Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed

Alterations of cellular electrophysiology and Ca2+-handling in patients with different forms of atrial fibrillation

Atrial Fibrillation (AF) is the most common clinical arrhythmia. When AF occurs after (cardiac) surgery it is referred to as postoperative AF (poAF). Though usually self-limiting, uncontrolled poAF can cause debilitating strokes and prolong hospital stay. A multifactorial pathophysiology involving triggered activity has been hypothesized, but the cellular and molecular mechanisms underlying poAF remain unknown and were the subject of the first part of this study. The second part of this study focused on inward-rectifier K+ currents like IK1, which stabilize the resting membrane potential. It has been suggested to contribute to the shorter action potentials that promote reentry in chronic AF (cAF). However, the exact magnitude of IK1 in cAF is unknown and was investigated. Membrane currents (whole-cell voltage clamp) and [Ca2+]i (Fluo-3) epifluorescence were measured in right-atrial cardiomyocytes from patients with sinus-rhythm (Ctl, n=58), poAF (n=46) or cAF (n=6). Protein expression was quantified by immunoblot. Amplitude of L-type Ca2+ current was unchanged, whereas the L-type Ca2+ current-triggered [Ca2+]i transient amplitude was reduced by 35% in poAF vs Ctl, likely contributing to the ~44% reduction in fractional cell shortening. Sarcoplasmic reticulum (SR) Ca2+ content, calculated by integrating Na+-Ca2+ exchange current during caffeine (10 mM)-induced SR Ca2+ release, was unchanged, consistent with the unaltered protein expression of the SR Ca2+ ATPase type-2a and its regulator phospholamban. When challenged with interleukin-1 as a postoperative trigger the frequency of potentially proarrhythmic spontaneous SR Ca2+ release events was increased, likely due to hyperphosphorylation of ryanodine receptor channels at Ser2014 (34%), whereas protein expression of the ryanodine receptor channel regulators triadin, junctin, junctophilin-2 and calsequestrin was unaltered. In cAF, basal inward-rectifier K+ current was increased, whereas the major effector of vagal nerve stimulation, the acetylcholine-activated K+ current IK,ACh, was reduced compared to Ctl patients. Application of the selective IK1-inbititor PA-6 (200 nM) to basal inward-rectifier K+ current unmasked a larger “pure” IK1 in cAF compared to Ctl patients. In summary, we discovered that poAF is associated with Ca2+-handling abnormalities that predispose patients to cellular triggered activity underlying poAF. We could demonstrate using PA-6; a novel inhibitor of IK1, that “pure” IK1 is larger in cAF. Our data improve our understanding of atrial cellular arrhythmogenic mechanisms in AF in general and in poAF in particular, potentially facilitating the development of improved therapeutic anti-AF strategies.Vorhofflimmern (VHF) ist die häufigste klinische Arrhythmie. Wenn es nach herzchirurgischen Eingriffen auftritt, wird es als postoperatives VHF (poVHF) bezeichnet. Wenngleich normalerweise selbstlimitierend, kann unkontrolliertes poVHF Schlaganfälle und einen verlängerten Krankenhausaufenthalt verursachen. PoVHF liegt vermutlich eine multifaktorielle Pathophysiologie zugrunde, wobei der „getriggerten“ Aktivität eine zentrale Rolle zukommen könnte. Die genauen zellulären Mechanismen des poVHF sind unbekannt und waren Gegenstand des ersten Teils dieser Arbeit. Der zweite Teil der Arbeit befasste sich mit einwärtsgleichrichtenden K+-Strömen wie dem IK1, die das negative Ruhemembranpotential aufrechterhalten. Es wird vermutet, dass diese zur Verkürzung der Aktionspotentiale beitragen und die Entstehung kreisender Erregungen („Reentry“) bei chronischem VHF (cVHF) begünstigen, wobei die genaue Größe des IK1 bei cVHF bisher unbekannt war. Membranströme („whole-cell voltage clamp“) und [Ca2+]i (Fluo-3) Epifluoreszenz wurden in rechtsatrialen Kardiomyozyten von Patienten mit Sinusrhythmus (Ktr, n=58), mit poVHF (n=46) oder mit cVHF (n=6) registriert. Die Proteinexpression wurde mittels Immunoblot quantifiziert. Die Amplitude des L-Typ Ca2+-Stromes war unverändert, während die L-Typ Ca2+-Strom ausgelöste [Ca2+]i-Transientamplitude beim poVHF um 35% reduziert war. Entsprechend war die relative Zellverkürzung um ~44% vermindert. Der Ca2+-Gehalt im sarkoplasmatischen Retikulum (SR), der durch Integration des Na+-Ca2+--Austauschstroms während der Koffein (10 mM)-ausgelösten Ca2+-Freisetzung aus dem SR berechnet wurde, war unverändert. Dies stimmte mit der unveränderten Proteinexpression der SR Ca2+-ATPase Typ-2a und dessen Regulator Phospholamban überein. Der Einsatz von Interleukin-1 als postoperativer Trigger steigerte die Frequenz spontaner proarrhythmischer Ca2+-Freisetzungen aus dem SR, möglicherweise aufgrund der Zunahme der Ser2014-Hyperphosphorylierung der Ryanodin-Rezeptorkanäle (~34%). Die Proteinexpression der Ryanodin-Rezeptorkanal-Regulatoren Triadin, Junctin, Junctophilin-2 und Calsequestrin blieb dagegen unverändert. Beim cVHF war der basale einwärtsgleichrichtende K+-Strom erhöht, während der Haupteffektor einer Stimulation von Nervus vagus, der Acetylcholin-aktivierte K+-Strom IK,ACh, reduziert war. Die Applikation des selektiven IK1-Inhibitors PA-6 (200 nM) beim cVHF konnte einen größeren "reinen" IK1 aufdecken. Zusammenfassend konnten wir nachweisen, dass poVHF mit Störungen der Ca2+-Homöostase assoziiert ist. Letzteres könnte das Auftreten von „getriggerter“ Aktivität als Auslöser von poVHF begünstigen. Wir konnten außerdem zeigen, dass PA-6; ein neuartiger Inhibitor von IK1, einen erhöhten IK1 beim cVHF demaskierte. Unsere Daten verbessern unser Verständnis der zellulären Mechanismen von III VHF, insbesondere von poVHF, und könnten zur Entwicklung von neuen therapeutischen Strategien für VHF führen

