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Sarcolemmal ATP-sensitive potassium (KATP) channels play a vital role in cardioprotection. Cardiac KATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of KATP channels through several signaling pathways. However, the direct functional effect of Cav3 on KATP channels is still poorly understood. Here, we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced KATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased KATP channel current in a concentration-dependent manner with an IC50 of â¼5 µM. The MAD1 segment prevented KATP channel recycling, thus decreasing KATP channel surface density and abolishing the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of KATP channel recycling, providing pharmacological potential in the treatment of diseases with KATP channel trafficking defects.NEW & NOTEWORTHY Cardiac KATP channels physically interact with caveolin-3 in caveolae. In this study, we investigated the functional effect of caveolin-3 on KATP channel activity and identified a novel segment (MAD1) in the C-terminus domain of Caveolin-3 that negatively regulates KATP channel surface density and current amplitude by impairing KATP channel recycling. The peptide corresponding to the MAD1 segment abolished the cardioprotective effect of ischemic preconditioning.
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Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 µM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.
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Sirtuins are NAD+-dependent deacetylases with beneficial roles in conditions relevant to human health, including metabolic disease, type II diabetes, obesity, cancer, aging, neurodegenerative diseases, and cardiac ischemia. Since ATP-sensitive K+ (KATP) channels have cardioprotective roles, we investigated whether they are regulated by sirtuins. Nicotinamide mononucleotide (NMN) was used to increase cytosolic NAD+ levels and to activate sirtuins in cell lines, isolated rat and mouse cardiomyocytes or insulin-secreting INS-1 cells. KATP channels were studied with patch clamping, biochemistry techniques, and antibody uptake experiments. NMN led to an increase in intracellular NAD+ levels and an increase in the KATP channel current, without significant changes in the unitary current amplitude or open probability. An increased surface expression was confirmed using surface biotinylation approaches. The rate of KATP channel internalization was diminished by NMN, which may be a partial explanation for the increased surface expression. We show that NMN acts via sirtuins since the increased KATP channel surface expression was prevented by blockers of SIRT1 and SIRT2 (Ex527 and AGK2) and mimicked by SIRT1 activation (SRT1720). The pathophysiological relevance of this finding was studied using a cardioprotection assay with isolated ventricular myocytes, in which NMN protected against simulated ischemia or hypoxia in a KATP channel-dependent manner. Overall, our data draw a link between intracellular NAD+, sirtuin activation, KATP channel surface expression, and cardiac protection against ischemic damage.
Assuntos
Diabetes Mellitus Tipo 2 , Sirtuínas , Ratos , Camundongos , Humanos , Animais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , NAD/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Miócitos Cardíacos/metabolismo , Trifosfato de Adenosina/metabolismo , Canais KATP/genética , Canais KATP/metabolismoRESUMO
ATP-sensitive K+ (KATP) channel couples membrane excitability to intracellular energy metabolism. Maintaining KATP channel surface expression is key to normal insulin secretion, blood pressure and cardioprotection. However, the molecular mechanisms regulating KATP channel internalization and endocytic recycling, which directly affect the surface expression of KATP channels, are poorly understood. Here we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and characterized Rab35 GTPase as a key regulator of KATP channel endocytic recycling. Electrophysiological recordings and surface biotinylation assays showed decreased KATP channel surface density with co-expression of a dominant negative Rab35 mutant (Rab35-DN), but not other recycling-related Rab GTPases, including Rab4, Rab11a and Rab11b. Immunofluorescence images revealed strong colocalization of Rab35-DN with recycling Kir6.2. Rab35-DN minimized the recycling rate of KATP channels. Rab35 also regulated KATP channel current amplitude in isolated adult cardiomyocytes by affecting its surface expression but not channel properties, which validated its physiologic relevance and the potential of pharmacologic target for treating the diseases with KATP channel trafficking defects.
