RESUMO
OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Neuropeptídeos/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Genótipo , Proteínas de Arcabouço Homer/metabolismo , Humanos , Hiperplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Fenótipo , Ligação Proteica , Transdução de Sinais , Transfecção , Canais de Potencial de Receptor Transitório/metabolismo , Remodelação VascularRESUMO
BACKGROUND: Spontaneously depolarizing nodal cells comprise the pacemaker of the heart. Intracellular calcium (Ca2+) plays a critical role in mediating nodal cell automaticity and understanding this so-called Ca2+ clock is critical to understanding nodal arrhythmias. We previously demonstrated a role for Jph2 (junctophilin 2) in regulating Ca2+-signaling through inhibition of RyR2 (ryanodine receptor 2) Ca2+ leak in cardiac myocytes; however, its role in pacemaker function and nodal arrhythmias remains unknown. We sought to determine whether nodal Jph2 expression silencing causes increased sinoatrial and atrioventricular nodal cell automaticity due to aberrant RyR2 Ca2+ leak. METHODS: A tamoxifen-inducible, nodal tissue-specific, knockdown mouse of Jph2 was achieved using a Cre-recombinase-triggered short RNA hairpin directed against Jph2 (Hcn4:shJph2). In vivo cardiac rhythm was monitored by surface ECG, implantable cardiac telemetry, and intracardiac electrophysiology studies. Intracellular Ca2+ imaging was performed using confocal-based line scans of isolated nodal cells loaded with fluorescent Ca2+ reporter Cal-520. Whole cell patch clamp was conducted on isolated nodal cells to determine action potential kinetics and sodium-calcium exchanger function. RESULTS: Hcn4:shJph2 mice demonstrated a 40% reduction in nodal Jph2 expression, resting sinus tachycardia, and impaired heart rate response to pharmacologic stress. In vivo intracardiac electrophysiology studies and ex vivo optical mapping demonstrated accelerated junctional rhythm originating from the atrioventricular node. Hcn4:shJph2 nodal cells demonstrated increased and irregular Ca2+ transient generation with increased Ca2+ spark frequency and Ca2+ leak from the sarcoplasmic reticulum. This was associated with increased nodal cell AP firing rate, faster diastolic repolarization rate, and reduced sodium-calcium exchanger activity during repolarized states compared to control. Phenome-wide association studies of the JPH2 locus identified an association with sinoatrial nodal disease and atrioventricular nodal block. CONCLUSIONS: Nodal-specific Jph2 knockdown causes increased nodal automaticity through increased Ca2+ leak from intracellular stores. Dysregulated intracellular Ca2+ underlies nodal arrhythmogenesis in this mouse model.
Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Camundongos , Cálcio/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Nó Sinoatrial , Trocador de Sódio e Cálcio/metabolismoRESUMO
BACKGROUND: Cardiac channelopathies such as catecholaminergic polymorphic tachycardia and long QT syndrome predispose patients to fatal arrhythmias and sudden cardiac death. As genetic testing has become common in clinical practice, variants of uncertain significance (VUS) in genes associated with catecholaminergic polymorphic ventricular tachycardia and long QT syndrome are frequently found. The objective of this study was to predict pathogenicity of catecholaminergic polymorphic ventricular tachycardia-associated RYR2 VUS and long QT syndrome-associated VUS in KCNQ1, KCNH2, and SCN5A by developing gene-specific machine learning models and assessing them using cross-validation, cellular electrophysiological data, and clinical correlation. METHODS: The GENe-specific EnSemble grId Search framework was developed to identify high-performing machine learning models for RYR2, KCNQ1, KCNH2, and SCN5A using variant- and protein-specific inputs. Final models were applied to datasets of VUS identified from ClinVar and exome sequencing. Whole cell patch clamp and clinical correlation of selected VUS was performed. RESULTS: The GENe-specific EnSemble grId Search models outperformed alternative methods, with area under the receiver operating characteristics up to 0.87, average precisions up to 0.83, and calibration slopes as close to 1.0 (perfect) as 1.04. Blinded voltage-clamp analysis of HEK293T cells expressing 2 predicted pathogenic variants in KCNQ1 each revealed an ≈80% reduction of peak Kv7.1 current compared with WT. Normal Kv7.1 function was observed in KCNQ1-V241I HEK cells as predicted. Though predicted benign, loss of Kv7.1 function was observed for KCNQ1-V106D HEK cells. Clinical correlation of 9/10 variants supported model predictions. CONCLUSIONS: Gene-specific machine learning models may have a role in post-genetic testing diagnostic analyses by providing high performance prediction of variant pathogenicity.
