Separation of early afterdepolarizations from arrhythmogenic substrate in the isolated perfused hypokalaemic murine heart through modifiers of calcium homeostasis

Aims: We resolved roles for early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in Langendorff‐perfused hypokalaemic murine hearts paced from the right ventricular epicardium. Methods: Left ventricular epicardial and endocardial monophasic action potentials (MAPs) and arrhythmogenic tendency were compared in the presence and absence of the L‐type Ca²⁺ channel blocker nifedipine (10 nm –1 μm ) and the calmodulin kinase type II inhibitor KN‐93 (2 μm ). Results: All the hypokalaemic hearts studied showed prolonged epicardial and endocardial MAPs, decreased epicardial‐endocardial APD₉₀ difference, EADs, triggered beats and ventricular tachycardia (VT) (n = 6). In all spontaneously beating hearts, 100 (but not 10) nm nifedipine reduced both the incidence of EADs and triggered beats from 66.9 ± 15.7% to 28.3 ± 8.7% and episodes of VT from 10.8 ± 6.3% to 1.2 ± 0.7% of MAPs (n = 6 hearts, P < 0.05); 1 μm nifedipine abolished all these phenomena (n = 6). In contrast programmed electrical stimulation (PES) still triggered VT in six of six hearts with 0, 10 and 100 nm but not 1 μm nifedipine. 1 μm nifedipine selectively reduced epicardial (from 66.1 ± 3.4 to 46.2 ± 2.5 ms) but not endocardial APD₉₀, thereby restoring ΔAPD₉₀ from ?5.9 ± 2.5 to 15.5 ± 3.2 ms, close to normokalaemic values. KN‐93 similarly reduced EADs, triggered beats and VT in spontaneously beating hearts to 29.6 ± 8.9% and 1.7 ± 1.1% respectively (n = 6) yet permitted PES‐induced VT (n = 6), in the presence of a persistently negative ΔAPD₉₀. Conclusions: These findings empirically implicate both EADs and triggered beats alongside arrhythmogenic substrate of ΔAPD₉₀ in VT pathogenesis at the whole heart level.     

Effects of potassium channel openers in the isolated perfused hypokalaemic murine heart

Aim: We explored the anti‐arrhythmic efficacy of K⁺ channel activation in the hypokalaemic murine heart using NS1643 and nicorandil, compounds which augment I_Kr and I_KATP respectively. Methods: Left ventricular epicardial and endocardial monophasic action potentials were compared in normokalaemic and hypokalaemic preparations in the absence and presence of NS1643 (30 μm ) and nicorandil (20 μm ). Results: Spontaneously beating hypokalaemic hearts (3 mm K⁺) all elicited early afterdepolarizations (EADs) and episodes of ventricular tachycardia (VT). Perfusion with NS1643 and nicorandil suppressed EADs and VT in 7 of 13 and five of six hypokalaemic hearts. Provoked arrhythmia studies using programmed electrical stimulation induced VT in all hypokalaemic hearts, but failed to do so in 7 of 13 and five of six hearts perfused with NS1643 and nicorandil respectively. These anti‐arrhythmic effects were accompanied by reductions in action potential duration at 90% repolarization (APD₉₀) and changes in the transmural gradient of repolarization, reflected in ΔAPD₉₀. NS1643 and nicorandil reduced epicardial APD₉₀ from 68.3 ± 1.1 to 56.5 ± 4.1 and 51.5 ± 1.5 ms, respectively, but preserved endocardial APD₉₀ in hypokalaemic hearts. NS1643 and nicorandil thus restored ΔAPD₉₀ from ?9.6 ± 4.3 ms under baseline hypokalaemic conditions to 3.9 ± 4.1 and 9.9 ± 2.1 ms, respectively, close to normokalaemic values. Conclusion: These findings demonstrate, for the first time, the anti‐arrhythmic efficacy of K⁺ channel activation in the setting of hypokalaemia. NS1643 and nicorandil are anti‐arrhythmic through the suppression of EADs, reductions in APD₉₀ and restorations of ΔAPD₉₀.     