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GTP Fosfo-Hidrolases , Canais KATP , Trifosfato de Adenosina/metabolismo , Transporte Biológico , GTP Fosfo-Hidrolases/metabolismo , Canais KATP/genética , Canais KATP/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Sarcolemmal/plasmalemmal ATP-sensitive K+ (KATP) channels have key roles in many cell types and tissues. Hundreds of studies have described how the KATP channel activity and ATP sensitivity can be regulated by changes in the cellular metabolic state, by receptor signaling pathways and by pharmacological interventions. These alterations in channel activity directly translate to alterations in cell or tissue function, that can range from modulating secretory responses, such as insulin release from pancreatic ß-cells or neurotransmitters from neurons, to modulating contractile behavior of smooth muscle or cardiac cells to elicit alterations in blood flow or cardiac contractility. It is increasingly becoming apparent, however, that KATP channels are regulated beyond changes in their activity. Recent studies have highlighted that KATP channel surface expression is a tightly regulated process with similar implications in health and disease. The surface expression of KATP channels is finely balanced by several trafficking steps including synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling, and degradation. This review aims to summarize the physiological and pathophysiological implications of KATP channel trafficking and mechanisms that regulate KATP channel trafficking. A better understanding of this topic has potential to identify new approaches to develop therapeutically useful drugs to treat KATP channel-related diseases.
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Células Secretoras de Insulina , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Canais KATP/genética , Canais KATP/metabolismo , Transporte ProteicoRESUMO
A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.
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Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , Células HEK293 , Humanos , Canais KATP/genética , Lipoilação/fisiologia , Mutagênese/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Cultura Primária de Células , Ratos , Receptores de Sulfonilureias/genéticaRESUMO
AIMS: ß-adrenergic receptors (ßARs) play pivotal roles in regulating cardiac excitation-contraction (E-C) coupling. Global signalling of ß1ARs up-regulates both the influx of Ca2+ through sarcolemmal L-type Ca2+ channels (LCCs) and the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptors (RyRs). However, we recently found that ß2AR stimulation meditates 'offside compartmentalization', confining ß1AR signalling into subsarcolemmal nanodomains without reaching SR proteins. In the present study, we aim to investigate the new question, whether and how compartmentalized ß1AR signalling regulates cardiac E-C coupling. METHODS AND RESULTS: By combining confocal Ca2+ imaging and patch-clamp techniques, we investigated the effects of compartmentalized ßAR signalling on E-C coupling at both cellular and molecular levels. We found that simultaneous activation of ß2 and ß1ARs, in contrast to global signalling of ß1ARs, modulated neither the amplitude and spatiotemporal properties of Ca2+ sparks nor the kinetics of the RyR response to LCC Ca2+ sparklets. Nevertheless, by up-regulating LCC current, compartmentalized ß1AR signalling synchronized RyR Ca2+ release and increased the functional reserve (stability margin) of E-C coupling. In circumstances of briefer excitation durations or lower RyR responsivity, compartmentalized ßAR signalling, by increasing the intensity of Ca2+ triggers, helped stabilize the performance of E-C coupling and enhanced the Ca2+ transient amplitude in failing heart cells. CONCLUSION: Given that compartmentalized ßAR signalling can be induced by stress-associated levels of catecholamines, our results revealed an important, yet unappreciated, heart regulation mechanism that is autoadaptive to varied stress conditions.
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Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cardiomegalia/metabolismo , Acoplamento Excitação-Contração , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Potenciais de Ação , Agonistas Adrenérgicos/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Cardiomegalia/fisiopatologia , Simulação por Computador , Modelos Animais de Doenças , Acoplamento Excitação-Contração/efeitos dos fármacos , Cinética , Masculino , Microscopia Confocal , Modelos Cardiovasculares , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
We investigated targeting mechanisms of Na+ and KATP channels to the intercalated disk (ICD) of cardiomyocytes. Patch clamp and surface biotinylation data show reciprocal downregulation of each other's surface density. Mutagenesis of the Kir6.2 ankyrin binding site disrupts this functional coupling. Duplex patch clamping and Angle SICM recordings show that INa and IKATP functionally co-localize at the rat ICD, but not at the lateral membrane. Quantitative STORM imaging show that Na+ and KATP channels are localized close to each other and to AnkG, but not to AnkB, at the ICD. Peptides corresponding to Nav1.5 and Kir6.2 ankyrin binding sites dysregulate targeting of both Na+ and KATP channels to the ICD, but not to lateral membranes. Finally, a clinically relevant gene variant that disrupts KATP channel trafficking also regulates Na+ channel surface expression. The functional coupling between these two channels need to be considered when assessing clinical variants and therapeutics.
