Isolated superfused rats atrial model for the investigation of atrial fibrillation mechanisms and treatment

Background One of the major barriers to an improved mechanistic understanding of atrial fibrillation (AF), and thus in the pipeline of drug development, has been a lack of appropriate tissue models, especially in small animals. Aim We propose an advanced anatomical ex-vivo model based on rat atria for acute assessment of AF susceptibility. This novel model could yield a better understanding of arrhythmia mechanisms as well as the development of potential therapeutic strategies for the prevention or termination of atrial arrhythmias. Methods Wistar rats atria (N=25) were isolated, flattened and pinned to a custom-made silicon plate. Atria were superfused with an oxygenized Tyrode's solution. Tissues were then loaded with a voltage-sensitive dye and mapped using a high-resolution optical mapping system. AF was induced with 1uM carbamylcholine (N=23) coupled with pacing maneuvers and treated with 30uM Vernakalant (N=10) or 10uM Flecainide (N=10). Finally, the feasibility of a new ablation technique (electroporation) was evaluated. Results Optical mapping results suggested that the superfusion procedure led to a fast atrial recovery. Sinus activity was conserved for all atria for a long period. All the anatomical landmarks were clearly visualized. The acquired optical signals were analyzed during sinus rhythm and pacing, which allowed the creation of detailed activation maps and measurements of action potential duration (APD) and conduction velocity (CV) at different pacing rates. The resulting APD restitution curves revealed electrical excitation at high pacing rates (cycle length between 50ms and 300ms) with a relatively flattened curve. AF was successfully induced and optically mapping confirmed the presence of reentrant activity. AF was successfully treated using Vernacalant and Flecainide. Finally, we demonstrated the feasibility of a new ablation approach (electroporation) for creation of a continuous linear lesion serving as a functional block. Conclusion The isolated superfused atria model, coupled with voltage-sensitive dyes, can be utilized for long-term high-resolution functional imaging of the atria during sinus rhythm, pacing and arrhythmogenic activity. This allows the study of the atrial electrophysiological properties, the mechanisms involved in AF initiation, perpetuation, and termination as well as the study of drug and new ablation modalities. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC) Spontaneous activation of isolated atria

Phosphodiesterases 2, 3 and 4 can decrease cardiac effects of H2-histamine-receptor activation in isolated atria of transgenic mice

Histamine exerts cAMP-dependent positive inotropic effects (PIE) and positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic mice which overexpress the human H2-receptor in the heart (=H2-TG). To determine whether these effects are antagonized by phosphodiesterases (PDEs), contractile studies were done in isolated left and right atrial preparations of H2-TG. The contractile effects of histamine were tested in the additional presence of the PDE-inhibitorserythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, 1 μM, PDE2-inhibitor) or cilostamide (1 μM, PDE3-inhibitor), rolipram (10 μM, a PDE4-inhibitor), and their combinations. Cilostamide (1 μM) and EHNA (1 μM), rolipram (1 μM), and EHNA (1 μM) and the combination of rolipram (0.1 μM) and cilostamide (1 μM) each increased the potency of histamine to elevate the force of contraction (FOC) in H2-TG. Cilostamide (1 μM) and rolipram (10 μM) alone increased and EHNA (1 μM) decreased alone, and their combination increased the potency of histamine to increase the FOC in H2-TG indicating that PDE3 and PDE4 regulate the inotropic effects of histamine in H2-TG. The PDE inhibitors (EHNA, cilostamide, rolipram) alone did not alter the potency of histamine to increase the heart beat in H2-TG whereas a combination of rolipram, cilostamide, and EHNA, or of rolipram and EHNA increased the potency of histamine to act on the beating rate. In summary, the data suggest that the PCE of histamine in H2-TG atrium involves PDE 2 and 4 activities, whereas the PIE of histamine are diminished by activity of PDE 3 and 4.

Abstract 418: Caveolar Disruption of L-type Calcium Channel and Ryanodine Receptor Facilitates Atrial Ectopy and Arrhythmogenesis in Heart Failure Mice

Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that trigger AF, are linked to discrete atrial regions that experience the highest remodeling and clinically used for AF ablation; however, mechanisms of their arrhythmogenic propensity remain elusive. We employed in vivo ECG telemetry, in vitro optical mapping and confocal imaging of Ca 2+ transients (CaT) from myocytes isolated from the right atrial appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10), caveolin-3 knockout (KO, n=6) and 8-weeks post-myocardial infarction HF (n=8) mice. HF and KO mice showed an increased susceptibility to pacing-induced AF and enhanced ectopy originated exclusively from ICR. Optical mapping in isolated atria showed prolongation of CaT rise up time (CaT-RT) in HF ICR, which suggested a remodeled coupling between L-type Ca 2+ channels (LTCCs) and ryanodine receptors (RyRs) in this specific region. In WT mice, RAA consists of structured myocytes with a prominent transverse-axial tubular system (TATS) while ICR myocytes don’t have TATS. In RAA, CaT-RT depends on LTCCs in TATS triggering RyR, while in ICR, all the LTCCs are localized in surface caveolae where they can activate subsarcolemmal RyRs and lead to a slow diffusion of Ca 2+ inside the cell interior. Downregulation of caveolae was observed specifically in HF ICR. To mimic this, we used cav3-KO mice. Triggered activities were observed in myocytes isolated from HF and KO ICR, which presumably underlie the ectopic activities in tissue level. These myocytes presented significantly unsynchronized sarcoplasmic reticulum (SR) Ca 2+ releases (synchronization index: 10.8±0.9 in WT vs 38.3±4.1 in HF vs 21.5±2.1 in KO, p <0.01 for HF and KO vs WT respectively) especially at the subsarcolemmal space that prolongs CaT-RT (62.2±4.1 ms in WT vs 122.5±12.8 ms in KO, p <0.01). In addition, failing ICR myocytes showed a higher occurrence and size of spontaneous Ca 2+ sparks which were linked to CaMKII activity and associated phosphorylation of RyR. Our findings demonstrate that in HF, caveolar disruption creates “hot spots” for arrhythmogenic ectopic activity emanated from discrete vulnerable regions of the right atrium which are associated with desynchronized SR Ca 2+ release and elevated fibrosis.

Pharmacological Evaluation of the Effects of Phenylcarbamic Acid Derivatives on Cardiovascular Functions in Rats

Four phenylcarbamic acid derivatives, (1-(4-fluorophenyl)- 4-[3-(4-methoxyphenylcarbamoyloxy)-2-hydroxypropyl]piperazinium chloride (1), (1-(2-methylphenyl)-4-[3-(4-methoxyphenylcarbamoyloxy)- 2-hydroxypropyl]piperazinium chloride) (2), (1-(2-methylphenyl)-4-[3-(4-ethoxyphenylcarbamoyloxy)- 2-hydroxypropyl]piperazinium chloride) (3) and (1-(3-trifluoromethylphenyl)-4-[3-(4-methoxyphenylcarbamoyloxy)- 2-hydroxypropyl]piperazinium chloride) (4) were investigated for their ability to affect various cardiovascular functions and to establish their chemical structure-biological activity relationship. The compounds were evaluated for their antiarrhythmic efficacy using ouabain-induced rhythm disturbances and the ability to inhibit the positive chronotropic effect of isoproterenol in isolated atria of Wistar rats. Electrocardiogram (ECG) parameters in isolated hearts of spontaneously hypertensive rats (SHR) perfused according to the Langendorff method and ability to decrease phenylephrine- -induced contraction of the aortic strips after repeated administration of the compounds were also analyzed. Only compound 3 delayed significantly the evaluated parameter of arrhythmogenicity and was able to antagonize the isoproterenol- induced positive chronotropic effect in normotensive rats’ atria. Similarly, in SHR rats, only compound 3 was able to decrease heart frequency significantly without influencing the duration of QT (time between the start of the Q wave and the end of the T wave) and QTc (frequency corrected QT) intervals. The evaluated endothelial function was improved after administration of compound 2. Fluorine-containing structures (1 and 4) were less effective compared to 2´-methylphenylpiperazine derivatives (2 and 3). The latter two compounds showed suitable efficacy, which supported their use for futher pharmacological research.

Simultaneous conduction mapping and intracellular membrane potential recording in isolated atria

We describe a novel approach for simultaneously determining regional differences in action potential (AP) morphology and tissue electrophysiological properties in isolated atria. The epicardial surface of rat atrial preparations was placed in contact with a multi-electrode array (9 × 10 silver chloride electrodes, 0.1 mm diameter and 0.1 mm pitch). A glass microelectrode (100 MΩ) was simultaneously inserted into the endocardial surface to record intracellular AP from either of 2 regions (A, B) during pacing from 2 opposite corners of the tissue. AP duration at 80% of repolarisation and its restitution curve was significantly different only in region A (p < 0.01) when AP was initiated at different stimulation sites. Alternans in AP duration and AP amplitude, and in conduction velocity were observed during 2 separate arrhythmic episodes. This approach of combining microelectrode array and intracellular membrane potential recording may provide new insights into arrhythmogenic mechanisms in animal models of cardiovascular disease.