Fahima Syeda

An introduction to murine models of atrial fibrillation

Understanding the mechanism of re-entrant arrhythmias in the past 30 years has allowed the development of almost curative therapies for many rhythm disturbances. The complex, polymorphic arrhythmias of atrial fibrillation (AF) and sudden death are, unfortunately, not yet well understood, and hence still in need of adequate therapy. AF contributes markedly to morbidity and mortality in aging Western populations. In the past decade, many genetically altered murine models have been described and characterized. Here, we review genetically altered murine models of AF; powerful tools that will enable a better understanding of the mechanisms of AF and the assessment of novel therapeutic interventions.

PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers

Background Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA). Objectives After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs. Methods LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na+)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials. Results Flecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c+/–). Resting membrane potential was more depolarized in Pitx2c+/– atria, and TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide’s effects between wild-type and Pitx2c+/– atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Nav1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized. Conclusions PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.

PITX2‐dependent gene regulation in atrial fibrillation and rhythm control

Atrial fibrillation (AF) is a common arrhythmia. Better prevention and treatment of AF are needed to reduce AF‐associated morbidity and mortality. There are several major mechanisms that cause AF in patients, including a genetic predisposition to develop AF. Genome‐wide association studies have identified genetic variants associated with AF populations, with the strongest hits clustering on chromosome 4q25, close to the gene for the homeobox transcription factor PITX2. The effect of these common gene variants on cardiac PITX2 mRNA is currently under study. PITX2 protein regulates right–left differentiation of the embryonic heart, thorax and aorta. PITX2 is expressed in the adult left atrium, but much less so in other heart chambers. Pitx2 deficiency results in electrical and structural remodelling, and impaired repair of the heart in murine models, all of which may influence AF through divergent mechanisms. PITX2 levels and single nucleotide polymorphisms on chromosome 4q25 may also be a predictor of the effectiveness of anti‐arrhythmic drug therapy.

An automated system using spatial oversampling for optical mapping in murine atria. Development and validation with monophasic and transmembrane action potentials.

We developed and validated a new optical mapping system for quantification of electrical activation and repolarisation in murine atria. The system makes use of a novel 2nd generation complementary metal-oxide-semiconductor (CMOS) camera with deliberate oversampling to allow both assessment of electrical activation with high spatial and temporal resolution (128 × 2048 pixels) and reliable assessment of atrial murine repolarisation using post-processing of signals. Optical recordings were taken from isolated, superfused and electrically stimulated murine left atria. The system reliably describes activation sequences, identifies areas of functional block, and allows quantification of conduction velocities and vectors. Furthermore, the system records murine atrial action potentials with comparable duration to both monophasic and transmembrane action potentials in murine atria.

Shear stress-induced angiogenesis in mouse muscle is independent of the vasodilator mechanism and quickly reversible.

Is modulation of skeletal muscle capillary supply by altering blood flow due to a presumptive shear stress response per se, or dependent on the vasodilator mechanism?

How ubiquitous is endothelial NOS

The ability to regulate vascular tone is an essential cardiovascular control mechanism, with nitric oxide (NO) assumed to be a ubiquitous smooth muscle relaxant. However, the literature contains reports of vasoconstrictor, vasodilator and no response to nitroergic stimulation in non-mammalian vertebrates. We examined functional (branchial artery myography), structural (immunohistochemistry of skeletal muscle), proteomic (Western analysis) and genomic (RT-PCR, sequence orthologues, syntenic analysis) evidence for endothelial NO synthase (NOS3) in model and non-model fish species. A variety of nitrodilators failed to elicit any changes in vascular tone, although a dilatation to exogenous cyclic GMP was noted. NOS3 antibody staining does not localise to endothelial markers in cryosections, and gives rise to non-specific staining of Western blots. Abundant NOS2 mRNA was found in all species but NOS3 was not found in any fish, while putative orthologues are not flanked by similar genes to NOS3 in humans. We conclude that NOS3 does not exist in fish, and that previous reports of its presence may reflect use of antibodies raised against mammalian epitopes.

A Regional Reduction in Ito and IKACh in the Murine Posterior Left Atrial Myocardium Is Associated with Action Potential Prolongation and Increased Ectopic Activity

Background The left atrial posterior wall (LAPW) is potentially an important area for the development and maintenance of atrial fibrillation. We assessed whether there are regional electrical differences throughout the murine left atrial myocardium that could underlie regional differences in arrhythmia susceptibility. Methods We used high-resolution optical mapping and sharp microelectrode recordings to quantify regional differences in electrical activation and repolarisation within the intact, superfused murine left atrium and quantified regional ion channel mRNA expression by Taqman Low Density Array. We also performed selected cellular electrophysiology experiments to validate regional differences in ion channel function. Results Spontaneous ectopic activity was observed during sustained 1Hz pacing in 10/19 intact LA and this was abolished following resection of LAPW (0/19 resected LA, P<0.001). The source of the ectopic activity was the LAPW myocardium, distinct from the pulmonary vein sleeve and LAA, determined by optical mapping. Overall, LAPW action potentials (APs) were ca. 40% longer than the LAA and this region displayed more APD heterogeneity. mRNA expression of Kcna4, Kcnj3 and Kcnj5 was lower in the LAPW myocardium than in the LAA. Cardiomyocytes isolated from the LAPW had decreased Ito and a reduced IKACh current density at both positive and negative test potentials. Conclusions The murine LAPW myocardium has a different electrical phenotype and ion channel mRNA expression profile compared with other regions of the LA, and this is associated with increased ectopic activity. If similar regional electrical differences are present in the human LA, then the LAPW may be a potential future target for treatment of atrial fibrillation.

205 Atrial Arrhythmia Susceptibility in Arrhythmogenic Right Ventricular Cardiomyopathy

Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder that causes sudden death and right ventricular heart failure in young athletes. Loss-of-function mutations in desmosomal proteins including plakoglobin deficiency are linked with ARVC and ventricular arrhythmias in trained mice. Chronic intensive endurance training increases susceptibility to atrial arrhythmias in rats. We tested whether heterozygous plakoglobin deficient (Plako+/-) mice that undergo moderate endurance training show increased atrial arrhythmia susceptibility compared to their wild type (WT) littermates. Methods Plako+/- and WT mice were group swim-trained for 6 d/week, gradually increased to 90 min/d, with a total average swimming times of 50h over 8 weeks. Echocardiography was carried out before and after the swim-training period. Hearts were rapidly excised and immediately perfused with Krebs-Henseleit solution and left atrial (LA) monophasic action potentials (MAPs) were recorded. Inter-atrial activation times (AT) and action potential durations (APDs) were measured during RA pacing at 100 ms fixed-rate cycle lengths. An 8-pulse S1 train, at 100 ms cycle length (CL), followed by a single extrastimulus, S2, was delivered to assess atrial arrhythmia inducibility (arrhythmias >1s). AT and APD90 values are expressed as mean ±SEM. Transmembrane action potentials (TAPs) from sedentary mice were recorded from isolated, superfused LA using floating glass microelectrodes at 100 ms paced CL. APD was measured as the average of 8 consecutive APs. AT and APD90 values are expressed as mean ±SEM. Results The number of hearts with atrial arrhythmias was greater in swim-trained Plako+/- (6/13 hearts,)than swim-trained WT (0/8 hearts) Hearts of trained Plako+/- mice were more susceptible to induced ventricular arrhythmias than trained WT mice (WT: 1/12; Plako+/-: 7/15 hearts, p < 0.05). Training increased left ventricular wall width by 13% in both WT and Plako+/- mice (WT: 0.58 ± 0.01 baseline vs. 0.66 ± 0.01 mm trained; Plako+/-: 0.58 ± 0.02 baseline vs. 0.66 ± 0.01 mm trained). Right ventricular size was increased in trained Plako+/- vs. trained WT, e.g. RV diameter measured parasternally was increased in Plako+/- (1.77 ± 0.03 mm) vs. WT (1.51 ± 0.03 mm) after training, p < 0.05. Atrial weight was unchanged between genotypes. Intra-atrial AT in sedentary mice measured using MAPs and TAPs were the same in both genotypes. Inter-atrial AT between the RA endocardial pacing site and the LA MAP electrode in trained mice also did not differ between genotypes. LA APD90 measured from MAPs in trained mice and MAPs and TAPs from sedentary mice, TAP amplitude, and TAP maximum upstroke velocity were unaffected by genotype. Thus far, we have not observed increased arrhythmia inducibility in sedentary Plako+/- mice. Conclusion Our observations suggest that defective mechanical cell-cell contact proteins predispose to moderate training-induced atrial arrhythmias. Abstract 205 Figure 1 Atrial arrhythmias >1 second

11 Arrhythmogenic Right Ventricular Cardiomyopathy-Like Phenotype Induced by Endurance Training and Prevented by Preload Reduction in Heterozygeous Desmoglein-2 Mutants

Arrhythmogenic right ventricular cardiomyopathy (ARVC) significantly contributes to sudden cardiac death in young otherwise healthy patients, especially endurance athletes. 5–10% of patients with ARVC harbour mutations in the extracellular domains of the desmoglein (DSG) 2 gene. To assess the role of DSG2 in ARVC pathomechanism, mice lacking exons 4–6 of the endogenous DSG2 gene (DSG2mt) were generated. Homozygous DSG2mt/mice developed dilatation of ventricles and pronounced fibrosis. Heterozygous DSG2mt/wt mutants did not show such morphological alterations. Methods To study whether exercise provokes a cardiac phenotype in DGSwt/mt mice, they were subjected to endurance training and compared with wild-type (WT) littermates. We also studied if preload reduction can prevent right ventricular dilation with training by concomitant therapy with nitrates and diuretics. Echocardiography was performed with 2% isoflurane + oxygen, using a small animal ultrasound unit. Right ventricular (RV) dimensions were increased in DSG2wt/mt after training compared to pre-training and compared to WT after training. Electrophysiological studies in isolated Langendorff DSGwt/mt and WT hearts from mice terminally anaesthetised with urethane (2 mg/kg) showed a correlation of the DSG2 mutation with increased arrhythmia inducibility after endurance training. Ventricular arrhythmias were induced by a single extra stimulus during right ventricular stimulation in 5 of 8 DSG2wt/mt, but in none of the 7 WT hearts (p = 0.03). Preload reduction prevented excessive right ventricular enlargement with endurance training in DSG2wt/mt. In conclusion, endurance training revealed an ARVC-like phenotype in heterozygous desmoglein mutant mice. Right ventricular dilation could be prevented by preload-reducing therapy.

Optical mapping design for murine atrial electrophysiology

Abstract Optical mapping is an important tool for assessment of cardiac electrophysiology. We demonstrate a system for quantification and automatic measurement of electrophysiological parameters in isolated cardiac tissue. The system makes use of voltage sensitive fluorescent dyes. These shift in wavelength in response to millivolt changes in potential across cell membranes located on the left atrium. Automated analysis of the pixel-wise measurements yields information on action potential durations and isochronal maps allowing for high throughput of data analysis. The algorithms that we propose reliably describe activation sequences and allow for quantification of conduction velocities.