The inward rectifier current inhibitor PA-6 terminates atrial fibrillation and does not cause ventricular arrhythmias in goat and dog models

Background and Purpose: The density of the inward rectifier current (IK1) increases in atrial fibrillation (AF), shortening effective refractory period and thus promoting atrial re-entry. The synthetic compound pentamidine analogue 6 (PA-6) is a selective and potent IK1 inhibitor. We tested PA-6 for anti-AF efficacy and potential proarrhythmia, using established models in large animals. Experimental Approach: PA-6 was applied i.v. in anaesthetized goats with rapid pacing-induced AF and anaesthetized dogs with chronic atrio-ventricular (AV) block. Electrophysiological and pharmacological parameters were determined. Key Results: PA-6 (2.5 mg·kg−1·10 min−1) induced cardioversion to sinus rhythm (SR) in 5/6 goats and prolonged AF cycle length. AF complexity decreased significantly before cardioversion. PA-6 accumulated in cardiac tissue with ratios between skeletal muscle : atrial muscle : ventricular muscle of approximately 1:8:21. In SR dogs, PA-6 peak plasma levels 10 min post infusion were 5.5 ± 0.9 μM, PA-6 did not induce significant prolongation of QTc and did not affect heart rate, PQ or QRS duration. In dogs with chronic AV block, PA-6 did not affect QRS but lengthened QTc during the experiment, but not chronically. PA-6 did not induce TdP arrhythmias in nine animals (0/9) in contrast to dofetilide (5/9). PA-6 (200 nM) inhibited IK1, but not IK,ACh, in human isolated atrial cardiomyocytes. Conclusion and Implications: PA-6 restored SR in goats with persistent AF and, in dogs with chronic AV block, prolonged QT intervals, without inducing TdP arrhythmias. Our results demonstrate cardiac safety and good anti-AF properties for PA-6

The inward rectifier current inhibitor PA-6 terminates atrial fibrillation and does not cause ventricular arrhythmias in goat and dog models

Background and Purpose: The density of the inward rectifier current (IK1) increases in atrial fibrillation (AF), shortening effective refractory period and thus promoting atrial re-entry. The synthetic compound pentamidine analogue 6 (PA-6) is a selective and potent IK1 inhibitor. We tested PA-6 for anti-AF efficacy and potential proarrhythmia, using established models in large animals. Experimental Approach: PA-6 was applied i.v. in anaesthetized goats with rapid pacing-induced AF and anaesthetized dogs with chronic atrio-ventricular (AV) block. Electrophysiological and pharmacological parameters were determined. Key Results: PA-6 (2.5 mg·kg−1·10 min−1) induced cardioversion to sinus rhythm (SR) in 5/6 goats and prolonged AF cycle length. AF complexity decreased significantly before cardioversion. PA-6 accumulated in cardiac tissue with ratios between skeletal muscle : atrial muscle : ventricular muscle of approximately 1:8:21. In SR dogs, PA-6 peak plasma levels 10 min post infusion were 5.5 ± 0.9 μM, PA-6 did not induce significant prolongation of QTc and did not affect heart rate, PQ or QRS duration. In dogs with chronic AV block, PA-6 did not affect QRS but lengthened QTc during the experiment, but not chronically. PA-6 did not induce TdP arrhythmias in nine animals (0/9) in contrast to dofetilide (5/9). PA-6 (200 nM) inhibited IK1, but not IK,ACh, in human isolated atrial cardiomyocytes. Conclusion and Implications: PA-6 restored SR in goats with persistent AF and, in dogs with chronic AV block, prolonged QT intervals, without inducing TdP arrhythmias. Our results demonstrate cardiac safety and good anti-AF properties for PA-6