Dataset Information

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

ABSTRACT: 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).

SUBMITTER: Gemel J

PROVIDER: S-EPMC4135452 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

Gemel Joanna J Simon Adria R AR Patel Dakshesh D Xu Qin Q Matiukas Arvydas A Veenstra Richard D RD Beyer Eric C EC

Journal of molecular and cellular cardiology 20140625

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 ...[more]

PMID: 24973497

Similar Datasets

Project description: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.

Project description:PurposeTo develop an accelerated three-dimensional (3D) late gadolinium enhancement (LGE) pulse sequence using balanced steady-state free precession readout with stack-of-stars k-space sampling and extra motion-state golden-angle radial sparse parallel (XD-GRASP) reconstruction and test the performance for detecting atrial scar and fibrosis in patients with atrial fibrillation (AF).Materials and methodsTwenty-five patients with AF (20 paroxysmal and five persistent; 65 years ± 7 [standard deviation]; 18 men) were imaged at 1.5 T using the proposed LGE sequence with 1.3 mm × 1.3 mm × 2-mm spatial resolution and predictable imaging time. The resulting images were compared with historic images of 25 patients with AF (18 paroxysmal and seven persistent; 67 years ± 10; 14 men) obtained using a reference 3D left atrial (LA) LGE sequence with 1.3 mm × 1.3 mm × 2.5-mm spatial resolution. Two readers visually graded the 3D LGE images (conspicuity, artifact, noise) on a five-point Likert scale (1 = worst, 3 = acceptable, 5 = best), in which the summed visual score (SVS) of 9 or greater was defined as clinically acceptable. Appropriate statistical analyses (Cohen κ coefficient, Mann-Whitney U test, t tests, and intraclass correlation) were performed, where a P value < .05 was considered significant.ResultsMean imaging time was significantly shorter (P < .01) for the proposed pulse sequence (5.9 minutes ± 1.3) than for the reference pulse sequence (10.6 minutes ± 2). Median SVS was significantly higher (P < .01) for the proposed (SVS = 11) than reference (SVS = 9.5) 3D LA LGE images. Interrater reproducibility in visual scores was higher for the proposed (κ = 0.78-1) than reference 3D LA LGE (κ = 0.44-0.75). Intrareader repeatability in fibrosis quantification was higher for the reference cohort (intraclass correlation coefficient [ICC] = 0.94) than the prospective cohort (ICC = 0.79).ConclusionThe proposed 3D LA LGE method produced clinically acceptable image quality with 1.5 mm × 1.5 mm × 2-mm nominal spatial resolution and 6-minute predictable imaging time for quantification of LA scar and fibrosis in patients with AF. Supplemental material is available for this article. © RSNA, 2020.

Dataset Information

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

Publications

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets