Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215–219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Non-valvular atrial fibrillation recurrence after sinus rhythm restoring at different follow-up periods: phenotype-genotype high-risk groups, considering rs10465885 polymorphism in connexin-40 gene

Funding Acknowledgements Type of funding sources: None. Purpose to provide risk stratification of non-valvular atrial fibrillation (AF) recurrence after sinus rhythm restoring (SRR) in patients (pts) with AF at different follow-up periods (3 months (AFr3m), 12 months (AFr12m) and 18 months [AFr18m]), based on phenotype-genotype high-risk groups, considering rs10465885 polymorphism in connexin-40 gene (SNP-Cx40). Methods. We enrolled 186 pts (mean age (55 ± 10) years; males 123 [66,1%]) with AF (paroxysmal – 86, persistent – 72, stable – 28 pts; first onset (FO) AF – 48 pts). Clinical, laboratory and echocardiographic data were analyzed. SNP-Cx40 was genotyped by real time PCR (T – reference, C – minor allele) in 112 pts. The genotypes were distributed as follows: TT – 25,9% (n = 29); CT – 49,1% (n = 55); CC – 25,0% (n = 28). SRR was performed in 112 cases (102 pts) with non-permanent AF: 30 – pharmacological cardioversion (PCV), 62 – direct-current cardioversion (DCV), 20 cases – radiofrequency ablation (RFA). AFr3m occurred in 53 (43,4%) of 122 available cases; AFr12m – 65,5% (76/116); AFr18m – 75,2% (79/105). The Artificial Neural networks (ANNs) analysis was performed to select the AF recurrence predictors. We considered the ANN activation function value (Y) and its relation to Y cut-off value (Ycrit). In case of Y > Ycrit, the AF recurrence risk was considered as «high». Results. We built three nonlinear ANN models (multilayer perceptrons) for AFr3m (Ycrit = 0,496), AFr12m (Ycrit = 0,503) and AFr18m (Ycrit = 0,720) risk prediction. In case of SNP-Cx40 CC genotype carriage, we determined the additional increase of AFr3m risk after PCV in pts with CHA2DS2-VASc score «0» and normal (Y = 0,629) or mildly decreased (Y = 0,616) left ventricular mid-wall fractional shortening, and in the case of its moderate decrease – both after PCV (Y = 0,585) and DCV (Y = 0,627). The CC genotype was associated with AFr12m high risk in pts without heart failure (HF) and mildly increased left atrial dimension (LAD) – both after PCV (FO AF with known precise event duration (PED); Y = 0,906) or DCV (FO AF with unknown PED; Y = 0,911). Additionally, CC genotype was associated with AFr12m high risk after RFA in pts without HF and normal or mildly increased LAD (Y = 0,912), and in pts with HF B or C1 stage (according to modified AHA/ACC classification) with moderately increased LAD (Y = 0,912). The high-risk groups of AFr18m in pts with CC genotype were as follows (Y = 0,913): after RFA in pts with recurrent AF and presence of episodes lasting ≥7 days; after PCA in case of FO AF with unknown PED and index episode lasting ≥1 month; after DCV in case of FO AF with unknown PED and index episode lasting ≥12 months. Conclusion. AFr3m, AFr12m and AFr18m, besides SNP-Cx40, were non-linearly associated with SRR type, and certain clinical and echocardiographic phenotypic parameters, which could be used for AF recurrence risk stratification, with the selection of phenotype-genotype high-risk groups, considering SNP-Cx40.

NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro

Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

Prolonged duration of repolarization and decreased conduction velocity in the atrial myocardium after hypothermic ischemia-reperfusion may be related to expressions of inward rectifier potassium channel 2.1 protein and connexin 40