Assuntos
Anquirinas/química , Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Sítios de Ligação , Biotinilação , Células HEK293 , Humanos , Mutagênese , Miocárdio/metabolismo , Técnicas de Patch-Clamp , RatosRESUMO
BACKGROUND: Genetic variation in ion channel genes ('channelopathies') are often associated with inherited arrhythmias and sudden death. Genetic testing ('molecular autopsies') of channelopathy genes can be used to assist in determining the likely causes of sudden unexpected death. However, different in silico approaches can yield conflicting pathogenicity predictions and assessing their impact on ion channel function can assist in this regard. METHODS AND RESULTS: We performed genetic testing of cases of sudden expected death in the New York City metropolitan area and found four rare or novel variants in ABCC9, which codes for the regulatory SUR2 subunit of KATP channels. All were missense variants, causing amino acid changes in the protein. Three of the variants (A355S, M941V, and K1379Q) were in cases of infants less than six-months old and one (H1305Y) was in an adult. The predicted pathogenicities of the variants were conflicting. We have introduced these variants into a human SUR2A cDNA, which we coexpressed with the Kir6.2 pore-forming subunit in HEK-293 cells and subjected to patch clamp and biochemical assays. Each of the four variants led to gain-of-function phenotypes. The A355S and M941V variants increased in the overall patch current. The sensitivity of the KATP channels to inhibitory 'cytosolic' ATP was repressed for the M941V, H1305Y and K1379Q variants. None of the variants had any effect on the unitary KATP channel current or the surface expression of KATP channels, as determined with biotinylation assays, suggesting that all of the variants led to an enhanced open state. CONCLUSIONS: All four variants caused a gain-of-function phenotype. Given the expression of SUR2-containing KATP channels in the heart and specialized cardiac conduction, vascular smooth muscle and respiratory neurons, it is conceivable that electrical silencing of these cells may contribute to the vulnerability element, which is a component of the triple risk model of sudden explained death in infants. The gain-of-function phenotype of these ABCC9 variants should be considered when assessing their potential pathogenicity.
Assuntos
Morte Súbita/etiologia , Mutação de Sentido Incorreto , Receptores de Sulfonilureias/genética , Adulto , Canalopatias/genética , DNA Complementar , Feminino , Mutação com Ganho de Função , Células HEK293 , Humanos , Lactente , Canais KATP/genética , Masculino , Cidade de Nova Iorque , Técnicas de Patch-Clamp , FenótipoRESUMO
RATIONALE: ßARs (ß-adrenergic receptors) are prototypical GPCRs (G protein-coupled receptors) that play a pivotal role in sympathetic regulation. In heart cells, ß1AR signaling mediates a global response, including both l-type Ca2+ channels in the sarcolemma/T tubules and RyRs (ryanodine receptors) in the SR (sarcoplasmic reticulum). In contrast, ß2AR mediates local signaling with little effect on the function of SR proteins. OBJECTIVE: To investigate the signaling relationship between ß1ARs and ß2ARs. METHOD AND RESULTS: Using whole-cell patch-clamp analyses combined with confocal Ca2+ imaging, we found that the activation of compartmentalized ß2AR signaling was able to convert the ß1AR signaling from global to local mode, preventing ß1ARs from phosphorylating RyRs that were only nanometers away from sarcolemma/T tubules. This offside compartmentalization was eliminated by selective inhibition of ß2AR, GRK2 (GPCR kinase-2), ßarr1 (ß-arrestin-1), and phosphodiesterase-4. A knockin rat model harboring mutations of the last 3 serine residues of the ß1AR C terminus, a component of the putative ßarr1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by ß2AR activation. CONCLUSIONS: ß2AR stimulation compartmentalizes ß1AR signaling into nanoscale local domains in a phosphodiesterase-4-dependent manner by targeting the C terminus of ß1ARs. This finding reveals a fundamental negative feed-forward mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient ß1AR response of sympathetic excitation.