Arrhythmogenic mechanisms in the isolated perfused hypokalaemic murine heart

Aim: Hypokalaemia is associated with a lethal form of ventricular tachycardia (VT), torsade de pointes, through pathophysiological mechanisms requiring clarification. Methods: Left ventricular endocardial and epicardial monophasic action potentials were compared in isolated mouse hearts paced from the right ventricular epicardium perfused with hypokalaemic (3 and 4 mm [K⁺]_o) solutions. Corresponding K⁺ currents were compared in whole‐cell patch‐clamped epicardial and endocardial myocytes. Results: Hypokalaemia prolonged epicardial action potential durations (APD) from mean APD₉₀s of 37.2 ± 1.7 ms (n = 7) to 58.4 ± 4.1 ms (n =7) and 66.7 ± 2.1 ms (n = 11) at 5.2, 4 and 3 mm [K⁺]_o respectively. Endocardial APD₉₀s correspondingly increased from 51.6 ± 1.9 ms (n = 7) to 62.8 ± 2.8 ms (n = 7) and 62.9 ± 5.9 ms (n = 11) giving reductions in endocardial–epicardial differences, ΔAPD₉₀, from 14.4 ± 2.6 to 4.4 ± 5.0 and ?3.4 ± 6.0 ms respectively. Early afterdepolarizations (EADs) occurred in epicardia in three of seven spontaneously beating hearts at 4 mm [K⁺]_o with triggered beats followed by episodes of non‐sustained VT in nine of 11 preparations at 3 mm . Programmed electrical stimulation never induced arrhythmic events in preparations perfused with normokalemic solutions yet induced VT in two of seven and nine of 11 preparations at 4 and 3 mm [K⁺]_o respectively. Early outward K⁺ current correspondingly fell from 73.46 ± 8.45 to 61.16±6.14 pA/pF in isolated epicardial but not endocardial myocytes (n = 9) (3 mm [K⁺]_o). Conclusions: Hypokalaemic mouse hearts recapitulate the clinical arrhythmogenic phenotype, demonstrating EADs and triggered beats that might initiate VT on the one hand and reduced transmural dispersion of repolarization reflected in ΔAPD₉₀ suggesting arrhythmogenic substrate on the other.     

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

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 μM). 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 (Δlatency), 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 Δlatency 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 Δlatency (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.     

Empirical correlation of triggered activity and spatial and temporal re-entrant substrates with arrhythmogenicity in a murine model for Jervell and Lange-Nielsen syndrome

KCNE1 encodes the β-subunit of the slow component of the delayed rectifier K⁺ current. The Jervell and Lange-Nielsen syndrome is characterized by sensorineural deafness, prolonged QT intervals, and ventricular arrhythmogenicity. Loss-of-function mutations in KCNE1 are implicated in the JLN2 subtype. We recorded left ventricular epicardial and endocardial monophasic action potentials (MAPs) in intact, Langendorff-perfused mouse hearts. KCNE1 ^−/− but not wild-type (WT) hearts showed not only triggered activity and spontaneous ventricular tachycardia (VT), but also VT provoked by programmed electrical stimulation. The presence or absence of VT was related to the following set of criteria for re-entrant excitation for the first time in KCNE1 ^−/− hearts: Quantification of APD₉₀, the MAP duration at 90% repolarization, demonstrated alterations in (1) the difference, ∆APD₉₀, between endocardial and epicardial APD₉₀ and (2) critical intervals for local re-excitation, given by differences between APD₉₀ and ventricular effective refractory period, reflecting spatial re-entrant substrate. Temporal re-entrant substrate was reflected in (3) increased APD₉₀ alternans, through a range of pacing rates, and (4) steeper epicardial and endocardial APD₉₀ restitution curves determined with a dynamic pacing protocol. (5) Nicorandil (20 μM) rescued spontaneous and provoked arrhythmogenic phenomena in KCNE1 ^−/− hearts. WTs remained nonarrhythmogenic. Nicorandil correspondingly restored parameters representing re-entrant criteria in KCNE1 ^−/− hearts toward values found in untreated WTs. It shifted such values in WT hearts in similar directions. Together, these findings directly implicate triggered electrical activity and spatial and temporal re-entrant mechanisms in the arrhythmogenesis observed in KCNE1 ^−/− hearts.     

