Use-dependent inhibition of hHCN4 by ivabradine and relationship with reduction in pacemaker activity.

BACKGROUND AND PURPOSE: Ivabradine, a specific and use-dependent I(f) inhibitor, exerts anti-ischaemic activity purely by reducing heart rate. The aim of this work was to characterize its effect on the predominant HCN channel isoform expressed in human sino-atrial nodes (hSAN), to determine its kinetics in HCN channels from multicellular preparations and rate-dependency of its action. EXPERIMENTAL APPROACH: RT-PCR analysis of the four HCN channel isoforms was carried out on RNAs from hSAN. Patch-clamp and intracellular recordings were obtained from CHO cells stably expressing hHCN4 and isolated SAN, respectively. Beating rate of rat isolated atria was followed using a transducer. KEY RESULTS: hHCN4 mRNAs were predominant in hSAN. Ivabradine induced a time-dependent inhibition of hHCN4 with an IC(50) of 0.5 microM. In rabbit SAN, ivabradine progressively reduced the frequency of action potentials: by 10% after 3 h at 0.1 microM, by 14% after 2 h at 0.3 microM and by 17% after 1.5 h at 1 microM. After 3h, ivabradine reduced the beating rate of rat right atria with an IC(30) of 0.2 microM. The onset of action of ivabradine was use-dependent rather than time-dependent with slower effects than caesium, an extracellular I (f) blocker. Ivabradine 3 microM decreased the frequency of action potentials in SAN from guinea-pig, rabbit and pig by 33%, 21% and 15% at 40 min, respectively. CONCLUSIONS AND IMPLICATIONS: The use-dependent inhibition of hHCN4 current by ivabradine probably contributes to its slow developing effect in isolated SAN and right atria and to its increased effectiveness in species with rapid SAN activity.

Peroxidation of docosahexaenoic acid is responsible for its effects on I TO and I SS in rat ventricular myocytes.

1 Exposure to docosahexaenoïc acid (DHA), a long-chain polyunsaturated fatty acid, is known to block several ionic currents such as the transient outward current I(TO). It has also been reported to activate certain potassium channels. It has been suggested that these effects, observed in single-cell experiments, participate in the antiarrhythmic properties of these compounds in vivo. 2 DHA is highly prone to peroxidation. To investigate the influence peroxidation may have on the effects of DHA on ion channels, we studied I(TO) and the steady-state outward current I(SS) in isolated rat ventricular myocytes under ruptured whole-cell patch-clamp conditions. 3 A measure of 10 micro M DHA alone reduced I(TO), evoked by a pulse to +70 mV, by 74.8+/-10.8% (n=7) and activated a delayed outward current with kinetic properties different from I(SS). 4 When an antioxidant, alpha-tocopherol (1 micro M), was added together with DHA, the blockade of I(TO) was reduced to 38.5+/-7.7% (n=8) and the delayed outward current was not activated. alpha-Tocopherol alone had no effect on these currents. 5 When an oxidant, hydrogen peroxide (1 micro M), was applied together with DHA, the blockade of I(TO) was almost complete (98.4+/-1.0%, n=7) and a large delayed outward current was activated. A measure of 1 micro M hydrogen peroxide alone had no effect on these currents. 6 Measurements of nonperoxidized DHA in experimental solutions confirmed the negative relation between DHA concentration and the effects on the currents. 7 We conclude that rather than DHA itself, it is the peroxidation products of DHA that block I(TO) and activate a delayed outward current in in vitro single-cell experiments. These findings have important implications for the extrapolation of in vitro experimental findings to the antiarrhythmic effects of DHA in vivo because, in vivo, peroxidation of DHA is unlikely to occur.

Cytochalasin D reduces Ca2+ sensitivity and maximum tension via interactions with myofilaments in skinned rat cardiac myocytes.

The F-actin disrupter cytochalasin D depresses cardiac contractility, an effect previously ascribed to the interaction of cytochalasin D with cytoskeletal actin. We have investigated the possibility that this negative inotropic effect is due to the interaction of cytochalasin D with sarcomeric actin of the thin filament. Confocal images of Triton X-100-skinned myocytes incubated with a fluorescent conjugate of cytochalasin D revealed a longitudinally striated pattern of binding, consistent with a myofibrillar rather than cytoskeletal structure.Tension-pCa relationships were determined at sarcomere lengths (SLs) of 2.0 and 2.3 [mu]m following 2 min incubation with 1 [mu]M cytochalasin D. Cytochalasin D significantly reduced the pCa for half-maximal activation (pCa50) at both SLs. The shift in pCa50 was significantly greater at a SL of 2.3 [mu]m compared with that at a SL of 2.0 [mu]m. Cytochalasin D had no effect on the Hill co-efficient at either SL. Cytochalasin D significantly reduced the maximum tension at both SLs. We suggest that the length-dependent decrease in myofilament Ca2+ sensitivity in response to cytochalasin D is due to a decrease in the affinity of troponin C for Ca2+. Cytochalasin D has been used for many years as the agent of choice for disruption of cytoskeletal actin. However, we have demonstrated for the first time an interaction of cytochalasin D with sarcomeric actin of the thin filament, which can account for the effects of cytochalasin D on cardiac contractility.