Assuntos
Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Adrenérgicos/farmacologia , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Masculino , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Transgênicos , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Determining the cause of unexplained death in all age groups, including infants, is a priority in forensic medicine. The triple risk model proposed for sudden infant death syndrome involves the intersection of three risks: (1) a critical developmental period in homeostatic control (2), exogenous stressors, and (3) a vulnerable infant. Even though sex and age factor into some forms of inherited arrhythmogenic deaths in young individuals and adults, more appropriate a dual-risk disease model for adults involves exogenous stressors and a vulnerable individual. The vulnerability aspect clearly has a genetic component as underscored by a number of recent large-scale and high-throughput genetic testing studies performed in attempt to define the causes of sudden unexplained death. These studies often focus on 'cardiac' and channelopathy genes. Genetic testing often identify lists of rare or ultra-rare nonsynonymous variants, classified according to the ACMG guidelines as 'pathogenic' or 'likely pathogenic', which may form the basis of diagnostic decisions and/or family counseling. However, computer algorithms used to categorize gene variants are not completely accurate and these variants are often not functionally tested to determine their pathogenicity. Due to conflicting computational predictions, a large number of variants are labeled as 'variants of uncertain significance' or VUS. Functional testing of these VUS can greatly assist to reclassify these VUS as 'likely benign' or 'likely pathogenic'. However, functional testing has its limits and by itself cannot be used to determine cause of death. Going forward, computer algorithms must be improved to take account of variants across multiple genes and efforts must be expanded to obtain clinical, familial and segregation data. Forensic genetic testing needs to be held to the same rigorous standards as defined by the NIH Clinical Genome Resource Consortium, where functional evaluation of a channelopathy variant is only one (but important) aspect of the overall picture.
Assuntos
Canalopatias/genética , Morte Súbita/etiologia , Variação Genética , Morte Súbita do Lactente/genética , Genética Forense , Predisposição Genética para Doença , Testes Genéticos , Humanos , LactenteRESUMO
ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.
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Proteínas de Transporte/metabolismo , Canais KATP/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Células HEK293 , Humanos , Canais KATP/genética , Camundongos , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Transporte Proteico , Ratos , Sarcolema/genéticaRESUMO
Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.
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Cálcio/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Placofilinas/genética , Transcrição Gênica , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Western Blotting , Expressão Gênica , Coração/fisiopatologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Placofilinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fracture healing, in which osteoclasts and osteoblasts play important roles, has drawn much clinical attention. Osteoclast deficiency or decreased osteoblast activity will impair fracture healing. TRPV1 is a member of the Ca2+ permeable cation channel subfamily, and pharmacological inhibition of TRPV1 prevents ovariectomy-induced bone loss, which makes TRPV1 a potential target for osteoporosis. However, whether long term TRPV1 inhibition or TRPV1 deletion will affect the fracture healing process is unclear. In this study, we found that the wild-type mice showed a well-remodeled fracture callus, whereas TRPV1 knockout mice still had an obvious fracture gap with unresorbed soft-callus 4 weeks post-fracture. The number of osteoclasts was reduced in the TRPV1 knockout fracture callus, and osteoclast formation and resorption activity were also impaired in vitro. TRPV1 deletion decreased the calcium oscillation frequency and peak cytoplasmic concentration in osteoclast precursors, subsequently reducing the expression and nuclear translocation of NFATc1 and downregulating DC-stamp, cathepsin K, and ATP6V. In addition, TRPV1 deletion caused reduced mRNA and protein expression of Runx2 and ALP in bone marrow stromal cells (BMSCs) and reduced calcium deposition in vitro. Our results suggest that TRPV1 deletion impairs fracture healing, and inhibited osteoclastogenesis and osteogenesis.