Atrial arrhythmogenesis in wild-type and Scn5a+/Δ murine hearts modelling LQT3 syndrome

Long QT(3) (LQT3) syndrome is associated with abnormal repolarisation kinetics, prolonged action potential durations (APD) and QT intervals and may lead to life-threatening ventricular arrhythmias. However, there have been few physiological studies of its effects on atrial electrophysiology. Programmed electrical stimulation and burst pacing induced atrial arrhythmic episodes in 16 out of 16 (16/16) wild-type (WT) and 7/16 genetically modified Scn5a+/Δ (KPQ) Langendorff-perfused murine hearts modelling LQT3 (P < 0.001 for both), and in 14/16 WT and 1/16 KPQ hearts (P < 0.001 for both; Fisher’s exact test), respectively. The arrhythmogenic WT hearts had significantly larger positive critical intervals (CI), given by the difference between atrial effective refractory periods (AERPs) and action potential durations at 90% recovery (APD₉₀), compared to KPQ hearts (8.1 and 3.2 ms, respectively, P < 0.001). Flecainide prevented atrial arrhythmias in all arrhythmogenic WT (P < 0.001) and KPQ hearts (P < 0.05). It prolonged the AERP to a larger extent than it did the APD₉₀ in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD₉₀ in both WT and KPQ groups. These findings, thus, demonstrate, for the first time, inhibitory effects of the KPQ mutation on atrial arrhythmogenesis and its modification by flecainide and quinidine. They attribute these findings to differences in the CI between WT and mutant hearts, in the presence or absence of these drugs. Thus, prolongation of APD₉₀ over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD₉₀ reduced such CI values and produced the opposite effect.     

Criteria for arrhythmogenicity in genetically-modified Langendorff-perfused murine hearts modelling the congenital long QT syndrome type 3 and the Brugada syndrome

The experiments investigated the applicability of two established criteria for arrhythmogenicity in Scn5a+/Δ and Scn5a+/− murine hearts modelling the congenital long QT syndrome type 3 (LQT3) and the Brugada syndrome (BrS). Monophasic action potentials (APs) recorded during extrasystolic stimulation procedures from Langendorff-perfused control hearts and hearts treated with flecainide (1 μM) or quinidine (1 or 10 μM) demonstrated that both agents were pro-arrhythmic in wild-type (WT) hearts, quinidine was pro-arrhythmic in Scn5a+/Δ hearts, and that flecainide was pro-arrhythmic whereas quinidine was anti-arrhythmic in Scn5a+/− hearts, confirming clinical findings. Statistical analysis confirmed a quadratic relationship between epicardial and endocardial AP durations (APDs) in WT control hearts. However, comparisons between plots of epicardial against endocardial APDs and this reference curve failed to correlate with arrhythmogenicity. Restitution curves, relating APD to diastolic interval (DI), were then constructed for the first time in a murine system and mono-exponential growth functions fitted to these curves. Significant (P < 0.05) alterations in the DI at which slopes equalled unity, an established indicator of arrhythmogenicity, now successfully predicted the presence or absence of arrhythmogenicity in all cases. We thus associate changes in the slopes of restitution curves with arrhythmogenicity in models of LQT3 and BrS.     

Epac activation, altered calcium homeostasis and ventricular arrhythmogenesis in the murine heart

The recently described exchange protein directly activated by cAMP (Epac) has been implicated in distinct protein kinase A-independent cellular signalling pathways. We investigated the role of Epac activation in adrenergically mediated ventricular arrhythmogenesis. In contrast to observations in control conditions (n = 20), monophasic action potentials recorded in 2 of 10 intrinsically beating and 5 of 20 extrinsically paced Langendorff-perfused wild-type murine hearts perfused with the Epac activator 8-pCPT-2′-O-Me-cAMP (8-CPT, 1 μM) showed spontaneous triggered activity. Three of 20 such extrinsically paced hearts showed spontaneous ventricular tachycardia (VT). Programmed electrical stimulation provoked VT in 10 of 20 similarly treated hearts (P < 0.001; n = 20). However, there were no statistically significant accompanying changes (P > 0.05) in left ventricular epicardial (40.7 ± 1.2 versus 44.0 ± 1.7 ms; n = 10) or endocardial action potential durations (APD₉₀; 51.8 ± 2.3 versus 51.9 ± 2.2 ms; n = 10), transmural (ΔAPD₉₀) (11.1 ± 2.6 versus 7.9 ± 2.8 ms; n = 10) or apico-basal repolarisation gradients, ventricular effective refractory periods (29.1 ± 1.7 versus 31.2 ± 2.4 ms in control and 8-CPT-treated hearts, respectively; n = 10) and APD₉₀ restitution characteristics. Nevertheless, fluorescence imaging of cytosolic Ca²⁺ levels demonstrated abnormal Ca²⁺ homeostasis in paced and resting isolated ventricular myocytes. Epac activation using isoproterenol in the presence of H-89 was also arrhythmogenic and similarly altered cellular Ca²⁺ homeostasis. Epac-dependent effects were reduced by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibition with 1 μM KN-93. These findings associate VT in an intact cardiac preparation with altered cellular Ca²⁺ homeostasis and Epac activation for the first time, in the absence of altered repolarisation gradients previously implicated in reentrant arrhythmias through a mechanism dependent on CaMKII activity.     