Assuntos
Síndrome do QT Longo , Taquicardia Ventricular , Arritmias Cardíacas/genética , Células HEK293 , Humanos , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/genética , Aprendizado de Máquina , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/genéticaRESUMO
RATIONALE: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein-coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. OBJECTIVE: Transient receptor potential canonical (TRPC) channels are G protein-coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. METHODS AND RESULTS: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1(-)(/)(-) mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1(-)(/)(-) mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1(-)(/)(-) mice. CONCLUSIONS: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.
Assuntos
Sinalização do Cálcio , Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Estresse Fisiológico , Canais de Cátion TRPC/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Cardiomegalia/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Insuficiência Cardíaca/genética , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR , Canais de Cátion TRPC/genéticaRESUMO
Aims: The actin-binding protein Drebrin is up-regulated in response to arterial injury and reduces smooth muscle cell (SMC) migration and proliferation through its interaction with the actin cytoskeleton. We, therefore, tested the hypothesis that SMC Drebrin inhibits angiotensin II-induced remodelling of the proximal aorta. Methods and results: Angiotensin II was administered via osmotic minipumps at 1000 ng/kg/min continuously for 28 days in SM22-Cre+/Dbnflox/flox (SMC-Dbn-/-) and control mice. Blood pressure responses to angiotensin II were assessed by telemetry. After angiotensin II infusion, we assessed remodelling in the proximal ascending aorta by echocardiography and planimetry of histological cross sections. Although the degree of hypertension was equivalent in SMC-Dbn-/- and control mice, SMC-Dbn-/- mice nonetheless exhibited 60% more proximal aortic medial thickening and two-fold more outward aortic remodelling than control mice in response to angiotensin II. Proximal aortas demonstrated greater cellular proliferation and matrix deposition in SMC-Dbn-/- mice than in control mice, as evidenced by a higher prevalence of proliferating cell nuclear antigen-positive nuclei and higher levels of collagen I. Compared with control mouse aortas, SMC-Dbn-/- aortas demonstrated greater angiotensin II-induced NADPH oxidase activation and inflammation, evidenced by higher levels of Ser-536-phosphorylated NFκB p65 subunits and higher levels of vascular cell adhesion molecule-1, matrix metalloproteinase-9, and adventitial macrophages. Conclusions: We conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.
Assuntos
Angiotensina II , Doenças da Aorta/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neuropeptídeos/metabolismo , Remodelação Vascular , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Pressão Arterial , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células HEK293 , Humanos , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , NADPH Oxidases/metabolismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Ca2+-activated ion channels shape membrane excitability and Ca2+ dynamics in response to cytoplasmic Ca2+ elevation. Compared to the Ca2+-activated K+ channels, known as BK and SK channels, the physiological importance of Ca2+-activated Cl- channels (CaCCs) in neurons has been largely overlooked. Here we report that CaCCs coexist with BK and SK channels in inferior olivary (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for the control of motor learning and timing. Ca2+ influx through the dendritic high-threshold voltage-gated Ca2+ channels activates CaCCs, which contribute to membrane repolarization of IO neurons. Loss of TMEM16B expression resulted in the absence of CaCCs in IO neurons, leading to markedly diminished action potential firing of IO neurons in TMEM16B knockout mice. Moreover, these mutant mice exhibited severe cerebellar motor learning deficits. Our findings thus advance the understanding of the neurophysiology of CaCCs and the ionic basis of IO neuron excitability.
