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.
Background Infection with the novel severe acute respiratory syndrome coronavirus 2 has been associated with a hypercoagulable state. Emerging data from China and Europe have consistently shown an increased incidence of venous thromboembolism (VTE). We aimed to identify the VTE incidence and early predictors of VTE at our high-volume tertiary care center. Methods We performed a retrospective cohort study of 147 patients who had been admitted to Temple University Hospital with coronavirus disease 2019 (COVID-19) from April 1, 2020 to April 27, 2020. We first identified the VTE (pulmonary embolism [PE] and deep vein thrombosis [DVT]) incidence in our cohort. The VTE and no-VTE groups were compared by univariable analysis for demographics, comorbidities, laboratory data, and treatment outcomes. Subsequently, multivariable logistic regression analysis was performed to identify the early predictors of VTE. Results The 147 patients (20.9% of all admissions) admitted to a designated COVID-19 unit at Temple University Hospital with a high clinical suspicion of acute VTE had undergone testing for VTE using computed tomography pulmonary angiography and/or extremity venous duplex ultrasonography. The overall incidence of VTE was 17% (25 of 147). Of the 25 patients, 16 had had acute PE, 14 had had acute DVT, and 5 had had both PE and DVT. The need for invasive mechanical ventilation (adjusted odds ratio, 3.19; 95% confidence interval, 1.07-9.55) and the admission D-dimer level ≥1500 ng/mL (adjusted odds ratio, 3.55; 95% confidence interval, 1.29-9.78) were independent markers associated with VTE. The all-cause mortality in the VTE group was greater than that in the non-VTE group (48% vs 22%; P = .007). Conclusion Our study represents one of the earliest reported from the United States on the incidence rate of VTE in patients with COVID-19. Patients with a high clinical suspicion and the identified risk factors (invasive mechanical ventilation, admission D-dimer level ≥1500 ng/mL) should be considered for early VTE testing. We did not screen all patients admitted for VTE; therefore, the true incidence of VTE could have been underestimated. Our findings require confirmation in future prospective studies.
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.
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 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 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 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).
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