A quantitative analysis of the effect of cycle length on arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts

The clinically established proarrhythmic effect of bradycardia and antiarrhythmic effect of lidocaine (10 μM) were reproduced in hypokalaemic (3.0 mM K⁺) Langendorff-perfused murine hearts paced over a range (80–180 ms) of baseline cycle lengths (BCLs). Action potential durations (at 90% repolarization, APD₉₀s), transmural conduction times and ventricular effective refractory periods (VERPs) were then determined from monophasic action potential records obtained during a programmed electrical stimulation procedure in which extrasystolic stimuli were interposed following regular stimuli at successively decreasing coupling intervals. A novel graphical analysis of epicardial and endocardial, local and transmural relationships between APD₉₀, corrected for transmural conduction time where appropriate, and VERP yielded predictions in precise agreement with the arrhythmogenic findings obtained over the entire range of BCLs studied. Thus, in normokalaemic (5.2 mM K⁺) hearts a statistical analysis confirmed that all four relationships were described by straight lines of gradients not significantly (P > 0.05) different from unity that passed through the origin and thus subtended constant critical angles, θ with the abscissa (45.8° ± 0.9°, 46.6° ± 0.5°, 47.6° ± 0.5° and 44.9° ± 0.8°, respectively). Hypokalaemia shifted all points to the left of these reference lines, significantly (P < 0.05) increasing θ at BCLs of 80–120 ms where arrhythmic activity was not observed (∼63°, ∼54°, ∼55° and ∼58°, respectively) and further significantly (P < 0.05) increasing θ at BCLs of 140–180 ms where arrhythmic activity was observed (∼68°, ∼60°, ∼61° and ∼65°, respectively). In contrast, the antiarrhythmic effect of lidocaine treatment was accompanied by a significant (P < 0.05) disruption of this linear relationship and decreases in θ in both normokalaemic (∼40°, ∼33°, ∼39° and ∼41°, respectively) and hypokalaemic (∼40°, ∼44°, ∼50° and ∼48°, respectively) hearts. This extended a previous approach that had correlated alterations in transmural repolarization gradients with arrhythmogenicity in murine models of the congenital long QT syndrome type 3 and hypokalaemia at a single BCL. Thus, the analysis in terms of APD₉₀ and VERP provided a more sensitive indication of the effect of lidocaine than one only considering transmural repolarization gradients and may be particularly applicable in physiological and pharmacological situations in which these parameters diverge.     

TNF‐α hyperpolarizes membrane potential and potentiates the response to nicotinic receptor stimulation in cultured rat myenteric neurones

Aim: Mechanically induced early afterdepolarization (EAD) is morphologically similar but different in the mechanisms with drug‐induced EAD, which lead to arrhythmia. Pacing suppresses the drug‐induced EAD and arrhythmia, however the effect of pacing on mechanically induced EAD and arrhythmia is not clear. This study addressed this issue in right ventricle (RV) of anaesthetized lambs. Methods: Six lambs were anaesthetized, and their hearts exposed. Nine monophasic action potential (MAP) electrodes were placed on RV apex, outflow and inflow regions, and recorded before, during, and after a 10 s occlusion of pulmonary artery at a number of pacing rates. Results: Pacing significantly reduced the baseline MAP duration at 90% repolarization (MAPD₉₀), decreased the reduction of MAPD at early repolarization at the peak of occlusion. Nonetheless, the percentage of reduction was not significantly different among them. Pacing was able to reduce the frequencies, size of mechanically induced EADs. MAPD₉₀ at the peak of occlusion was all shortened during pacing rather than some lengthened at intrinsic rate. Therefore, the dispersion of MAPD₉₀ at the peak of occlusion reduced from 86 ± 6 ms at intrinsic rate to 42 ± 4 ms at 120 beats min⁻¹ , 38 ± 3 ms at 150 beats min⁻¹ and 26 ± 3 ms at 170 beats min⁻¹. Ultimately, pacing reduced/suppressed mechanically induced premature ventricular beats. These alterations were inversely related to heart rates. Conclusion: Pacing reduces/suppresses both stretch‐induced EADs and arrhythmia. These modulations are remarkably similar to those on other EADs by the pacing.     

