are poorly understood. In the skeletal muscle, the major peripheral site of glucose utilization, we observe an increase in miR-133b in heart failure mice, which reduces the insulin-sensitive glucose transporter (GLUT4), glucose uptake, and metabolism in C2C12 and in myocytes. The antagomir for miR-133b restores GLUT4 protein and markers of metabolism in skeletal myocytes from heart failure mice demonstrating that miR-133b is an exciting target for systemic insulin resistance in heart failure and an important player in the cross talk between the heart and the periphery in the heart failure syndrome.
Assuntos
Insuficiência Cardíaca , Resistência à Insulina , MicroRNAs , Camundongos , Animais , Resistência à Insulina/genética , Antagomirs/metabolismo , Músculo Esquelético/metabolismo , Glucose/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Insulina/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismoRESUMO
Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.
Assuntos
Mama/patologia , Cálcio/metabolismo , Mecanotransdução Celular , NADPH Oxidase 2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Microtúbulos/metabolismo , NADPH Oxidase 2/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Taxa de Sobrevida , Canais de Cátion TRPM/genética , Microambiente TumoralRESUMO
The majority of voltage-gated ion channels contain a defined voltage-sensing domain and a pore domain composed of highly conserved amino acid residues that confer electrical excitability via electromechanical coupling. In this sense, the voltage-gated proton channel (Hv1) is a unique protein in that voltage-sensing, proton permeation and pH-dependent modulation involve the same structural region. In fact, these processes synergistically work in concert, and it is difficult to separate them. To investigate the process of Hv1 voltage sensor trapping, we follow voltage-sensor movements directly by leveraging mutations that enable the measurement of Hv1 channel gating currents. We uncover that the process of voltage sensor displacement is due to two driving forces. The first reveals that mutations in the selectivity filter (D160) located in the S1 transmembrane interact with the voltage sensor. More hydrophobic amino acids increase the energy barrier for voltage sensor activation. On the other hand, the effect of positive charges near position 264 promotes the formation of salt bridges between the arginines of the voltage sensor domain, achieving a stable conformation over time. Our results suggest that the activation of the Hv1 voltage sensor is governed by electrostatic-hydrophobic interactions, and S4 arginines, N264 and selectivity filter (D160) are essential in the Ciona-Hv1 to understand the trapping of the voltage sensor.
Assuntos
Antifibrinolíticos , Ciona , Animais , Prótons , Aminoácidos , ArgininaRESUMO
Pregnancies complicated by preexisting maternal diabetes mellitus are associated with a higher risk of birth defects in infants, known as diabetic embryopathy. The common defects seen in the central nervous system result from failure of neural tube closure. The formation of neural tube defects (NTDs) is associated with excessive programmed cell death (apoptosis) in the neuroepithelium under hyperglycemia-induced intracellular stress conditions. The early cellular response to hyperglycemia remains to be identified. We hypothesize that hyperglycemia may disturb intracellular calcium (Ca2+) homeostasis, which perturbs organelle function and apoptotic regulation, resulting in increased apoptosis and embryonic NTDs. In an animal model of diabetic embryopathy, we performed Ca2+ imaging and observed significant increases in intracellular Ca2+ ([Ca2+]i) in the embryonic neural epithelium. Blocking T-type Ca2+ channels with mibefradil, but not L-type with verapamil, significantly blunted the increases in [Ca2+]i, implicating an involvement of channel type-dependent Ca2+ influx in hyperglycemia-perturbed Ca2+ homeostasis. Treatment of diabetic pregnant mice with mibefradil during neurulation significantly reduced NTD rates in the embryos. This effect was associated with decreases in apoptosis, alleviation of endoplasmic reticulum stress, and increases of anti-apoptotic factors. Taken together, our data suggest an important role of Ca2+ influx in hyperglycemia-induced NTDs and of T-type Ca2+ channels as a potential target to prevent birth defects in diabetic pregnancies.