Assuntos
Cerebelo/fisiologia , Canais de Cloreto/fisiologia , Aprendizagem/fisiologia , Destreza Motora/fisiologia , Núcleo Olivar/metabolismo , Potenciais de Ação/fisiologia , Animais , Anoctaminas , Cálcio/metabolismo , Cerebelo/citologia , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/fisiopatologia , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Núcleo Olivar/citologia , Células de Purkinje/fisiologiaRESUMO
BACKGROUND: Sinus bradycardia is frequently observed in patients treated with crizotinib, a receptor tyrosine kinase inhibitor used for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We investigated whether crizotinib could influence heart rate (HR) through direct cardiac effects. METHODS: The direct effect of crizotinib on HR was studied using ECG analysis of Langendorff-perfused mouse hearts. The whole-cell patch clamp technique was used to measure the effects of crizotinib on the hyperpolarization-activated funny current, If, in mouse sinoatrial node cells (SANCs) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) activity in HEK-293 cells stably expressing human HCN4. RESULTS: Crizotinib resulted in a dose-dependent reduction in HR in isolated intact mouse hearts with a half maximal inhibitory concentration (IC50) of 1.7 ± 0.4 µmol/L. Because ECG analysis revealed that crizotinib (0-5 µmol/L) resulted in significant reductions in HR in isolated mouse hearts without changes in PR, QRS, or QT intervals, we performed whole-cell patch clamp recordings of SANCs which showed that crizotinib inhibited If which regulates cardiac pacemaker activity. Crizotinib resulted in diminished current density of HCN4, the major molecular determinant of If, with an IC50 of 1.4 ± 0.3 µmol/L. Crizotinib also slowed HCN4 activation and shifted the activation curve to the left towards more hyperpolarized potentials. CONCLUSIONS: Our results suggest that crizotinib's effects on HCN4 channels play a significant role in mediating its observed effects on HR.
RESUMO
The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and ß-arrestin2, thereby regulating ß-adrenergic receptor (ßAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects ßAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac ß1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable ßAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued ß1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of ßAR signaling.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Contração Miocárdica/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Animais , Ecocardiografia , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Coração/diagnóstico por imagem , Coração/fisiologia , Hemodinâmica/efeitos dos fármacos , Isoproterenol/farmacologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
The homeodomain transcription factor Pdx-1 has important roles in pancreatic development and ß-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, cooverexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly ß-cell proliferation, whereas Pdx-1 stimulates both α- and ß-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1-stimulated but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 but not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and small interfering RNA (siRNA)-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1.
Assuntos
Células Secretoras de Glucagon/metabolismo , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Canais de Cátion TRPC/genética , Transativadores/genética , Adenoviridae/genética , Animais , Proliferação de Células , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Células Secretoras de Glucagon/citologia , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6 , Transativadores/metabolismoRESUMO
Immediately after birth, skeletal muscle must undergo an enormous period of growth and differentiation that is coordinated by several intertwined growth signaling pathways. How these pathways are integrated remains unclear but is likely to involve skeletal muscle contractile activity and calcium (Ca(2+)) signaling. Here, we show that Ca(2+) signaling governed by stromal interaction molecule 1 (STIM1) plays a central role in the integration of signaling and, therefore, muscle growth and differentiation. Conditional deletion of STIM1 from the skeletal muscle of mice (mSTIM1(-/-) mice) leads to profound growth delay, reduced myonuclear proliferation, and perinatal lethality. We show that muscle fibers of neonatal mSTIM1(-/-) mice cannot support the activity-dependent Ca(2+) transients evoked by tonic neurostimulation, even though excitation contraction coupling (ECC) remains unperturbed. In addition, disruption of tonic Ca(2+) signaling in muscle fibers attenuates downstream muscle growth signaling, such as that of calcineurin, mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AKT. Based on our findings, we propose a model wherein STIM1-mediated store-operated calcium entry (SOCE) governs the Ca(2+) signaling required for cellular processes that are necessary for neonatal muscle growth and differentiation.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/genética , Glicoproteínas de Membrana/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/fisiologia , Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Sistema de Sinalização das MAP Quinases , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/crescimento & desenvolvimento , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Molécula 1 de Interação Estromal , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.
