The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells.

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 µM ORM-10962. Mechanistic simulations were performed by Maltsev-Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 µM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

The Inhibition of the Small-Conductance Ca2+-Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation.

Sinus pacemaking is based on tight cooperation of intracellular Ca2+ handling and surface membrane ion channels. An important player of this synergistic crosstalk could be the small-conductance Ca2+-activated K+-channel (ISK) that could contribute to the sinoatrial node (SAN) pacemaking driven by the intracellular Ca2+ changes under normal conditions and beta-adrenergic activation, however, the exact role is not fully clarified. SK2 channel expression was verified by immunoblot technique in rabbit SAN cells. Ionic currents and action potentials were measured by patch-clamp technique. The ECG R-R intervals were obtained by Langendorff-perfusion method on a rabbit heart. Apamin, a selective inhibitor of SK channels, was used during the experiments. Patch-clamp experiments revealed an apamin-sensitive current. When 100 nM apamin was applied, we found no change in the action potential nor in the ECG R-R interval. In experiments where isoproterenol was employed, apamin increased the cycle length of the SAN action potentials and enhanced the ECG R-R interval. Apamin did not amplify the cycle length variability or ECG R-R interval variability. Our data indicate that ISK has no role under normal condition, however, it moderately contributes to the SAN automaticity under beta-adrenergic activation.

The development of L-type Ca2+ current mediated alternans does not depend on the restitution slope in canine ventricular myocardium.

Cardiac alternans have crucial importance in the onset of ventricular fibrillation. The early explanation for alternans development was the voltage-driven mechanism, where the action potential (AP) restitution steepness was considered as crucial determining factor. Recent results suggest that restitution slope is an inadequate predictor for alternans development, but several studies still claim the role of membrane potential as underlying mechanism of alternans. These controversial data indicate that the relationship of restitution and alternans development is not completely understood. APs were measured by conventional microelectrode technique from canine right ventricular papillary muscles. Ionic currents combined with fluorescent measurements were recorded by patch-clamp technique. APs combined with fluorescent measurements were monitored by sharp microelectrodes. Rapid pacing evoked restitution-independent AP duration (APD) alternans. When non-alternating AP voltage command was used, Ca2+i-transient (CaT) alternans were not observed. When alternating rectangular voltage pulses were applied, CaT alternans were proportional to ICaL amplitude alternans. Selective ICaL inhibition did not influence the fast phase of APD restitution. In this study we found that ICaL has minor contribution in shaping the fast phase of restitution curve suggesting that ICaL-if it plays important role in the alternans mechanism-could be an additional factor that attenuates the reliability of APD restitution slope to predict alternans.

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue.

BACKGROUND AND PURPOSE: The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN). EXPERIMENTAL APPROACH: Currents were measured by patch-clamp, Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model. KEY RESULTS: ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed. CONCLUSION AND IMPLICATIONS: We confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between If and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.