Assuntos
Cálcio/metabolismo , Hiperglicemia/complicações , Defeitos do Tubo Neural/etiologia , Gravidez em Diabéticas/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Feminino , Doenças Fetais/etiologia , Doenças Fetais/metabolismo , Glucose/metabolismo , Hiperglicemia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Defeitos do Tubo Neural/metabolismo , GravidezRESUMO
Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.
Assuntos
Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio , Proteínas do Citoesqueleto/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Vasoconstrição , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Animais , Aorta/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Feminino , Regulação da Expressão Gênica , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/ultraestrutura , Receptores de LDL/deficiência , Receptores de LDL/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Técnicas de Cultura de Tecidos , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Skeletal muscle fibers have been used to examine a variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential (AP) propagation has been measured to assess the integrity and function of skeletal muscle. In this work, we utilize 1-(3-sulfonatopropyl)-4[ß[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine, a potentiometric dye, and mag-fluo-4, a low-affinity intracellular Ca2+indicator, to noninvasively and reliably measure AP conduction velocity in skeletal muscle. We used remote extracellular bipolar electrodes to generate an alternating polarity electric field that initiates an AP at either end of the fiber. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers and high-speed line scans, we determine the conduction velocity to be â¼0.4 m/s. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions and confirmed that the conduction velocity is significantly reduced in elevated [K+]o. Because our recorded velocities for FDB fibers were much slower than previously reported for other muscle groups, we compared the conduction velocity in FDB fibers to that of extensor digitorum longus (EDL) fibers and measured a significantly faster velocity in EDL fibers than FDB fibers. As a basis for this difference in conduction velocity, we found a similarly higher level of expression of Na+ channels in EDL than in FDB fibers. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by applying tetrodotoxin and have constructed a circuit model of a skeletal muscle fiber to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how various pathologies and mutations affect skeletal muscle AP propagation. Our work demonstrates the utility of using 1-(3-sulfonatopropyl)-4[ß[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine or mag-fluo-4 to noninvasively measure AP initiation and conduction.
Assuntos
Potenciais de Ação , Fibras Musculares Esqueléticas/fisiologia , Potássio/metabolismo , Sódio/metabolismo , Animais , Estimulação Elétrica , Feminino , Camundongos , Fibras Musculares Esqueléticas/citologiaRESUMO
Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.
Assuntos
Sinalização do Cálcio , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Histona Desacetilases/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas S100/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Ativação Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/genéticaRESUMO
INTRODUCTION: Respiratory and locomotor skeletal muscle dysfunction are common findings in chronic obstructive pulmonary disease (COPD); however, the mechanisms that cause muscle impairment in COPD are unclear. Because Ca2+ signaling in excitation-contraction (E-C) coupling is important for muscle activity, we hypothesized that Ca2+ dysregulation could contribute to muscle dysfunction in COPD. METHODS: Intercostal and flexor digitorum brevis muscles from control and cigarette smoke-exposed mice were investigated. We used single cell Ca2+ imaging and Western blot assays to assess Ca2+ signals and E-C coupling proteins. RESULTS: We found impaired Ca2+ signals in muscle fibers from both muscle types, without significant changes in releasable Ca2+ or in the expression levels of E-C coupling proteins. CONCLUSIONS: Ca2+ dysregulation may contribute or accompany respiratory and locomotor muscle dysfunction in COPD. These findings are of significance to the understanding of the pathophysiological course of COPD in respiratory and locomotor muscles. Muscle Nerve 56: 282-291, 2017.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Pé/inervação , Fibras Musculares Esqueléticas/fisiologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/efeitos adversos , Potenciais de Ação/fisiologia , Poluentes Atmosféricos/toxicidade , Animais , Calmodulina/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas S100/metabolismoRESUMO
The α1 and ß1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca(2+) release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the ß1a subunit and this effect can be mimicked by a peptide (ß1a490-524) corresponding to the 35-residue C-terminal tail of the ß1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the ß1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (ß1a490-508) is sufficient to reproduce activating effects of ß1a490-524. Here we examined the effects of ß1a490-508 on Ca(2+) release and Ca(2+) currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. ß1a490-508 (25 nM) increased the peak Ca(2+) release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of ß1a490-508 in fibers. ß1a490-508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of ß1a490-508 peptide was used as negative control and produced negligible effects on Ca(2+) release flux and RyR1 activity. Our results show that the ß1a490-508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Canais de Cálcio Tipo L/química , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.
