RESUMO
Normal atrial conduction requires similar abundances and homogeneous/overlapping distributions of two connexins (Cx40 and Cx43). The remodeling of myocyte connections and altered electrical conduction associated with atrial fibrillation (AF) likely involves perturbations of these connexins. We conducted a comprehensive series of experiments to examine the abundances and distributions of Cx40 and Cx43 in the atria of AF patients. Atrial appendage tissues were obtained from patients with lone AF (paroxysmal or chronic) or normal controls. Connexins were localized by double label immunofluorescence confocal microscopy, and their overlap was quantified. Connexin proteins and mRNAs were quantified by immunoblotting and qRT-PCR. PCR amplified genomic DNA was sequenced to screen for connexin gene mutations or polymorphisms. Immunoblotting showed reductions of Cx40 protein (to 77% or 49% of control values in samples from patients with paroxysmal and chronic AF, respectively), but no significant changes of Cx43 protein levels in samples from AF patients. The extent of Cx43 immunostaining and its distribution relative to N-cadherin were preserved in the AF patient samples. Although there was variability of Cx40 staining among paroxysmal AF patients, all had some fields with substantial Cx40 heterogeneity and reduced overlap with Cx43. Cx40 immunostaining was severely reduced in all chronic AF patients. qRT-PCR showed no change in Cx43 mRNA levels, but reductions in total Cx40 mRNA (to <50%) and Cx40 transcripts A (to ~50%) and B (to <25%) as compared to controls. No Cx40 coding region mutations were identified. The frequency of promoter polymorphisms did not differ between AF patient samples and controls. Our data suggest that reduced Cx40 levels and heterogeneity of its distribution (relative to Cx43) are common in AF. Multiple mechanisms likely lead to reductions of functional Cx40 in atrial gap junctions and contribute to the pathogenesis of AF in different patients.
Assuntos
Fibrilação Atrial/metabolismo , Conexinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Caderinas/metabolismo , Estudos de Casos e Controles , Conexina 43/metabolismo , Conexinas/genética , Feminino , Junções Comunicantes , Átrios do Coração/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Regiões Promotoras Genéticas , Proteína alfa-5 de Junções ComunicantesRESUMO
Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several of these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by the transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20-31% of wild type levels. After the inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition).
Assuntos
Conexinas/genética , Átrios do Coração/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Linhagem Celular Tumoral , Conexinas/metabolismo , Cicloeximida/farmacologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/patologia , Humanos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Oligopeptídeos/farmacologia , Técnicas de Patch-Clamp , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica , Inibidores da Síntese de Proteínas/farmacologia , Proteólise , Transdução de Sinais , Transgenes , Ubiquitinação , Proteína alfa-5 de Junções ComunicantesRESUMO
Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (Vj) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of Vj gating properties and increased the gap junction channel conductance (γj) by 20-30 pS. While co-expression of the two mutations had similar effects on Vj gating, it synergistically increased γj by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.