Chromatin organization, spatial localization of heterochromatin sequences and nucleolar activity, change towards final maturation in porcine oocytes

International audienceThe chromatin conformation of oocytes is subjected to large-scale modifications correlated to transcriptional silencing during final maturation. These modifications seem essential both for completion of the meiosis and subsequent embryonic developmental success. However, in pig, knowledge about spatial heterochromatin organization and nucleolar transcriptional activity toward the completion of oocyte growth are still lacking. In the present study, 300 porcine cumulus-oocyte-complexes (COCs) were recovered from ovaries and then divided into 2 groups, pre-categorized by supravital brilliant-cresyl-blue (BCB) staining, defining either fully mature (BCB+) and still maturating (BCB-) oocytes. Three dimension nuclear organization of heterochromatin was determined by either 3D-immunofluorescence (n=140) using specific antibodies against H3K9me3 and centromeric proteins (CENPs) and by 3D-DNA-FISH experiments (n=140) using fluorescent oligonucleotides specific for porcine meta- and acrocentric chromosomes. Nucleolar activity was detected using antibodies upstream binding factor (UBF). All images were acquired using laser scanning confocal microscpy. Finally, oocytes (n=20) were prepared for TEM according to standard protocol. Qualitative assessment of cellular ultrastructure by TEM revealed distinct differences between BCB+ and BCB- oocytes and supported BCB-staining as viable method for rough categorization regarding maturation status. All chromatin conformations were detected: from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) and their intermediate conformations (pNSN, pSN) in both BCB groups. However, the BCB+ group contained a higher percentage of oocytes expressing chromatin conformations categorized as mature (pSN and SN), whereas the opposite was true for the BCB- group. UBF-activity was only present in NSN and pNSN categorized oocytes and detected significantly more often in the BCB- group. The distribution of centromeric (CENP) and pericentromeric chromatin (H3K9me3) as well as repeated sequences DNA-FISH signal displayed distinct changes in their 3D-organization between NSN and SN conformation, characterized by significant signal-condensation around the nucleolus in SN oocytes. Altogether, these results indicate that oocytes from the BCB+ group, i.e. oocytes with higher competency for embryonic development, display specific heterochromatin organization patterns related to final oocyte maturation

Increased fibroblast accumulation in the equine heart following persistent atrial fibrillation

BACKGROUND: Fibroblasts maintain the extracellular matrix homeostasis and may couple to cardiomyocytes through gap junctions and thereby increase the susceptibility to slow conduction and cardiac arrhythmias, such as atrial fibrillation (AF). In this study, we used an equine model of persistent AF to characterize structural changes and the role of fibroblasts in the development of an arrhythmogenic substrate for AF. MATERIAL AND METHODS: Eleven horses were subjected to atrial tachypacing until self-sustained AF developed and were kept in AF for six weeks. Horses in sinus rhythm (SR) served as control. In terminal open-chest experiments conduction velocity (CV) was measured. Tissue was harvested and stained from selected sites. Automated image analysis was performed to assess fibrosis, fibroblasts, capillaries and various cardiomyocyte characteristics. RESULTS: Horses in SR showed a rate-dependent slowing of CV, while in horses with persistent AF this rate-dependency was completely abolished (CV•basic cycle length relation p = 0.0295). Overall and interstitial amounts of fibrosis were unchanged, but an increased fibroblast count was found in left atrial appendage, Bachmann's bundle, intraatrial septum and pulmonary veins (p < 0.05 for all) in horses with persistent AF. The percentage of α-SMA expressing fibroblasts remained the same between the groups. CONCLUSION: Persistent AF resulted in fibroblast accumulation in several regions, particularly in the left atrial appendage. The increased number of fibroblasts could be a mediator of altered electrophysiology during AF. Targeting the fibroblast proliferation and differentiation could potentially serve as a novel therapeutic target slowing down the structural remodeling associated with AF

Effect of selective I-K,I-ACh inhibition by XAF-1407 in an equine model of tachypacing-induced persistent atrial fibrillation