RESUMO
Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.
Assuntos
Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Núcleo Celular/genética , Estimulação Elétrica/métodos , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismoRESUMO
The CaV1.1 voltage-gated Ca2+ channel carries L-type Ca2+ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, CaV1.1 was the first voltage-gated Ca2+ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how CaV1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca2+ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.
Assuntos
Canais de Cálcio Tipo L , Músculo Esquelético , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/metabolismo , Acoplamento Excitação-Contração/fisiologia , Potenciais da Membrana/fisiologia , Retículo Sarcoplasmático/metabolismo , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels' activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.
Assuntos
Cisteína , Músculo Esquelético , Camundongos , Animais , Cisteína/química , Músculo Esquelético/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Canais Iônicos , Fluorometria/métodos , MamíferosRESUMO
In skeletal muscle, CaV 1.1 serves as the voltage sensor for both excitation-contraction coupling (ECC) and L-type Ca2+ channel activation. We have recently adapted the technique of action potential (AP) voltage clamp (APVC) to monitor the current generated by the movement of intramembrane voltage sensors (IQ ) during single imposed transverse tubular AP-like depolarization waveforms (IQAP ). We now extend this procedure to monitoring IQAP , and Ca2+ currents during trains of tubular AP-like waveforms in adult murine skeletal muscle fibers, and compare them with the trajectories of APs and AP-induced Ca2+ release measured in other fibers using field stimulation and optical probes. The AP waveform remains relatively constant during brief trains (<1 sec) for propagating APs in non-V clamped fibers. Trains of 10 AP-like depolarizations at 10 Hz (900 ms), 50 Hz (180 ms), or 100 Hz (90 ms) did not alter IQAP amplitude or kinetics, consistent with previous findings in isolated muscle fibers where negligible charge immobilization occurred during 100 ms step depolarizations. Using field stimulation, Ca2+ release did exhibit a considerable decline from pulse to pulse during the train, also consistent with previous findings, indicating that the decline of Ca2+ release during a short train of APs is not correlated to modification of charge movement. Ca2+ currents during single or 10 Hz trains of AP-like depolarizations were hardly detectable, were minimal during 50 Hz trains, and became more evident during 100 Hz trains in some fibers. Our results verify predictions on the behavior of the ECC machinery in response to AP-like depolarizations and provide a direct demonstration that Ca2+ currents elicited by single AP-like waveforms are negligible, but can become more prominent in some fibers during short high-frequency train stimulation that elicits maximal isometric force.
Assuntos
Fibras Musculares Esqueléticas , Músculo Esquelético , Camundongos , Animais , Potenciais de Ação/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Acoplamento Excitação-Contração , CálcioRESUMO
Voltage-gated proton channels (Hv1) are important regulators of the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice and have been proposed as a potential therapeutic target to alleviate dysregulated immunosuppression in tumors. However, till date, there is a lack of evidence regarding the functioning of the Hvcn1 and reports on mHv1 isoform diversity in mice and MDSCs. A computational prediction has suggested that the Hvcn1 gene may express up to six transcript variants, three of which are translated into distinct N-terminal isoforms of mHv1: mHv1.1 (269 aa), mHv1.2 (269 + 42 aa), and mHv1.3 (269 + 4 aa). To validate this prediction, we used RT-PCR on total RNA extracted from MDSCs, and the presence of all six predicted mRNA variances was confirmed. Subsequently, the open-reading frames (ORFs) encoding for mHv1 isoforms were cloned and expressed in Xenopus laevis oocytes for proton current recording using a macro-patch voltage clamp. Our findings reveal that all three isoforms are mammalian mHv1 channels, with distinct differences in their activation properties. Specifically, the longest isoform, mHv1.2, displays a right-shifted conductance-voltage (GV) curve and slower opening kinetics, compared to the mid-length isoform, mHv1.3, and the shortest canonical isoform, mHv1.1. While mHv1.3 exhibits a V0.5 similar to that of mHv1.1, mHv1.3 demonstrates significantly slower activation kinetics than mHv1.1. These results suggest that isoform gating efficiency is inversely related to the length of the N-terminal end. To further explore this, we created the truncated mHv1.2 ΔN20 construct by removing the first 20 amino acids from the N-terminus of mHv1.2. This construct displayed intermediate activation properties, with a V0.5 value lying intermediate of mHv1.1 and mHv1.2, and activation kinetics that were faster than that of mHv1.2 but slower than that of mHv1.1. Overall, these findings indicate that alternative splicing of the N-terminal exon in mRNA transcripts encoding mHv1 isoforms is a regulatory mechanism for mHv1 function within MDSCs. While MDSCs have the capability to translate multiple Hv1 isoforms with varying gating properties, the Hvcn1 gene promotes the dominant expression of mHv1.1, which exhibits the most efficient gating among all mHv1 isoforms.
