Calmodulin kinase II initiates arrhythmogenicity during metabolic acidification in murine hearts.

AIM: The multifunctional signal molecule calmodulin kinase II (CaMKII) has been associated with cardiac arrhythmogenesis under conditions where its activity is chronically elevated. Recent studies report that its activity is also acutely elevated during acidosis. We test a hypothesis implicating CaMKII in the arrhythmogenesis accompanying metabolic acidification. METHODS: We obtained monophasic action potential recordings from Langendorff-perfused whole heart preparations and single cell action potentials (AP) using whole-cell patch-clamped ventricular myocytes. Spontaneous sarcoplasmic reticular (SR) Ca(2+)release events during metabolic acidification were investigated using confocal microscope imaging of Fluo-4-loaded ventricular myocytes. RESULTS: In Langendorff-perfused murine hearts, introduction of lactic acid into the Krebs-Henseleit perfusate resulted in abnormal electrical activity and ventricular tachycardia. The CaMKII inhibitor, KN-93 (2 microm), reversibly suppressed this spontaneous arrhythmogenesis during intrinsic rhythm and regular 8 Hz pacing. However, it failed to suppress arrhythmia evoked by programmed electrical stimulation. These findings paralleled a CaMKII-independent reduction in the transmural repolarization gradients during acidosis, which previously has been associated with the re-entrant substrate under other conditions. Similar acidification produced spontaneous AP firing and membrane potential oscillations in patch-clamped isolated ventricular myocytes when pipette solutions permitted cytosolic Ca(2+) to increase following acidification. However, these were abolished by both KN-93 and use of pipette solutions that held cytosolic Ca(2+) constant during acidosis. Acidosis also induced spontaneous Ca(2+) waves in isolated intact Fluo-4-loaded myocytes studied using confocal microscopy that were abolished by KN-93. CONCLUSION: These findings together implicate CaMKII-dependent SR Ca(2+) waves in spontaneous arrhythmic events during metabolic acidification.

Novel consequences of voltage-dependence to G-protein-coupled P2Y1 receptors.

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.

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 Ca(2+) channel blocker nifedipine (10 nm-1 microm) and the calmodulin kinase type II inhibitor KN-93 (2 microm). RESULTS: All the hypokalaemic hearts studied showed prolonged epicardial and endocardial MAPs, decreased epicardial-endocardial APD(90) 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 microm 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 microm nifedipine. 1 microm nifedipine selectively reduced epicardial (from 66.1 +/- 3.4 to 46.2 +/- 2.5 ms) but not endocardial APD(90), thereby restoring DeltaAPD(90) 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 DeltaAPD(90). CONCLUSIONS: These findings empirically implicate both EADs and triggered beats alongside arrhythmogenic substrate of DeltaAPD(90) in VT pathogenesis at the whole heart level.

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 APD(90)s 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 APD(90)s 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, DeltaAPD(90), 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 DeltaAPD(90) suggesting arrhythmogenic substrate on the other.