Assuntos
Diferenciação Celular , Consolidação da Fratura , Deleção de Genes , Osteoblastos/patologia , Osteoclastos/patologia , Canais de Cátion TRPV/genética , Animais , Densidade Óssea , Calo Ósseo/patologia , Sinalização do Cálcio , Cartilagem/patologia , Contagem de Células , Regulação para Baixo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo , Microtomografia por Raio-XRESUMO
Aims: The heart contraction is controlled by the Ca2+-induced Ca2+ release (CICR) between L-type Ca2+ channels and ryanodine receptors (RyRs). The FK506-binding protein FKBP12.6 binds to RyR subunits, but its role in stabilizing RyR function has been debated for long. Recent reports of high-resolution RyR structure show that the HD2 domain that binds to the SPRY2 domain of neighbouring subunit in FKBP-bound RyR1 is detached and invisible in FKBP-null RyR2. The present study was to test the consequence of FKBP12.6 absence on the in situ activation of RyR2. Methods and results: Using whole-cell patch-clamp combined with confocal imaging, we applied a near threshold depolarization to activate a very small fraction of LCCs, which in turn activated RyR Ca2+ sparks stochastically. FKBP12.6-knockout and FK506/rapamycin treatments increased spark frequency and LCC-RyR coupling fidelity without altering LCC open probability. Neither FK506 nor rapamycin further altered LCC-RyR coupling fidelity in FKBP12.6-knockout cells. In loose-seal patch-clamp experiments, the LCC-RyR signalling kinetics, indexed by the delay for a LCC sparklet to trigger a RyR spark, was accelerated after FKBP12.6 knockout and FK506/rapamycin treatments. These results demonstrated that RyRs became more sensitive to Ca2+ triggers without FKBP12.6. Isoproterenol (1 µM) further accelerated the LCC-RyR signalling in FKBP12.6-knockout cells. The synergistic sensitization of RyRs by catecholaminergic signalling and FKBP12.6 dysfunction destabilized the CICR system, leading to chaotic Ca2+ waves and ventricular arrhythmias. Conclusion: FKBP12.6 keeps the RyRs from over-sensitization, stabilizes the potentially regenerative CICR system, and thus may suppress the life-threatening arrhythmogenesis.
Assuntos
Arritmias Cardíacas/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Miócitos Cardíacos/metabolismo , Receptor Cross-Talk , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/deficiência , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Genótipo , Isoproterenol/farmacologia , Cinética , Masculino , Potenciais da Membrana , Camundongos Knockout , Microscopia Confocal , Modelos Moleculares , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptor Cross-Talk/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Sirolimo/farmacologia , Processos Estocásticos , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo/antagonistas & inibidores , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
The elementary Ca2+ release events, Ca2+ sparks, has been found for a quarter of century. However, the molecular regulation of the spark generator, the ryanodine receptor (RyR) on the sarcoplasmic reticulum, remains obscure. Although each subunit of the RyR homotetramer has a site for FK506-binding protein (FKBP), the role of FKBPs in modifying RyR Ca2+ sparks has been debated for long. One of the reasons behind the controversy is that most previous studies detect spontaneous sparks, where the mixture with out-of-focus events and local wavelets prevents an accurate characterization of Ca2+ sparks. In the present study, we detected Ca2+ sparks triggered by single L-type Ca2+ channels (LCCs) under loose-seal patch clamp conditions in FK506-treated or FKBP12.6 knockout cardiomyocytes. We found that FKBP dissociation both by FK506 and by rapamycin decreased the Ca2+ spark amplitude in ventricular cardiomyocytes. This change was neither due to decreased releasable Ca2+ in the sarcoplasmic reticulum, nor explained by changed RyR sensitivity. Actually FK506 increased the LCC-RyR coupling probability and curtailed the latency for an LCC to trigger a RyR Ca2+ spark. FKBP12.6 knockout had similar effects as FK506/rapamycin treatment, indicating that the decreased spark amplitude was attributable to the dissociation of FKBP12.6 rather than FKBP12. We also explained how decreased amplitude of spontaneous sparks after FKBP dissociation sometimes appears to be increased or unchanged due to inappropriate data processing. Our results provided firm evidence that without the inter-RyR coordination by functional FKBP12.6, the RyR recruitment during a Ca2+ spark would be compromised despite the sensitization of individual RyRs.
