Abstract:

The electrophysiological mechanism of the sinus node automaticity was previously considered exclusively regulated by the so-called "funny current". However, parallel investigations increasingly emphasized the importance of the Ca-homeostasis and Na/Ca exchanger (NCX). Recently, increasing experimental evidence, as well as insight through mechanistic modeling demonstrates the crucial role of the exchanger in sinus node pacemaking. NCX had a key role in the exciting story of discovery of sinus node pacemaking mechanisms, which recently settled with a consensus on the coupled-clock mechanism after decades of debate. This review focuses on the role of the Na/Ca exchanger from the early results and concepts to recent advances and attempts to give a balanced summary of the characteristics of the local, spontaneous, and rhythmic Ca releases, the molecular control of the NCX and its role in the fight-or-flight response. Transgenic animal models and pharmacological manipulation of intracellular Ca concentration and/or NCX demonstrate the pivotal function of the exchanger in sinus node automaticity. We also highlight where specific hypotheses regarding NCX function have been derived from computational modeling and require experimental validation. Nonselectivity of NCX inhibitors and the complex interplay of processes involved in Ca handling render the design and interpretation of these experiments challenging.

Abstract:

Background and Purpose: The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca handling and sarcolemmal ion channels. An important player of this crosstalk is the Na/Ca 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 I-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN). Experimental Approach: Currents were measured by patch-clamp, Ca-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 . SAN model. Key Results: ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (I) was previously inhibited and , the effect of I was augmented when the Ca 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 I 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 I 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.

Abstract:

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.