BACKGROUND AND PURPOSE: Inhibition of the G‐protein gated ACh‐activated inward rectifier potassium current, I (K,ACh) may be an effective atrial selective treatment strategy for atrial fibrillation (AF). Therefore, the anti‐arrhythmic and electrophysiological properties of a novel putatively potent and highly specific I (K,ACh) inhibitor, XAF‐1407 (3‐methyl‐1‐[5‐phenyl‐4‐[4‐(2‐pyrrolidin‐1‐ylethoxymethyl)‐1‐piperidyl]thieno[2,3‐d]pyrimidin‐6‐yl]azetidin‐3‐ol), were characterised for the first time in vitro and investigated in horses with persistent AF. EXPERIMENTAL APPROACH: The pharmacological ion channel profile of XAF‐1407 was investigated using cell lines expressing relevant ion channels. In addition, eleven horses were implanted with implantable cardioverter defibrillators enabling atrial tachypacing into self‐sustained AF. The electrophysiological effects of XAF‐1407 were investigated after serial cardioversions over a period of 1 month. Cardioversion success, drug‐induced changes of atrial tissue refractoriness, and ventricular electrophysiology were assessed at baseline (day 0) and days 3, 5, 11, 17, and 29 after AF induction. KEY RESULTS: XAF‐1407 potently and selectively inhibited K(ir)3.1/3.4 and K(ir)3.4/3.4, underlying the I (K,ACh) current. XAF‐1407 treatment in horses prolonged atrial effective refractory period as well as decreased atrial fibrillatory rate significantly (~20%) and successfully cardioverted AF, although with a decreasing efficacy over time. XAF‐1407 shortened atrioventricular‐nodal refractoriness, without effect on QRS duration. QTc prolongation (4%) within 15 min of drug infusion was observed, however, without any evidence of ventricular arrhythmia. CONCLUSION AND IMPLICATIONS: XAF‐1407 efficiently cardioverted sustained tachypacing‐induced AF of short duration in horses without notable side effects. This supports I (K,ACh) inhibition as a potentially safe treatment of paroxysmal AF in horses, suggesting potential clinical value for other species including humans

Effect of selective I

Background and Purpose: Inhibition of the G-protein gated ACh-activated inward rectifier potassium current, IK,ACh may be an effective atrial selective treatment strategy for atrial fibrillation (AF). Therefore, the anti-arrhythmic and electrophysiological properties of a novel putatively potent and highly specific IK,ACh inhibitor, XAF-1407 (3-methyl-1-[5-phenyl-4-[4-(2-pyrrolidin-1-ylethoxymethyl)-1-piperidyl]thieno[2,3-d]pyrimidin-6-yl]azetidin-3-ol), were characterised for the first time in vitro and investigated in horses with persistent AF. Experimental Approach: The pharmacological ion channel profile of XAF-1407 was investigated using cell lines expressing relevant ion channels. In addition, eleven horses were implanted with implantable cardioverter defibrillators enabling atrial tachypacing into self-sustained AF. The electrophysiological effects of XAF-1407 were investigated after serial cardioversions over a period of 1 month. Cardioversion success, drug-induced changes of atrial tissue refractoriness, and ventricular electrophysiology were assessed at baseline (day 0) and days 3, 5, 11, 17, and 29 after AF induction. Key Results: XAF-1407 potently and selectively inhibited Kir3.1/3.4 and Kir3.4/3.4, underlying the IK,ACh current. XAF-1407 treatment in horses prolonged atrial effective refractory period as well as decreased atrial fibrillatory rate significantly (~20%) and successfully cardioverted AF, although with a decreasing efficacy over time. XAF-1407 shortened atrioventricular-nodal refractoriness, without effect on QRS duration. QTc prolongation (4%) within 15 min of drug infusion was observed, however, without any evidence of ventricular arrhythmia. Conclusion and Implications: XAF-1407 efficiently cardioverted sustained tachypacing-induced AF of short duration in horses without notable side effects. This supports IK,ACh inhibition as a potentially safe treatment of paroxysmal AF in horses, suggesting potential clinical value for other species including humans