RESUMO
Right heart failure is the major cause of death in Pulmonary Artery Hypertension (PAH) patients but is not a current, specific therapeutic target. Pre-clinical studies have shown that adrenoceptor blockade can improve cardiac function but the mechanisms of action within right ventricular (RV) myocytes are unknown. We tested whether the ß1-adrenoceptor blocker metoprolol could improve RV myocyte function in an animal model of PAH, by attenuating adverse excitation-contraction coupling remodeling. PAH with RV failure was induced in rats by monocrotaline injection. When PAH was established, animals were given 10â¯mg/kg/day metoprolol (MCTâ¯+â¯BB) or vehicle (MCT). The median time to the onset of heart failure signs was delayed from 23â¯days (MCT), to 31â¯days (MCTâ¯+â¯BB). At 23⯱â¯1â¯days post-injection, MCTâ¯+â¯BB showed improved in vivo cardiac function, measured by echocardiography. RV hypertrophy was reduced despite persistent elevated afterload. RV myocyte contractility during field stimulation was improved at higher pacing frequencies in MCTâ¯+â¯BB. Preserved t-tubule structure, more uniform evoked Ca2+ release, increased SERCA2a expression and faster ventricular repolarization (measured in vivo by telemetry) may account for the improved contractile function. Sarcoplasmic reticulum Ca2+ overload was prevented in MCTâ¯+â¯BB myocytes resulting in fewer spontaneous Ca2+ waves, with a lower pro-arrhythmic potential. Our novel finding of attenuation of defects in excitation contraction coupling by ß1-adrenoceptor blockade with delays in the onset of HF, identifies the RV as a promising therapeutic target in PAH. Moreover, our data suggest existing therapies for left ventricular failure may also be beneficial in PAH induced RV failure.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Cálcio/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Metoprolol/uso terapêutico , Miócitos Cardíacos/metabolismo , Artéria Pulmonar/fisiopatologia , Disfunção Ventricular Direita/tratamento farmacológico , Antagonistas de Receptores Adrenérgicos beta 1/administração & dosagem , Análise de Variância , Animais , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Insuficiência Cardíaca/metabolismo , Hipertensão Pulmonar/diagnóstico por imagem , Hipertrofia Ventricular Direita/tratamento farmacológico , Masculino , Metoprolol/administração & dosagem , Ratos , Ratos Wistar , Volume Sistólico/efeitos dos fármacos , Disfunção Ventricular Direita/diagnóstico por imagemRESUMO
Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.
Assuntos
Cavéolas , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Animais , Cavéolas/fisiologia , Separação Celular , Células Cultivadas , Capacitância Elétrica , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , Modelos Biológicos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Coelhos , Sarcolema/fisiologia , Propriedades de Superfície , Fixação de TecidosRESUMO
The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation-contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation. Rat ventricular myocytes were treated with methyl-ß-cyclodextrin (MßCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MßCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MßCD treatment. Selective activation of α-, ß1-, and ß2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes. We typically identified 600-850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.
Assuntos
Agonistas Adrenérgicos/farmacologia , Cavéolas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Proteoma/isolamento & purificação , Proteômica/métodos , Antagonistas Adrenérgicos/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais , beta-Ciclodextrinas/farmacologiaRESUMO
The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a â¼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Fosfoproteínas/química , Aciltransferases , Motivos de Aminoácidos , Animais , Membrana Celular/enzimologia , Cães , Endocitose , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Lipoilação , Camundongos , Miocárdio/metabolismo , Plasticidade Neuronal , Fosfolipídeos/química , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Sódio/química , Especificidade por Substrato , SinapsesRESUMO
Increased physical activity is recommended for the general population and for patients with many diseases because of its health benefits but can be contraindicated if it is thought to be a risk for serious cardiovascular events. One such condition is pulmonary artery hypertension (PAH). PAH and right ventricular failure was induced in rats by a single injection of monocrotaline (MCT). MCT rats with voluntary access to a running wheel ran on average 2 km/day. The time for half the animals to develop heart failure signs (median survival time) was 28 days (exercise failure group), significantly longer than sedentary animals (sedentary failure group, 23 days). The contractility of single failing myocytes in response to increasing demand (stimulation frequency) was significantly impaired compared with that in both sedentary control and exercising control myocytes. However, myocytes from exercising MCT rats, tested at 23 days (exercise + MCT group), showed responses intermediate to the control (sedentary control and exercising control) and failing (sedentary failure and exercise failure) groups. We conclude that voluntary exercise is beneficial to rats with heart failure induced by PAH, and this is evidence to support the consideration of appropriate exercise regimes for potentially vulnerable groups.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Esforço Físico , Animais , Células Cultivadas , Insuficiência Cardíaca/etiologia , Hipertensão Pulmonar/complicações , Masculino , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Artéria Pulmonar/fisiopatologia , Ratos , Ratos WistarRESUMO
BACKGROUND: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. RESULTS: In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. CONCLUSION: Phospholemman oligomers exist in cardiac muscle. SIGNIFICANCE: Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.