Refractory dispersion promotes conduction disturbance and arrhythmias in a Scn5a +/? mouse model

Accentuated right ventricular (RV) gradients in action potential duration (APD) have been implicated in the arrhythmogenicity observed in Brugada syndrome in studies assuming that ventricular effective refractory periods (VERPs) vary in concert with APDs. The present experiments use a genetically modified mouse model to explore spatial heterogeneities in VERP that in turn might affect conduction velocity, thereby causing arrhythmias. Activation latencies, APDs and VERPs recorded during programmed S1S2 protocols were compared in RV and left ventricular (LV) epicardia and endocardia of Langendorff-perfused wild-type (WT) and Scn5a (+/-) hearts. Scn5a (+/-) and WT hearts showed similar patterns of shorter VERPs in RV than LV epicardia, and in epicardia than endocardia. However, Scn5a (+/-) hearts showed longer VERPs, despite shorter APD(90)s, than WT in all regions examined. The pro- and anti-arrhythmic agents flecainide and quinidine increased regional VERPs despite respectively decreasing and increasing the corresponding APD(90)s particularly in Scn5a (+/-) RV epicardia. In contrast, Scn5a (+/-) hearts showed greater VERP gradients between neighbouring regions, particularly RV transmural gradients, than WT (9.1 ± 1.1 vs. 5.7 ± 0.5 ms, p < 0.05, n = 12). Flecainide increased (to 21 ± 0.9 ms, p < 0.05, n = 6) but quinidine decreased (to 4.5 ± 0.5 ms, p < 0.05, n = 6) these gradients, particularly across the Scn5a (+/-) RV. Finally, Scn5a (+/-) hearts showed greater conduction slowing than WT following S2 stimuli, particularly with flecainide administration. Rather than arrhythmogenesis resulting from increased transmural repolarization gradients in an early, phase 2, reentrant excitation mechanism, the present findings implicate RV VERP gradients in potential reentrant mechanisms involving impulse conduction slowed by partial refractoriness.     

In vivo temporal and spatial distribution of depolarization and repolarization and the illusive murine T wave

This study assessed in vivo temporal and spatial electrophysiological properties of murine hearts and the effect of manipulation of transmural action potential durations (APDs) on T wave morphology. Monophasic action potentials (MAPs) were acquired from multiple left ventricular sites. All MAPs exhibited a plateau phase, with a spike and dome appearance being present in epicardial recordings. Activation occurred from endocardial apex to epicardial apex and apex to base while repolarization occurred from base (shortest 90 η₀ level of repolarization (MAP₉₀), 95.4 ± 8.9 ms) to apex and epicardium to endocardium (longest MAP₉₀, 110.77 ± 10.6 ms). The peak of phase 0 of the epicardial base MAP correlated with the return to baseline of the initial and usually dominant waveform of the QRS and the onset of the second usually smaller wave, which clearly occurred in early repolarization, thus establishing where depolarization ended and repolarization began on the murine ECG. This second waveform was similar to the J wave seen in larger animals. Despite temporal and spatial electrophysiological similarities, a T wave is frequently not seen on a murine ECG. There are several determinants of T wave morphology, including transmural activation time, slope of phase 3 repolarization and differences in epicardial, endocardial and M cell APDs. Experimental manipulation of murine transmural gradients by shortening epicardial MAP₉₀ to 84% of endocardial MAP₉₀ the epicardial/endocardial ratio in larger mammals when a positive T wave is present, resulted in a positive murine T wave. Thus, manipulation of the transmural gradients such that they are similar to larger mammals can result in T waves with similar morphology.     