Objectives: The study aimed to determine the role of inward rectifier potassium channel 2.1 protein and connexin 40 expressions in regulating the duration of repolarization and conduction velocity of right atrial myocardium in rats following hypothermic ischemia-reperfusion. Methods: The Langendorff isolated rat cardiac perfusion models were divided into control (C) and hypothermic ischemia-reperfusion groups, with 8 models in group C and 16 models in group ischemia-reperfusion. Depending on the incidence of atrial arrhythmia after reperfusion, the models in group ischemia-reperfusion were further divided into reperfusion non-atrial arrhythmia or reperfusion atrial arrhythmia subgroup. Right atrial monophasic action potential duration at 50% and 90% of repolarization after 30 minutes of continuous perfusion in group C and group ischemia-reperfusion (T0), 105 minutes of continuous perfusion in group C or after 15 minutes of reperfusion in group ischemia-reperfusion (T1) and 120 minutes of continuous perfusion in group C or 30 minutes of reperfusion in group ischemia-reperfusion (T2) were recorded. Right atrial conduction velocity and effective refractory period were recorded at T2. Then, the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 in the right atrial myocardium were detected. Results: Monophasic action potential duration at 50% and 90% were higher at T1 and T2 than those at T0 in subgroup reperfusion atrial arrhythmia (p < 0.05); monophasic action potential duration at 50% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); monophasic action potential duration at 90% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); effective refractory period in subgroup reperfusion atrial arrhythmia was greater than that in group C and subgroup reperfusion non-atrial arrhythmia, and the conduction velocity and the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 were significantly lower than group C and subgroup reperfusion non-atrial arrhythmia (p < 0.05). Conclusions: The prolonged duration of repolarization and a decrease in conduction velocity of the atrial myocardium occur in rats after hypothermic ischemia-reperfusion. These observed effects may be related to the downregulated expressions of connexin 40 and inward rectifier potassium channel 2.1.

P1079Non-valvular atrial fibrillation recurrence after sinus rhythm restoring at 1-year follow-up: predictors and risk stratification considering rs10465885 polymorphism in connexin-40 gene

Purpose to establish the predictors of non-valvular atrial fibrillation (AF) recurrence after sinus rhythm restoring (SRR) in patients (pts) with AF at 1-year follow-up (AFr1y), and provide AFr1y risk stratification, considering single nucleotide polymorphism rs10465885 in connexin-40 gene (SNP-Cx40). Methods. We enrolled 186 pts before the age of 65 years (mean age (55 ± 10) years; males 123 [66,1%]) with AF (paroxysmal – 86, persistent – 72, stable – 28 pts; first onset (FO) AF – 48 pts). Clinical, laboratory and echocardiographic data were analyzed. SNP-Cx40 was genotyped by real time polymerase chain reaction (T – reference, C – minor allele) in 112 pts. Genotype distribution of SNP-Cx40 was as follows: TT – 25,9% (n = 29); CT – 49,1% (n = 55); CC – 25,0% (n = 28). SRR was performed in 112 cases (102 pts) with non-permanent AF: 30 – pharmacological cardioversion (PCV), 62 – direct-current cardioversion (DCV), 20 cases – radiofrequency ablation (RFA). AFr1y occurred in 74 (66,1%) of 112 cases. We performed Artificial Neural networks (ANNs) analysis to select the AFr1y predictors. While AFr1y risk stratification, we considered the activation function value (Y) of the certain ANN model and its relation to Y cut-off value (Ycrit). In case of Y &gt; Ycrit, the AFr1y risk was considered as «high». Results. Genetic algorithm Input Selection revealed the set of parameters, associated with AFr1y, included SRR type, SNP-Cx40, and baseline clinical (body mass index, global cardiovascular risk, heart failure (HF) stage, CHA2DS2-VASc score, AF anamnesis duration, average AF event duration, FO AF), laboratory (fasting glucose level, estimated glomerular filtration rate, red cell distribution width, total serum cholesterol level) and echocardiographic (left atrial dimension (LAD), left ventricular (LV) mid-wall fractional shortening degree, LV hypertrophy degree) characteristics. In order to obtain the maximal reduction of predictors, we built non-linear multilayer perceptron model (MLP5) on the basis of set of 5 the most sensitive parameters, included SRR type, SNP-Cx40, HF stage, LAD and FO AF. The area under curve for MLP5 (0,808 [CI 0,723-0,876]) was higher than 0,5 (p &lt; 0,001), with Ycrit = 0,503. The rs10465885 CC genotype was associated with AFr1y high risk in pts without HF and mildly increased LAD – both after PCV (FO AF with known precise event duration (PED); Y = 0,906) or DCV (FO AF with unknown PED; Y = 0,911). Additionally, rs10465885 CC genotype was associated with AFr1y high risk after RFA in pts without HF and normal or mildly increased LAD (Y = 0,912), and in pts with HF B or C1 stage (according to modified AHA/ACC classification) with moderately increased LAD (Y = 0,912). Conclusions. AFr1y was non-linearly associated with SRR type, SNP-Cx40 and certain phenotypic parameters, including HF stage, FO AF and LAD. These parameters could be used for AFr1y risk stratification, with the selection of phenotype-genotype high risk groups, considering SNP-Cx40.