Assuntos
Proteínas de Transporte , Estresse Oxidativo , Animais , Linhagem Celular , Dissulfetos , Proteínas de Arcabouço Homer , Humanos , Camundongos , Doenças Musculares/etiologia , Mutação , Miofibrilas/patologia , Doenças Neurodegenerativas/etiologia , Polimerização , Isoformas de Proteínas , SolubilidadeRESUMO
It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.
Assuntos
Cálcio/metabolismo , Glicoproteínas de Membrana/fisiologia , Animais , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Inativação Gênica , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Modelos Genéticos , Contração Muscular , Músculos/metabolismo , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Molécula 1 de Interação EstromalRESUMO
Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Deleção de Genes , Distrofias Musculares/fisiopatologia , Canais de Cátion TRPC/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteínas de Arcabouço Homer , Camundongos , Camundongos Knockout , Contração Muscular , Distrofias Musculares/genética , Distrofias Musculares/patologia , Ligação Proteica , Canais de Cátion TRPC/genéticaRESUMO
Some mutations of the sodium channel gene Na(V1.5) are multifunctional, causing combinations of LQTS, Brugada syndrome and progressive cardiac conduction system disease (PCCD). The combination of Brugada syndrome and PCCD is uncommon, although they both result from a reduction in the sodium current. We hypothesize that slow conduction is sufficient to cause S-T segment elevation and undertook a combined experimental and theoretical study to determine whether conduction slowing alone can produce the Brugada phenotype. Deletion of lysine 1479 in one of two positively charged clusters in the III/IV inter-domain linker causes both syndromes. We have examined the functional effects of this mutation using heterologous expression of the wild-type and mutant sodium channel in HEK-293-EBNA cells. We show that DeltaK1479 shifts the potential of half-activation, V(1/2m), to more positive potentials (V(1/2m) = -36.8 +/- 0.8 and -24.5 +/- 1.3 mV for the wild-type and DeltaK1479 mutant respectively, n = 11, 10). The depolarizing shift increases the extent of depolarization required for activation. The potential of half-inactivation, V(1/2h), is also shifted to more positive potentials (V(1/2h) = -85 +/- 1.1 and -79.4 +/- 1.2 mV for wild-type and DeltaK1479 mutant respectively), increasing the fraction of channels available for activation. These shifts are quantitatively the same as a mutation that produces PCCD only, G514C. We incorporated experimentally derived parameters into a model of the cardiac action potential and its propagation in a one dimensional cable (simulating endo-, mid-myocardial and epicardial regions). The simulations show that action potential and ECG changes consistent with Brugada syndrome may result from conduction slowing alone; marked repolarization heterogeneity is not required. The findings also suggest how Brugada syndrome and PCCD which both result from loss of sodium channel function are sometimes present alone and at other times in combination.
Assuntos
Síndrome de Brugada/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Ativação do Canal Iônico , Rim/fisiopatologia , Modelos Cardiovasculares , Proteínas Musculares/metabolismo , Síndromes de Pré-Excitação/fisiopatologia , Canais de Sódio/metabolismo , Potenciais de Ação , Linhagem Celular , Simulação por Computador , Humanos , Cinética , Proteínas Musculares/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5 , Canais de Sódio/genética , Relação Estrutura-AtividadeRESUMO
Noroviruses are one of the major causes of nonbacterial gastroenteritis epidemics in humans. Recent studies on norovirus receptors show that different noroviruses recognize different human histo-blood group antigens (HBGAs), and eight receptor binding patterns of noroviruses have been identified. The P domain of the norovirus capsids is directly involved in this recognition. To determine the precise locations and receptor binding modes of HBGA carbohydrates on the viral capsids, a recombinant P protein of a GII-4 strain norovirus, VA387, was cocrystallized with synthetic type A or B trisaccharides. Based on complex crystal structures observed at a 2.0-A resolution, we demonstrated that the receptor binding site lies at the outermost end of the P domain and forms an extensive hydrogen-bonding network with the saccharide ligand. The A and B trisaccharides display similar binding modes, and the common fucose ring plays a key role in this interaction. The extensive interface between the two protomers in a P dimer also plays a crucial role in the formation of the receptor binding interface.