Adenosine induces prolonged anti‐β‐adrenergic effects in guinea‐pig papillary muscle

A sustained anti‐β‐adrenergic effect of adenosine has been reported. This study was initiated to investigate this topic and especially elucidate the role of protein kinase C (PKC). Contractile force amplitude and action potential duration at 90% repolarization (APD₉₀) were measured in guinea‐pig papillary muscles before and after 5 min challenge with 5 nm isoproterenol. Protocols contained 30 min exposure to the test agents adenosine 33 μm (ado), adenosine + PKC‐inhibitor bisindolylmaleimide 20 nM (ado + BIM), PKC‐activator 1,2‐dioctanoyl‐sn‐glycerol 10 μm (DOG) and α‐agonist phenylephrine 5 μm (phe). Isoproterenol was given at the end of test exposure and after 15 min washout. Results are mean ± SEM of percentage‐change, P ≤ 0.05 considered significant and labelled *. The first isoproterenol challenge significantly increased contractile force (27 ± 7%*) in the control group. Responses in the test groups were 2 ± 4 (ado), 1 ± 5 (ado + BIM), 14 ± 4* (DOG), 0 ± 2% (phe). After washout of adenosine, DOG and phenylephrine, isoproterenol induced 3 ± 8 (ado), 23 ± 5* (ado + BIM), 13 ± 5* (DOG), 15 ± 7% (phe) increase in test groups compared with 22 ± 5%* increase in contractile force in the control group. After 45 min washout of adenosine the inotropic response was still significantly reduced compared with control (29 ± 4 vs. 79 ± 8%*). Isoproterenol stimulation shortened APD₉₀ in controls at both time points (5 ± 1%* and 4 ± 1%*), with no significant shortening in test groups. Adenosine induces sustained anti‐β‐adrenergic effects on contractile force as well as APD₉₀. A role for PKC in signal transduction is supported with respect to contractile force.     

Regional variations in action potential alternans in isolated murine Scn5a+/− hearts during dynamic pacing

Aim: Clinical observations suggest that alternans in action potential (AP) characteristics presages breakdown of normal ordered cardiac electrical activity culminating in ventricular arrhythmogenesis. We compared such temporal nonuniformities in monophasic action potential (MAP) waveforms in left (LV) and right ventricular (RV) epicardia and endocardia of Langendorff-perfused murine wild-type (WT), and Scn5a^+/− hearts modelling Brugada syndrome (BrS) for the first time. Methods: A dynamic pacing protocol imposed successively incremented steady pacing rates between 5.5 and 33 Hz. A signal analysis algorithm detected sequences of >10 beats showing alternans. Results were compared before and following the introduction of flecainide (10 μm ) and quinidine (5 μm ) known to exert pro- and anti-arrhythmic effects in BrS. Results: Sustained and transient amplitude and duration alternans were both frequently followed by ventricular ectopic beats and ventricular tachycardia or fibrillation. Diastolic intervals (DIs) that coincided with onsets of transient (tr) or sustained (ss) alternans in MAP duration (DI*) and amplitude (DI′) were determined. Kruskal–Wallis tests followed by Bonferroni-corrected Mann–Whitney U-tests were applied to these DI results sorted by recording site, pharmacological conditions or experimental populations. WT hearts showed no significant heterogeneities in any DI. Untreated Scn5a^+/− hearts showed earlier onsets of transient but not sustained duration alternans in LV endocardium compared with RV endocardium or LV epicardium. Flecainide administration caused earlier onsets of both transient and sustained duration alternans selectively in the RV epicardium in the Scn5a^+/− hearts. Conclusion: These findings in a genetic model thus implicate RV epicardial changes in the arrhythmogenicity produced by flecainide challenge in previously asymptomatic clinical BrS.     

Scn3b knockout mice exhibit abnormal sino-atrial and cardiac conduction properties

Aim: In contrast to extensive reports on the roles of Na_v1.5 α-subunits, there have been few studies associating the β-subunits with cardiac arrhythmogenesis. We investigated the sino-atrial and conduction properties in the hearts of Scn3b^−/− mice. Methods: The following properties were compared in the hearts of wild-type (WT) and Scn3b^−/− mice: (1) mRNA expression levels of Scn3b, Scn1b and Scn5a in atrial tissue. (2) Expression of the β₃ protein in isolated cardiac myocytes. (3) Electrocardiographic recordings in intact anaesthetized preparations. (4) Bipolar electrogram recordings from the atria of spontaneously beating and electrically stimulated Langendorff-perfused hearts. Results: Scn3b mRNA was expressed in the atria of WT but not Scn3b^−/− hearts. This was in contrast to similar expression levels of Scn1b and Scn5a mRNA. Immunofluorescence experiments confirmed that the β₃ protein was expressed in WT and absent in Scn3b^−/− cardiac myocytes. Lead I electrocardiograms from Scn3b^−/− mice showed slower heart rates, longer P wave durations and prolonged PR intervals than WT hearts. Spontaneously beating Langendorff-perfused Scn3b^−/− hearts demonstrated both abnormal atrial electrophysiological properties and evidence of partial or complete dissociation of atrial and ventricular activity. Atrial burst pacing protocols induced atrial tachycardia and fibrillation in all Scn3b^−/− but hardly any WT hearts. Scn3b^−/− hearts also demonstrated significantly longer sinus node recovery times than WT hearts. Conclusion: These findings demonstrate, for the first time, that a deficiency in Scn3b results in significant atrial electrophysiological and intracardiac conduction abnormalities, complementing the changes in ventricular electrophysiology reported on an earlier occasion.     

