Background— Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. Methods and Results— Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na v 1.5, K v 4.3, K v 1.5, ERG, K ir 2.1, K ir 6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca v 1.3, Ca v 3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K v 4.2, K ir 6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. Conclusions— Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

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The majority of Na + channels in the heart are composed of the tetrodotoxin (TTX)-resistant (K D , 2-6 µM) Na v 1.5 isoform; however, recently it has been shown that TTX-sensitive (K D , 1-10 nM) neuronal Na + channel isoforms (Na v 1.1, Na v 1.3 and Na v 1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na + currents under physiological conditions and the expression of cardiac and neuronal Na + channel isoforms. We identified two distinct Na + current components, TTX resistant and TTX sensitive. At 37• C, TTX-resistant i Na and TTX-sensitive i Na started to activate at ∼ −70 and ∼ −60 mV, and peaked at −30 and −10 mV, with a current density of 22 ± 3 and 18 ± 1 pA pF −1 , respectively. TTX-sensitive i Na inactivated at more positive potentials as compared to TTX-resistant i Na . Using action potential clamp, TTX-sensitive i Na was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Na v 1.5, and the TTX-sensitive neuronal isoform, Na v 1.1, were observed: Na v 1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Na v 1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nM) of TTX, which block TTX-sensitive i Na , slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 µM) of TTX, which block TTX-resistant i Na as well as TTX-sensitive i Na , slowed both pacemaking and SA node conduction. It is concluded that two Na + channel isoforms are important for the functioning of the SA node: neuronal (putative Na v 1.1) and cardiac Na v 1.5 isoforms are involved in pacemaking, although the cardiac Na v 1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.

Abstract-Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K m values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia. Key Words: nucleoside Ⅲ adenosine Ⅲ transport Ⅲ ischemia Ⅲ pH T he purine nucleoside adenosine is produced by the action of both endo-and ecto-nucleotidases on adenine nucleotides in the heart and plays key roles in the regulation of coronary blood flow and myocardial O 2 supply-demand balance. 1-4 For example, action of adenosine on A 2A receptors on vascular smooth muscle and endothelial cells causes coronary vasodilatation. 1,5 In contrast, the negative inotropic and dromotropic effects of adenosine on the heart are mediated primarily by A 1 receptors. 2 Similarly, the negative chromotropic effect of adenosine involves action of A 1 receptors in the sinoatrial (SA) node on the inwardly rectifying potassium channel current I K-Ado and the hyperpolarization-activated pacemaker current I f . 2,6 Endogenous adenosine, acting on mitochondrial K ATP channels via A 1 and A 3 receptors, also makes a major contribution to the phenomenon of ischemic preconditioning. 5,7 Extracellular adenosine concentrations in the heart are governed both by action of ecto-5Ј-nucleotidase on adenine nucleotides released from cells and by transporter-mediated flux of adenosine across cell membranes. 3,4 Although most adenosine production occurs intracellularly, under normoxic conditions, metabolism maintains a low intracellular concentration and, therefore, the net flux of adenosine is into cardiomyocytes and endothelial cells. Under such conditions, administration of transport inhibitors increases extracellular concentrations of adenosine, leading to vasodilatation. 8 However, increased adenine nucleotide breakdown and inhibition of adenosine kinase duri...