Evaluation of genes encoding for the transient outward current (Ito) identifies the KCND2 gene as a cause of J-wave syndrome associated with sudden cardiac death.

BACKGROUND: J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experimental evidence supports a transient outward current (I(to))-mediated mechanism of J-wave formation. This study aimed to determine the frequency of genetic mutations in genes encoding the I(to) in patients with J waves on ECG. METHODS AND RESULTS: Comprehensive mutational analysis was performed on I(to)-encoding KCNA4, KCND2, and KCND3 genes, as well as the previously described J-wave-associated KCNJ8 gene, in 51 unrelated patients with ECG evidence defining a J-wave syndrome. Only patients with a resuscitated cardiac arrest or type 1 Brugada ECG pattern were included for analysis. A rare genetic mutation of the KCND2 gene, p.D612N, was identified in a single patient. Co-expression of mutant and wild-type KCND2 with KChIP2 in HEK293 cells demonstrated a gain-of-function phenotype, including an increase in peak I(to) density of 48% (P<0.05) in the heterozygous state. Using computer modeling, this increase in Ito resulted in loss of the epicardial action potential dome, predicting an increased ventricular transmural Ito gradient. The previously described KCNJ8-S422L mutation was not identified in this cohort of patients with ECG evidence of J-wave syndrome. CONCLUSIONS: These findings are the first to implicate the KCND2 gene as a novel cause of J-wave syndrome associated with sudden cardiac arrest. However, genetic defects in I(to)-encoding genes seem to be an uncommon cause of sudden cardiac arrest in patients with apparent J-wave syndromes.

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes.

BACKGROUND AND PURPOSE: The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. EXPERIMENTAL APPROACH: Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. KEY RESULTS: In the presence of 1-10 µM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 µM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 µM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 µM), changing Bmax but not Kd . CONCLUSION AND IMPLICATIONS: Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Lanthanum suppresses arachidonic acid-induced cell death and mitochondrial depolarization in PC12 cells.

Within the framework of studying the mechanisms of acute toxicity of arachidonic acid and the role of ambient cations, we have investigated the effects of extracellular La(3+) on arachidonic acid-induced death (lactate dehydrogenase release) and mitochondrial depolarization (rhodamine 123 fluorescence) in PC12 cells. Micromolar La(3+) profoundly suppressed arachidonic acid toxicity and this effect was dependent on the presence of other cations. Whereas in the cation-free solution 10-20 microM La(3+) protected most cells from death caused by a 2 hour-long exposure to 20 microM arachidonic acid, the cytoprotective effect of 100 microM La(3+) was reduced to approximately 70% in the presence of a normal complement of monovalent cations and was hardly detectable with 5 mM Ca(2+) in the bath. Increasing the concentration of arachidonic acid could defeat La(3+) cytoprotection. In fluorescence experiments, arachidonic acid caused a decrease in the mitochondrial membrane potential, with the rate and extent of depolarization increasing with an increase in the concentration of arachidonic acid. La(3+) countered the depolarizing effect of arachidonic acid in a manner consistent with a decrease in the effective arachidonic acid concentration. The results suggest that extracellular cations modulate cellular effects of arachidonic acid by reducing its ability to pass through the plasma membrane, possibly by binding the fatty acid. The similarities of the La(3+) effects on arachidonic acid-induced cell death and arachidonic acid-induced mitochondrial depolarization strongly support the causal relations between the two events and suggest that mitochondria are the primary target of arachidonic acid at the cellular level.

The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells.

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.

Ca2+ influx is not involved in acute cytotoxicity of arachidonic acid.

Arachidonic acid (AA; 20:4, n-6) has been implicated in cell damage in the brain under ischemia-reperfusion and other pathological conditions. In our experiments, PC12 cells exposed to >10 microM AA died within 1-2 hr, as assessed by the LDH release assay. Since AA is known to induce Ca2+/cation-permeable conductance in the plasma membrane, we investigated whether Ca2+ influx plays a role in this acute cell death. We found that extracellular Ca2+ was not required for the toxic effect of AA. In fact, the removal of extracellular Ca2+ dramatically accelerated its development: the half-time of the toxic effect of 40 microM AA decreased from 70.1 +/- 0.3 min in the presence of 5 mM Ca2+ to 7.4 +/- 0.3 min in the Ca-free solution. The extent of cell killing depended only weakly on AA concentration and ion composition, remaining within the 70-95% range. The AA-induced acute death was not affected by inhibitors of AA metabolism (nordihydroguaiaretic acid, indomethacin, proadifen), whereas some antioxidants tested (deferoxamine and ellagic acid), but not all (melatonin), partially suppressed it. Also, it was not affected by changes in the extracellular ionic strength or mimicked by an acetylenic analog of AA 5,8,11,14-eicosatetraynoic acid. We conclude that lethal injuries sustained by cells during short exposures to AA were caused by the fatty acid itself and were not mediated by the AA-induced influx of Ca2+/cations. Moreover, direct physical effects of AA on the plasma membrane (changes in membrane fluidity or detergent-like action) were also excluded.

Ion dependence of cytotoxicity of carmustine against PC12 cells.

Cytotoxicity is a major complication of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU) therapy for treatment of brain tumors and lymphomas. Using the lactate dehydrogenase (LDH) cell death assay in PC12 cells, we studied the role in this phenomenon of transmembrane ion fluxes that could be activated following inhibition by carmustine of glutathione reductase. The cytotoxic effect of carmustine developed during 4-6 h, with the EC50 of 27 microM. It depended on the extracellular Ca2+ concentration and substantially decreased upon Ca2+ removal. An almost complete suppression of toxicity was achieved when, additionally, monovalent cations were also replaced with impermeant organic cations. A similar loss of toxicity occurred in the presence of Ca2+ when extracellular Cl- was replaced with impermeable gluconate. Various blockers of cation and Cl- channels, as well as antioxidants also protected cells from carmustine. We conclude that carmustine toxicity against PC12 cells requires an influx of Ca2+ ions, supposedly through redox-sensitive cation channels.