RESUMO
With advanced aging, there is a decline in innate cardiovascular function. This decline is not general in nature. Instead, specific changes occur that impact the basic cardiovascular function, which include alterations in biochemical pathways and ion channel function. This review focuses on a particular ion channel that couple the latter two processes, namely the KATP channel, which opening is promoted by alterations in intracellular energy metabolism. We show that the intrinsic properties of the KATP channel changes with advanced aging and argue that the channel can be further modulated by biochemical changes. The importance is widespread, given the ubiquitous nature of the KATP channel in the cardiovascular system where it can regulate processes as diverse as cardiac function, blood flow and protection mechanisms against superimposed stress, such as cardiac ischemia. We highlight questions that remain to be answered before the KATP channel can be considered as a viable target for therapeutic intervention.
RESUMO
Myocardial ischemia remains the primary cause of morbidity and mortality in the United States. Ischemic preconditioning (IPC) is a powerful form of endogenous protection against myocardial infarction. We studied alterations in KATP channels surface density as a potential mechanism of the protection of IPC. Using cardiac-specific knockout of Kir6.2 subunits, we demonstrated an essential role for sarcolemmal KATP channels in the infarct-limiting effect of IPC in the mouse heart. With biochemical membrane fractionation, we demonstrated that sarcolemmal KATP channel subunits are distributed both to the sarcolemma and intracellular endosomal compartments. Global ischemia causes a loss of sarcolemmal KATP channel subunit distribution and internalization to endosomal compartments. Ischemia-induced internalization of KATP channels was prevented by CaMKII inhibition. KATP channel subcellular redistribution was also observed with immunohistochemistry. Ischemic preconditioning before the index ischemia reduced not only the infarct size but also prevented KATP channel internalization. Furthermore, not only did adenosine mimic IPC by preventing infarct size, but it also prevented ischemia-induced KATP channel internalization via a PKC-mediated pathway. We show that preventing endocytosis with dynasore reduced both KATP channel internalization and strongly mitigated infarct development. Our data demonstrate that plasticity of KATP channel surface expression must be considered as a potentially important mechanism of the protective effects of IPC and adenosine.
Assuntos
Precondicionamento Isquêmico Miocárdico , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sarcolema/metabolismo , Adenosina/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Endocitose , Endossomos/metabolismo , Hidrazonas/farmacologia , Preparação de Coração Isolado , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Sarcolema/efeitos dos fármacos , Fatores de TempoRESUMO
RATIONALE: During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. OBJECTIVE: To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. METHODS AND RESULTS: In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. CONCLUSIONS: MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Acoplamento Excitação-Contração/efeitos dos fármacos , Insuficiência Cardíaca/prevenção & controle , Hipertrofia Ventricular Esquerda/tratamento farmacológico , MicroRNAs/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Estenose Aórtica Subvalvar/complicações , Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/fisiologia , Modelos Cardiovasculares , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Oligonucleotídeos Antissenso/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/fisiologia , Retículo Sarcoplasmático/ultraestruturaRESUMO
RATIONALE: Failing cardiomyocytes exhibit decreased efficiency of excitation-contraction (E-C) coupling. The downregulation of junctophilin-2 (JP2), a protein anchoring the sarcoplasmic reticulum to T-tubules, has been identified as a major mechanism underlying the defective E-C coupling. However, the regulatory mechanism of JP2 remains unknown. OBJECTIVE: To determine whether microRNAs regulate JP2 expression. METHODS AND RESULTS: Bioinformatic analysis predicted 2 potential binding sites of miR-24 in the 3'-untranslated regions of JP2 mRNA. Luciferase assays confirmed that miR-24 suppressed JP2 expression by binding to either of these sites. In the aortic stenosis model, miR-24 was upregulated in failing cardiomyocytes. Adenovirus-directed overexpression of miR-24 in cardiomyocytes decreased JP2 expression and reduced Ca(2+) transient amplitude and E-C coupling gain. CONCLUSIONS: MiR-24-mediated suppression of JP2 expression provides a novel molecular mechanism for E-C coupling regulation in heart cells and suggests a new target against heart failure.