The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts

Ian N. Sabir 0 James A. Fraser 0 Matthew J. Killeen 0 Andrew A. Grace 0 Christopher L.-H. Huang 0 0 A. A. Grace Department of Biochemistry, University of Cambridge , Tennis Court Road, Cambridge CB2 1QW, UK 1 ) Physiological Laboratory, University of Cambridge , Downing Street, Cambridge CB2 3EG, UK The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic (3.0 mM K+) Langendorff-perfused murine hearts before and after exposure to lidocaine (10 M). Novel limiting criteria for local and transmural, epicardial, and endocardial re-excitation involving action potential duration (at 90% repolarization, APD90), ventricular effective refractory period (VERP), and transmural conduction time (latency), where appropriate, were applied to normokalemic (5.2 mM K+) and hypokalemic hearts. Hypokalemia increased epicardial APD90 from 46.6 1.2 to 53.1 0.7 ms yet decreased epicardial VERP from 41 4 to 29 1 ms, left endocardial APD90 unchanged (58.2 3.7 to 56.9 4.0 ms) yet decreased endocardial VERP from 48 4 to 29 2 ms, and left latency unchanged (1.6 1.4 to 1.1 1.1 ms; eight normokalemic and five hypokalemic hearts). These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization. Hypokalemia thus shifted all re-excitation criteria in the positive direction. In contrast, hypokalemia spared epicardial APD90 (54.8 2.7 to 60.6 2.7 ms), epicardial VERP (84 5 to 81 7 ms), endocardial APD90 (56.6 4.2 to 63.7 6.4 ms), endocardial VERP (80 2 to 84 4 ms), and latency (12.5 6.2 to 7.6 3.4 ms; five hearts in each case) in lidocaine-treated hearts. Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction, again precisely agreeing with the arrhythmogenic findings. In contrast, established analyses invoking transmural dispersion of repolarization failed to account for any of these findings. We thus establish novel, more general, criteria predictive of arrhythmogenicity that may be particularly useful where APD90 might diverge sharply from VERP. - Hypokalemia exerts important clinical effects on cardiac function that in some respects resemble those seen in the congenital long-QT syndromes (LQTS). Thus, both conditions result in electrocardiographic QT prolongation [12, 23] and premature ventricular depolarizations (PVDs), which may result in the initiation of an arrhythmic activity [41, 52]. In contrast to the cardiac effects of hypokalemia, arrhythmic activity in LQTS has been extensively studied and has often been attributed to after-depolarizations occurring against a background of re-entrant substrate [2, 36, 44]. Re-entry may take place as a result of inhomogeneities producing regions of conduction block, which lead to wave-break and circus movement [21, 37] or altered repolarization gradients, which lead to wave reflection [1]. In this situation, depolarization propagates from active cells into previously active adjacent regions, establishing reentrant circuits. These may become established either locally or over larger regions of the myocardium, such as across the thickness of the myocardial wall. Tendencies to transmural re-entrant excitation in models of LQTS have been previously analyzed in terms of transmural dispersions of repolarization (TDR) obtained from the positive part of the difference between respective endocardial and epicardial stimulation to repolarization times [36, 44, 45]. In human LQTS, increases in the interval between the peak and full recovery of electrocardiographic precordial T waves (Tpeak to Tend), previously shown to reflect TDR [54], are indeed associated with arrhythmic activity [33]. Certainly, recent reports correlate Tpeak to Tend to arrhythmic risk more closely than more widely accepted indicators such as corrected QT interval and QT dispersion [53]. However, such re-excitation may also be limited by recovery from refractoriness; re-entrant excitation would require this to precede the return of the membrane potential to threshold [40]. Certainly, class 1 antiarrhythmic drugs such as lidocaine are known to increase ventricular effective refractory period (VERP) [28]. Yet, such use of spatial differences in action potential repolarization times to quantify arrhythmic substrate neither explicitly considers changes in VERP nor applies such criteria to potential local as opposed to transmural reexcitation. This paper associates for the first time the proarrhythmic effect of hypokalemia with a significant decrease in VERP, despite contrasting prolongation of action potentials, in agreement with computer-modeling studies of action potential waveforms using established data on the various effects of hypokalemia on ionic conductivity properties of ventricular myocytes. Furthermore, it associates the antiarrhythmic effects of lidocaine with a significant increase in VERP, despite having little effect on action potential duration, in agreement with clinical observations. Analyses using TDR were insufficiently sensitive to account for any of these arrhythmogenic findings. This study accordingly established more general novel criteria that would provide necessary conditions for local and transmural and epicardial and endocardial re-excitation incorporating not only action potential duration but also VERP and conduction times that may be particularly useful when action potential duration differs sharply from VERP. These criteria successfully accounted for all the arrhythmogenic findings. Materials and methods Mice were housed in an animal facility at 211C with 12 h light/dark cycles. Animals were fed sterile chow (RM3 Maintenance Diet, SDS, Witham, Essex, UK) and had free access to water. Wild-type 129 Sv mice aged 3 6 months were used in the experiments. All procedures complied with UK Home Office regulations (Animals [Scientific Procedures] Act 1986). All solutions were based on bicarbonate-buffered KrebsHenseleit solution (mM: NaCl 119, NaHCO3 25, KCl 4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and Napyruvate 2; pH adjusted to 7.4) bubbled with 95% O2/5% CO2 (British Oxygen Company, Manchester, UK). Hypokalemic (3.0 mM K+) solutions were prepared by reducing the quantity of KCl added. Lidocaine-containing normokalemic and hypokalemic solutions were prepared by adding lidocaine (SigmaAldrich, Poole, UK) to a final concentration of 10 M. A Langendorff-perfusion protocol previously adapted for murine hearts [4] was used. In brief, mice were killed by cervical dislocation (Schedule 1: UK Animals [Scientific Procedures] Act 1986), and hearts were then quickly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution. A short section of aorta was cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled with the same solution using an aneurysm clip (Harvard Apparatus, E (...truncated)