Assuntos
Ventrículos do Coração/citologia , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Motivos de Aminoácidos , Animais , Cavéolas/metabolismo , Fixadores/química , Formaldeído/química , Ventrículos do Coração/metabolismo , Imunoprecipitação , Complexos Multiproteicos/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , RatosRESUMO
Researchers in biomedical sciences must continually re-evaluate their investment in experiments using laboratory animals. Our group is interested in various signalling pathways that underlie physiological and pathophysiological functioning of the mammalian heart. Two important resources for this type of work are isolated cardiomyocytes and homogenized or preserved sections of whole myocardium. In order to improve our experimental approach ethically, we devised an adaptation of the Langendorff whole-heart retrograde perfusion technique that allows the isolation of adult rat ventricular cardiomyocytes and processing/storage of myocardium from the same heart. This could result in a 50% reduction in the number of animals required for certain experiments. We believe that a generalized adoption of this method would represent a meaningful reduction of animal use in our field of research and, furthermore, strengthen data sets by permitting correlation between myocyte function and various parameters of myocardial biochemistry/structure in individual hearts. This approach is of particular relevance for studies of cardiac pathology, given the cost and time involved in generating experimental disease models.
Assuntos
Animais de Laboratório/fisiologia , Procedimentos Cirúrgicos Cardíacos/métodos , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Masculino , Miocárdio , Ratos , Ratos WistarRESUMO
Compartmentation of signalling allows multiple stimuli to achieve diverse cellular responses with only a limited pool of second messengers. This spatial control of signalling is achieved, in part, by cellular structures which bring together elements of a particular cascade. One such structure is the caveola, a flask-shaped lipid raft. Caveolae are well-recognised as signalosomes, platforms for assembly of signalling complexes of receptors, effectors and their targets, which can facilitate efficient and specific cellular responses. Here we extend this simple model and present evidence to show how the protein and lipid profiles of caveolae, as well as their characteristic morphology, define their roles in creating local signalling domains in the cardiac myocyte. This article is part of a Special Issue entitled "Local Signaling in Myocytes."
Assuntos
Cavéolas/química , Cavéolas/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Animais , Caveolinas/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologiaRESUMO
Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of ß1-, but not ß2-, adrenoceptors (ARs). Compartmentation of ß2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of ß2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-ß-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca(2+) current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser(16). Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during ß2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed ß2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of ß2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit ß2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled "Local Signaling in Myocytes".
Assuntos
Cavéolas/metabolismo , AMP Cíclico/biossíntese , Miócitos Cardíacos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Retículo Sarcoplasmático/enzimologia , Transdução de Sinais , Animais , Cálcio/metabolismo , Cavéolas/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Morfolinas/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Transporte Proteico , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
ß(1)-Adrenergic receptors (ß(1)ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. ß(1)ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, ß(1)AR activation enhanced the L-type Ca(2+) current, intracellular Ca(2+) transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-ß-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by ß(1)ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. ß(1)AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected ß(1)AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between ß(1)AR and EPR responses. They also suggest that ß(1)AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.