Positive inotropic and sustained anti‐β‐adrenergic effect of diadenosine pentaphosphate in human and guinea pig hearts. Role of dinucleotide receptors and adenosine receptors

Aim: Diadenosine polyphosphates are present intracellularly and in extracellular fluid due to release from secretory vesicles in platelets, chromaffin cells and other cells. This study investigates effects of diadenosine pentaphosphate (AP₅A) on heart muscle function. Methods: Contractile force amplitude and action potential duration at 90% repolarization (APD₉₀) were measured after challenge with AP₅A 50 μm or isoproterenol 50–70 nM in guinea pig papillary muscles. Isoproterenol was given immediately after AP₅A‐exposure or after 45 min washout. AP₅A was combined with antagonists to the purinergic P2 receptor (suramin 100 μm ), the dinucleotide receptor [diinosine pentaphosphate 30 μm (IP₅I)] or adenosine receptors [8‐(P‐sulfophenyl) theophylline 50 μm (8‐SPT)]. Results: Results are %‐change (mean ± SEM) from value before exposure. AP₅A increased contractile force by 22 ± 3%* (*P < 0.05), and IP₅I abolished this. AP₅A prolonged APD₉₀ by 7 ± 2%*. AP₅A significantly reduced response to isoproterenol acutely from 31 ± 4* (controls) to 9 ± 4% and after 45 min washout from 61 ± 14* (controls) to 16 ± 5%. 8‐SPT abolished the sustained effect. Increase in contractile force by AP₅A was confirmed in human atria trabecula preparations. Conclusion: AP₅A increased contractile force and prolonged APD₉₀. Contractile force increased by stimulation of the dinucleotide receptor in guinea pig myocardium. The sustained anti‐β‐adrenergic effect of AP₅A was due to adenosine receptor stimulation.     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

 Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca²⁺ entry via Na-Ca exchange current (I _NaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1–5 day), juvenile (JV, 10–14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD₉₀: 108–378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (Q _Exout) and inward (Q _Exin) Ni²⁺-sensitive charge movement in response to AP prolongation. Q _Exout and Q _Exin measured during age-appropriate APs declined postnatally [Q _EXout: NB (2 day) 0.19 ± 0.02, JV (10 day) 0.10 ± 0.01, AD 0.04 ± 0.002; Q _EXin: NB –0.2 ± 0.01, JV –0.11 ± 0.02; AD –0.04 ± 0.003 pC/pF] despite the significantly shorter APD₉₀ of newborn myocytes (NB 122 ± 10; AD 268 ± 22 ms). When Ca²⁺ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 ± 0.5, JV 1.2 ± 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth. Received: 23 September 1997 / Received after revision: 2 January 1998 / Accepted: 5 January 1998     

Electrophysiological determinants of hypokalaemia‐induced arrhythmogenicity in the guinea‐pig heart