Deletion of Sphingosine 1-Phosphate Receptor 1 in cardiomyocytes during development leads to abnormal ventricular conduction and fibrosis

ABSTRACTSphingosine 1-phosphate receptor 1 (S1P1, encoded by S1pr1) is a G protein-coupled receptor that signals in multiple cell types including endothelial cells and cardiomyocytes. Cardiomyocyte-specific deletion of S1pr1 during development leads to ventricular noncompaction, with one-third of mutant mice surviving to adulthood. Adult survivors of embryonic cardiomyocyte S1pr1 deletion showed cardiac hypertrabeculation consistent with ventricular noncompaction. Surprisingly, systolic function in mutant mice was preserved through at least one year of age. Cardiac conduction was abnormal in cardiomyocyte S1pr1 mutant mice, with prolonged QRS intervals in mutants as compared with littermate control mice. Immunostaining of hearts from S1pr1 mutant embryos displayed a zone of intermediate Connexin 40 expression in the trabecular myocardium. However, we observed no significant differences in Connexin 40 and Connexin 43 immunostaining in hearts from adult survivors of embryonic cardiomyocyte S1pr1 deletion, which suggests normalized development of the ventricular conduction system in mutant mice. By contrast, the adult survivors of embryonic cardiomyocyte S1pr1 deletion showed increased cardiac fibrosis as compared with littermate controls. These results demonstrate that ventricular hypertrabeculation caused by embryonic deletion of cardiomyocyte S1pr1 leads to cardiac fibrosis, which contributes to abnormal ventricular conduction. These results also reveal conduction abnormalities in the setting of hypertrabeculation with normal systolic function, which may be of clinical relevance in humans with ventricular hypertrabeculation.

Rho Kinase Activity, Connexin 40, and Atrial Fibrillation: Mechanistic Insights from End-Stage Renal Disease on Dialysis Patients

Evidence on cellular/molecular mechanisms leading to atrial fibrillation (AF) are scanty. Increased expression of Rho kinase (ROCK) and myosin-phosphatase-target subunit-1 (MYPT-1), ROCK activity’s marker, were shown in AF patients, which correlated with connexin 40 (Cx40) expression, membrane protein of heart gap junctions, key for rapid action potential’s cell–cell transfer. AF is the most frequent arrhythmia in dialysis patients who present increased MYPT-1 phosphorylation, which correlates with left ventricular (LV) mass. Given ROCK’s established role in cardiovascular–renal remodeling, induction of impaired cell-to-cell coupling/potential conduction promoting AF initiation/perpetuation, we evaluated in dialysis patients with AF, MYPT-1 phosphorylation, Cx40 expression, and their relationships to support their involvement in AF. Mononuclear cells’ MYPT-1 phosphorylation, Cx40 expression, and the ROCK inhibitor fasudil’s effect were assessed in dialysis patients with AF (DPAFs), dialysis patients with sinus rhythm (DPs), and healthy subjects (C) (western blot). M-mode echocardiography assessed LV mass and left atrial systolic volume. DPAF’s phospho-MYPT-1 was increased vs. that of DPs and C (1.57 ± 0.17 d.u. vs. 0.69 ± 0.04 vs. 0.51 ± 0.05 respectively, p < 0.0001). DP’s phospho-MYPT-1 was higher vs. that of C, p = 0.009. DPAF’s Cx40 was higher vs. that of DPs and C (1.23 ± 0.12 vs. 0.74 ± 0.03 vs. 0.69 ± 0.03, p < 0.0001). DPAF’s phospho-MYPT-1 correlated with Cx40 (p < 0.001), left atrial systolic volume (p = 0.013), and LV mass (p = 0.014). In DPAFs, fasudil reduced MYPT-1 phosphorylation (p < 0.01) and Cx40 expression (p = 0.03). These data point toward ROCK and Cx40’s role in the mechanism(s) leading to AF in dialysis patients. Exploration of the ROCK pathway in AF could contribute to AF generation’s mechanistic explanations and likely identify potential pharmacologic targets for translation into treatment.