Assuntos
Cálcio/metabolismo , Colesterol/deficiência , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Alprostadil/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Cavéolas/metabolismo , Caveolina 3/metabolismo , Compartimento Celular/fisiologia , Colesterol/metabolismo , Isoproterenol/metabolismo , Masculino , Microdomínios da Membrana/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca(2+) triggers sarcolemmal Ca(2+) influx via store-operated Ca(2+) entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca(2+)] either within the resealed t-system ([Ca(2+)](t-sys)) or within the cytosol. In normal fibers, halothane (0.5 mM) failed to initiate SR Ca(2+) release or Ca(2+)(t-sys) depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca(2+) release and Ca(2+)(t-sys) depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca(2+) release took the form of a propagated wave, which was temporally coupled to a wave of Ca(2+)(t-sys) depletion. SOCE was potently inhibited by "extracellular" application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca(2+) depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca(2+) release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca(2+) pool may contribute to the maintained rise in cytosolic [Ca(2+)] that underlies MH.
Assuntos
Cálcio/metabolismo , Hipertermia Maligna/metabolismo , Músculo Esquelético/metabolismo , Western Blotting , Halotano/farmacologia , Humanos , Técnicas In Vitro , Microscopia Confocal , Músculo Esquelético/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
This study aimed to identify a mechanism for statin-induced myopathy that explains its prevalence and selectivity for skeletal muscle, and to understand its interaction with moderate exercise. Statin-associated adverse muscle symptoms reduce adherence to statin therapy; this limits the effectiveness of statins in reducing cardiovascular risk. The issue is further compounded by perceived interactions between statin treatment and exercise. This study examined muscles from individuals taking statins and rats treated with statins for 4 weeks. In skeletal muscle, statin treatment caused dissociation of the stabilizing protein FK506 binding protein (FKBP12) from the sarcoplasmic reticulum (SR) calcium (Ca2+) release channel, the ryanodine receptor 1, which was associated with pro-apoptotic signaling and reactive nitrogen species/reactive oxygen species (RNS/ROS)-dependent spontaneous SR Ca2+ release events (Ca2+ sparks). Statin treatment had no effect on Ca2+ spark frequency in cardiac myocytes. Despite potentially deleterious effects of statins on skeletal muscle, there was no impact on force production or SR Ca2+ release in electrically stimulated muscle fibers. Statin-treated rats with access to a running wheel ran further than control rats; this exercise normalized FKBP12 binding to ryanodine receptor 1, preventing the increase in Ca2+ sparks and pro-apoptotic signaling. Statin-mediated RNS/ROS-dependent destabilization of SR Ca2+ handling has the potential to initiate skeletal (but not cardiac) myopathy in susceptible individuals. Importantly, although exercise increases RNS/ROS, it did not trigger deleterious statin effects on skeletal muscle. Indeed, our results indicate that moderate exercise might benefit individuals who take statins.
RESUMO
Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the beta(2) adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-beta-cyclodextrin (M beta C) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to beta(2) AR stimulation (with salbutamol in the presence of 1 microM atenolol). Ser(16) phosphorylation of PLB (pPLB), Ser(22,23) phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of M beta C to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with beta(2) AR stimulation (P>0.05), but following caveolar disruption a 60-70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P<0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with beta(2) AR stimulation to a global signal that targets proteins of the sarcoplasmic reticulum and myofilaments, with functional sequelae. The role of caveolae in spatial control of cAMP may be relevant to perturbation of beta AR signalling in cardiovascular disease.
Assuntos
Cavéolas/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Sarcolema/metabolismo , Sistemas do Segundo Mensageiro , Citoesqueleto de Actina/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Animais , Atenolol/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Doenças Cardiovasculares/metabolismo , Caveolina 2/metabolismo , Caveolina 3/metabolismo , Colesterol/metabolismo , Citosol , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Troponina I/metabolismoRESUMO
BACKGROUND AND PURPOSE: Statins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca2+ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. EXPERIMENTAL APPROACH: RyR1 and RyR2 single-channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage-clamp conditions. LC-MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy-acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca2+ release. KEY RESULTS: Hydroxy acid simvastatin (active at HMG-CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca2+ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro-drug form of simvastatin (inactive at HMG-CoA reductase) also activated RyR1, suggesting that the HMG-CoA inhibitor pharmacophore was not responsible for RyR1 activation. CONCLUSION AND IMPLICATIONS: Simvastatin interacts with RyR1 to increase SR Ca2+ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca2+ -dependent arrhythmias and sudden cardiac death.