Aim: Hypokalaemia is an independent risk factor contributing to arrhythmic death in cardiac patients. In the present study, we explored the mechanisms of hypokalaemia‐induced tachyarrhythmias by measuring ventricular refractoriness, spatial repolarization gradients, and ventricular conduction time in isolated, perfused guinea‐pig heart preparations. Methods: Epicardial and endocardial monophasic action potentials from distinct left ventricular (LV) and right ventricular (RV) recording sites were monitored simultaneously with volume‐conducted electrocardiogram (ECG) during steady‐state pacing and following a premature extrastimulus application at progressively reducing coupling stimulation intervals in normokalaemic and hypokalaemic conditions. Results: Hypokalaemic perfusion (2.5 mm K⁺ for 30 min) markedly increased the inducibility of tachyarrhythmias by programmed ventricular stimulation and rapid pacing, prolonged ventricular repolarization and shortened LV epicardial and endocardial effective refractory periods, thereby increasing the critical interval for LV re‐excitation. Hypokalaemia increased the RV‐to‐LV transepicardial repolarization gradients but had no effect on transmural dispersion of APD₉₀ and refractoriness across the LV wall. As determined by local activation time recordings, the LV‐to‐RV transepicardial conduction and the LV transmural (epicardial‐to‐endocardial) conduction were slowed in hypokalaemic heart preparations. This change was attributed to depressed diastolic excitability as evidenced by increased ventricular pacing thresholds. Conclusion: These findings suggest that hypokalaemia‐induced arrhythmogenicity is attributed to shortened LV refractoriness, increased critical intervals for LV re‐excitation, amplified RV‐to‐LV transepicardial repolarization gradients and slowed ventricular conduction in the guinea‐pig heart.     

Origin of complex behaviour of spatially discordant alternans in a transgenic rabbit model of type 2 long QT syndrome

Enhanced dispersion of repolarization has been proposed as an important mechanism in long QT related arrhythmias. Dispersion can be dynamic and can be augmented with the occurrence of spatially out-of-phase action potential duration (APD) alternans (discordant alternans; DA). We investigated the role of tissue heterogeneity in generating DA using a novel transgenic rabbit model of type 2 long QT syndrome (LQT2). Littermate control (LMC) and LQT2 rabbit hearts ( n = 5 for each) were retrogradely perfused and action potentials were mapped from the epicardial surface using di-4-ANEPPS and a high speed CMOS camera. Spatial dispersion (ΔAPD and Δslope of APD restitution) were both increased in LQT2 compared to LMC (ΔAPD: 34 ± 7 ms vs. 23 ± 6 ms; Δslope:1.14 ± 0.23 vs. 0.59 ± 0.19). Onset of DA under a ramp stimulation protocol was seen at longer pacing cycle length (CL) in LQT2 compared to LMC hearts (206 ± 24 ms vs. 156 ± 5 ms). Nodal lines between regions with APD alternans out of phase from each other were correlated with conduction velocity (CV) alternation in LMC but not in LQT2 hearts. In LQT2 hearts, larger APD dispersion was associated with onset of DA at longer pacing CL. At shorter CLs, closer to ventricular fibrillation induction (VF), nodal lines in LQT2 ( n = 2 out of 5) showed persistent complex beat-to-beat changes in nodal line formation of DA associated with competing contribution from CV restitution and tissue spatial heterogeneity, increasing vulnerability to conduction block. In conclusion, tissue heterogeneity plays a significant role in providing substrate for ventricular arrhythmia in LQT2 rabbits by facilitating DA onset and contributing to unstable nodal lines prone to reentry formation.     

血小板活化因子对豚鼠心室肌细胞钾电流及动作电位的影响

 目的：研究血小板活化因子(PAF)对豚鼠心室肌细胞钾电流及动作电位的影响。 方法:应用全细胞膜片钳技术，记录豚鼠心室肌细胞动作电位及钾电流(I_K 与I_K1)。 结果:当电极内液ATP浓度为5 mmol/L，1 μmol/L PAF使APD₉₀由对照的(225.8±23.3)ms延长至(352.8±29.8)ms(n=5, P<0.05)；使I_K尾电流在指令电压 +30 mV 时由对照的(173.5±16.7)pA降为(152.1±11.5)pA(P<0.05, n=4)；使I_K1在指令电压 -120 mV 时从(-6.1±1.3)nA降为(-5.6±1.1)nA(P<0.05, n=5)；当电极内液ATP 为0 mmol/L，APD₉₀明显缩短，1 μmol/L PAF使APD₉₀由对照的(153.0±24.6)ms缩短为(88.2±19.4)ms (n=5, P<0.01)，而用1 μmol/L格列本脲 ( I_KATP特异性阻滞剂)预处理后，恢复了PAF可显著延长动作电位时程的作用。 结论: PAF使缺血区K_ATP开放，动作电位时程缩短，却可抑制正常区I_K 与I_K1，使动作电位延长，从而放大了缺血区与正常区的不均一性，这可能与缺血时心律失常的发生有关。