RESUMO
Reactive oxygen species (ROS) have been linked to oxidation and nuclear efflux of class IIa histone deacetylase 4 (HDAC4) in cardiac muscle. Here we use HDAC-GFP fusion proteins expressed in isolated adult mouse flexor digitorum brevis muscle fibers to study ROS mediation of HDAC localization in skeletal muscle. H(2)O(2) causes nuclear efflux of HDAC4-GFP or HDAC5-GFP, which is blocked by the ROS scavenger N-acetyl-l-cysteine (NAC). Repetitive stimulation with 100-ms trains at 50 Hz, 2/s ("50-Hz trains") increased ROS production and caused HDAC4-GFP or HDAC5-GFP nuclear efflux. During 50-Hz trains, HDAC5-GFP nuclear efflux was completely blocked by NAC, but HDAC4-GFP nuclear efflux was only partially blocked by NAC and partially blocked by the calcium-dependent protein kinase (CaMK) inhibitor KN-62. Thus, during intense activity both ROS and CaMK play roles in nuclear efflux of HDAC4, but only ROS mediates HDAC5 nuclear efflux. The 10-Hz continuous stimulation did not increase the rate of ROS production and did not cause HDAC5-GFP nuclear efflux but promoted HDAC4-GFP nuclear efflux that was sensitive to KN-62 but not NAC and thus mediated by CaMK but not by ROS. Fibers from NOX2 knockout mice lacked ROS production and ROS-dependent nuclear efflux of HDAC5-GFP or HDAC4-GFP during 50-Hz trains but had unmodified Ca(2+) transients. Our results demonstrate that ROS generated by NOX2 could play important roles in muscle remodeling due to intense muscle activity and that the nuclear effluxes of HDAC4 and HDAC5 are differentially regulated by Ca(2+) and ROS during muscle activity.
Assuntos
Histona Desacetilases/metabolismo , Glicoproteínas de Membrana/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Acetilcisteína/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares de Contração Rápida/efeitos dos fármacos , NADPH Oxidase 2 , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Ca2+ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca2+ influx, to mediate disparate functions is diversity of HVACC pore-forming α1 and auxiliary CaVß1-CaVß4 subunits. Selective CaVα1 blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on CaVß isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of CaVß isoforms. Here, we identify a nanobody (nb.E8) that selectively binds CaVß1 SH3 domain and inhibits CaVß1-associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through CaVß1-reconstituted CaV1/CaV2 and native CaV1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca2+ influx and excitation-transcription coupling in hippocampal neurons, but was inert against CaVß2-associated CaV1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of CaVß1 signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.
Assuntos
Canais de Cálcio , Miócitos Cardíacos , Canais de Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Domínios de Homologia de src , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismoRESUMO
In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²âº release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²44²-Pro²477 of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²âº release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²âº release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²âº current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.
Assuntos
Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Compostos de Anilina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Diálise , Fluorescência , Técnicas In Vitro , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Dinâmica não Linear , Técnicas de Patch-Clamp , Conformação Proteica , Fatores de Tempo , Xantenos/metabolismoRESUMO
In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.