Assuntos
Cálcio/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Sinvastatina/análogos & derivados , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Ovinos , Sinvastatina/farmacologiaRESUMO
During vertebrate evolution there has been a shift in the way in which the heart varies cardiac output (the product of heart rate and stroke volume). While mammals, birds, and amphibians increase cardiac output through large increases in heart rate and only modest increases (approximately 30%) in stroke volume, fish and some reptiles use modest increases in heart rate and very large increases in stroke volume (up to 300%). The cellular mechanisms underlying these fundamentally different approaches to cardiac output modulation are unknown. We hypothesized that the divergence between volume modulation and frequency modulation lies in the response of different vertebrate myocardium to stretch. We tested this by progressively stretching individual cardiac myocytes from the fish heart while measuring sarcomere length (SL), developed tension, and intracellular Ca2+ ([Ca2+]i) transients. We show that in fish cardiac myocytes, active tension increases at SLs greater than those previously demonstrated for intact mammalian myocytes, representing a twofold increase in the functional ascending limb of the length-tension relationship. The mechanism of action is a length-dependent increase in myofilament Ca2+ sensitivity, rather than changes in the [Ca2+]i transient or actin filament length in the fish cell. The capacity for greater sarcomere extension in fish myocardium may be linked to the low resting tension that is developed during stretch. These adaptations allow the fish heart to volume modulate and thus underpin the fundamental difference between the way fish and higher vertebrates vary cardiac output.
Assuntos
Débito Cardíaco/fisiologia , Miócitos Cardíacos/fisiologia , Oncorhynchus mykiss/fisiologia , Animais , Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Estimulação Elétrica , Feminino , Coração/fisiologia , Masculino , Miocárdio/citologia , Miócitos Cardíacos/citologia , Ratos , Ratos Wistar , Sarcômeros/efeitos dos fármacos , Sarcômeros/fisiologiaRESUMO
OBJECTIVE: Caveolae, flask shaped invaginations of the cell membrane, influence signalling cascades in many cell types. We have tested the hypothesis that caveolae modulate excitation-contraction coupling (ECC) and beta-adrenergic stimulation in the adult cardiac myocyte. METHODS: Shortening, [Ca(2+)](i) and L-type Ca(2+) current (I(Ca,L)) were recorded in rat ventricular myocytes. Caveolae were disrupted with methyl-beta-cyclodextrin (MbetaC). RESULTS: Shortening and [Ca(2+)](i) transient amplitude were reduced in myocytes treated with MbetaC. MbetaC did not alter the density or characteristics of I(Ca,L) or the sarcoplasmic reticulum (SR) Ca(2+) load, but significantly reduced fractional SR Ca(2+) release. The inotropic response of myocytes to beta(1)-adrenoceptor stimulation was insensitive to caveolae disruption. By contrast, the increase in shortening, [Ca(2+)](i) transient and I(Ca,L) seen following beta(2) stimulation was markedly enhanced (3-5 fold) following MbetaC treatment, and the effect on I(Ca,L) could be mimicked by dialyzing cells with an antibody to caveolin 3. When the G(alphai) pathway was disabled with pertussis toxin (PTX), control cells showed a similar response to beta(2) stimulation as seen in MbetaC-treated myocytes, whereas MbetaC-treated cells were insensitive to PTX. CONCLUSIONS: Caveolae modulate ECC via the efficiency of the Ca(2+)-induced Ca(2+) release process, rather than Ca(2+) influx. Our data are also consistent with the hypothesis that interaction of G(i) protein cascade components with caveolin in the caveolae is necessary for effective signalling by this pathway. This suggests that changes in caveolin expression in the adult heart seen during aging and in disease will have consequences for baseline cardiac function and beta-adrenergic responsiveness.
Assuntos
Cavéolas/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/fisiologia , Antagonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Animais , Cálcio/metabolismo , Cavéolas/efeitos dos fármacos , Caveolina 3/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/metabolismo , Colesterol/farmacologia , Eletrofisiologia , Ventrículos do Coração , Líquido Intracelular/metabolismo , Isoproterenol/farmacologia , Masculino , Microscopia Confocal , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Propanolaminas/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins' myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here, we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca2+]i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser23,24. Statin treatment increased expression of eNOS and Ser1177 phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolins 1 and 3, and increased (P = 0.06) NO metabolites, consistent with enhanced NO production. It is well-established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance.
RESUMO
Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.The mechanisms that regulate the body's response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.