Assuntos
Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/metabolismo , Norovirus/química , Norovirus/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Trissacarídeos/química , Trissacarídeos/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Norovirus/genética , Oligossacarídeos de Cadeias Ramificadas , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/metabolismoRESUMO
AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, we have solved the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin (Protein Data Bank ID code 2FOT). The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes.
Assuntos
Calmodulina/química , Espectrina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calmodulina/metabolismo , Calpaína/química , Calpaína/metabolismo , Bovinos , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrina/metabolismoRESUMO
Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.
Assuntos
Brônquios/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Sequência de Aminoácidos , Animais , Brônquios/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/fisiologia , Células Cultivadas , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ratos , Ratos Wistar , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologiaRESUMO
beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Insuficiência Cardíaca/fisiopatologia , Coração/efeitos dos fármacos , Receptores Adrenérgicos beta 3/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Animais , Canais de Cálcio Tipo L/fisiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Coração/fisiologia , Insuficiência Cardíaca/induzido quimicamente , Ventrículos do Coração/citologia , Isoproterenol/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 3/fisiologiaRESUMO
The new myofilament Ca2+ sensitizer levosimendan (LSM) is a positive inotropic and vasodilatory agent. Its beneficial effects have been demonstrated at rest in congestive heart failure (CHF). However, its effect during exercise (Ex) in CHF is unknown. We assessed the effects of LSM on left ventricular (LV) dynamics at rest and during Ex in eight conscious, instrumented dogs with pacing-induced CHF. After CHF, with dogs at rest, LSM decreased arterial elastance (Ea) and increased LV contractile performance as assessed by the slope of LV pressure-volume (P-V) relation. LSM caused a >60% increase in the peak rate of mitral flow (dV/dtmax) due to decreases in minimal LV pressure and the time constant of LV relaxation (tau). LV arterial coupling, quantified as the ratio of end-systolic elastance (Ees) to Ea, was increased from 0.47 to 0.85%. LV mechanical efficiency, determined as the ratio of stroke work to total P-V area, was improved from 0.54 +/- 0.09 to 0.61 +/- 0.07. These beneficial effects persisted during Ex after CHF. Compared with CHF Ex dogs, treatment with LSM prevented Ex-induced abnormal increases in mean left atrial pressure and end-diastolic pressure and decreased Ees/Ea. With LSM treatment during CHF Ex, the early diastolic portion of the LV P-V loop was shifted downward with decreased minimal LV pressure and tau values and a further augmented dV/dtmax. Ees/Ea improved, and mechanical efficiency further increased from 0.61 +/- 0.07 to 0.67 +/- 0.07, which was close to the value reached during normal Ex. After CHF, LSM produced arterial vasodilatation; improved LV relaxation and diastolic filling; increased contractility, LV arterial coupling, and mechanical efficiency; and normalized the response to Ex.
Assuntos
Cardiotônicos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Hidrazonas/farmacologia , Esforço Físico/fisiologia , Piridazinas/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Diástole/efeitos dos fármacos , Cães , Insuficiência Cardíaca/fisiopatologia , Marca-Passo Artificial , Descanso/fisiologia , Simendana , Sístole/efeitos dos fármacosRESUMO
We studied an infant with severe neonatal hemolytic anemia and hyperbilirubinemia that evolved into a partially compensated ellipto-poikilocytic anemia. His father had typical elliptocytosis. Their erythrocyte membranes demonstrated structural and functional defects in spectrin. Genetic studies revealed that the proband and his father were heterozygous for an alpha-spectrin mutation, Ile24Thr, in the alpha beta spectrin self-association binding site. The proband also carried the low expression allele alpha(LELY) in trans, influencing the clinical phenotype. The importance of isoleucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all alpha spectrins from Drosophila to humans. Molecular modeling demonstrated that replacement of a hydrophobic isoleucine with a hydrophilic threonine disrupts highly conserved hydrophobic interactions in the interior of the spectrin triple helix critical for spectrin function.