Abstract-Inwardly rectifying Kϩ (Kir) channels are responsible for maintaining endothelial membrane potential and play a key role in endothelium-dependent vasorelaxation. In this study, we show that endothelial Kir channels are suppressed by hypercholesterolemic levels of lipoproteins in vitro and by serum hypercholesterolemia in vivo. Specifically, exposing human aortic endothelial cells to acetylated low-density lipoprotein or very low density lipoprotein resulted in a time-and concentration-dependent decrease in Kir current that correlated with the degree of cholesterol loading. The suppression was fully reversible by cholesterol depletion. Furthermore, a decrease in Kir current resulted in depolarization of endothelial membrane potential. Most important, the flow sensitivity of Kir currents was also impaired by cholesterol loading. Specifically, flow-induced increase in Kir current was suppressed by 70%, and flow-induced hyperpolarization was almost completely abrogated. Furthermore, we show that hypercholesterolemia in vivo also strongly suppresses endothelial Kir currents and causes a shift in endothelial membrane potential, as determined by comparing the currents in aortic endothelial cells freshly isolated from healthy or hypercholesterolemic pigs. Therefore, we suggest that suppression of Kir current is one of the important factors in hypercholesterolemia-induced endothelial dysfunction. (Circ Res. 2006;98:1064-1071.) Key Words: K channels Ⅲ cholesterol Ⅲ lipoproteins Ⅲ flow Ⅲ vasodilatation N umerous studies have shown that a major risk factor for the development of cardiovascular disease is plasma hypercholesterolemia, elevation of low-and very low-density lipoproteins (LDL and VLDL), as well as the oxidized modification of LDL, oxidized LDL (oxLDL). 1,2 Endothelial dysfunction develops in the early stages of cardiovascular disease and is a strong predictor of its development. 3 A hallmark of endothelial dysfunction in vivo is impairment of flow-mediated vasodilatation (FMV). Indeed, several studies have shown that plasma hypercholesterolemia impairs FMV both in humans and in animal models of atherosclerosis. 4,5 However, molecular mechanisms that underlie hypercholesterolemia-induced endothelial dysfunction are poorly understood.Our studies focus on the impact of hypercholesterolemia on endothelial inwardly rectifying K ϩ (Kir) channels, the major determinants of endothelial membrane potential 6,7 and putative flow sensors. 8 -10 The resting membrane potential in endothelial cells ranges between approximately Ϫ30 and Ϫ80 mV depending on a specific endothelial cell type. 7 Aortic endothelial cells typically have resting potentials in a strongly hyperpolarizing range (approximately Ϫ60 mV/Ϫ80 mV) both in culture 8,[11][12][13] and in intact aortas, 14 consistent with a major contribution of K ϩ channels. Several types of K ϩ channels are expressed in endothelial cells: Kir channels that maintain stable membrane potential under resting condition and are responsible for flow-induced hyperpolarization an...

Background Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting over two million patients in the US alone. Despite decades of research, surprisingly little is known regarding the molecular pathways underlying the pathogenesis of AF. ANK2 encodes ankyrin-B, a multifunctional adapter molecule implicated in membrane targeting of ion channels, transporters, and signaling molecules in excitable cells. Methods and Results Here, we report early-onset AF in patients harboring loss-of-function mutations in ANK2. In mice, we show that ankyrin-B-deficiency results in atrial electrophysiological dysfunction and increased susceptibility to AF. Moreover, ankyrin-B+/− atrial myocytes display shortened action potentials, consistent with human AF. Ankyrin-B is expressed in atrial myocytes, and we demonstrate its requirement for the membrane targeting and function of a subgroup of voltage-gated Ca2+ channels (Cav1.3) responsible for low-voltage activated L-type Ca2+current. Ankyrin-B directly associates with Cav1.3, and this interaction is regulated by a short, highly-conserved motif specific to Cav1.3. Moreover, loss of ankyrin-B in atrial myocytes results in decreased Cav1.3 expression, membrane localization, and function sufficient to produce shortened atrial action potentials and arrhythmias. Finally, we demonstrate reduced ankyrin-B expression in atrial samples of patients with documented AF, further supporting an association between ankyrin-B and AF. Conclusions These findings support that reduced ankyrin-B expression or mutations in ANK2 are associated with atrial fibrillation. Additionally, our data demonstrate a novel pathway for ankyrin-B-dependent regulation of Cav1.3 channel membrane targeting and regulation in atrial myocytes.

Ankyrins are critical components of ion channel and transporter signaling complexes in the cardiovascular system. Over the past five years, ankyrin dysfunction has been linked with abnormal ion channel and transporter membrane organization and fatal human arrhythmias. Loss-of-function variants in the ankyrin-B gene (ANK2) cause "ankyrin-B syndrome" (previously called type 4 long QT syndrome), manifested by a complex cardiac phenotype including ventricular arrhythmias and sudden cardiac death. More recently, dysfunction in the ankyrin-B-based targeting pathway has been linked with a highly penetrant and severe form of human sinus node disease. Ankyrin-G (a second ankyrin gene product) is required for normal expression, membrane localization, and biophysical function of the primary cardiac voltage-gated sodium channel, Na v 1.5. Loss of the ankyrin-G/ Na v 1.5 interaction is associated with human cardiac arrhythmia (Brugada syndrome). Finally, in the past year ankyrin dysfunction has been associated with more common arrhythmia and cardiovascular disease phenotypes. Specifically, large animal studies reveal striking remodeling of ankyrin-B and associated proteins following myocardial infarction. Additionally, the ANK2 locus has been linked with QT c interval variability in the general human population. Together, these findings identify a host of unanticipated and exciting roles for ankyrin polypeptides in cardiac function. More broadly, these findings illustrate the importance of local membrane organization for normal cardiac physiology.

Ankyrin-B is a multifunctional adapter protein responsible for localization and stabilization of select ion channels, transporters, and signaling molecules in excitable cells including cardiomyocytes. Ankyrin-B dysfunction has been linked with highly penetrant sinoatrial node (SAN) dysfunction and increased susceptibility to atrial fibrillation. While previous studies have identified a role for abnormal ion homeostasis in ventricular arrhythmias, the molecular mechanisms responsible for atrial arrhythmias and SAN dysfunction in human patients with ankyrin-B syndrome are unclear. Here, we develop a computational model of ankyrin-B dysfunction in atrial and SAN cells and tissue to determine the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human patients with ankyrin-B syndrome. Our simulations predict that defective membrane targeting of the voltage-gated L-type Ca(2+) channel Cav1.3 leads to action potential shortening that reduces the critical atrial tissue mass needed to sustain reentrant activation. In parallel, increased fibrosis results in conduction slowing that further increases the susceptibility to sustained reentry in the setting of ankyrin-B dysfunction. In SAN cells, loss of Cav1.3 slows spontaneous pacemaking activity, whereas defects in Na(+)/Ca(2+) exchanger and Na(+)/K(+) ATPase increase variability in SAN cell firing. Finally, simulations of the intact SAN reveal a shift in primary pacemaker site, SAN exit block, and even SAN failure in ankyrin-B-deficient tissue. These studies identify the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human disease. Importantly, ankyrin-B dysfunction involves changes at both the cell and tissue levels that favor the common manifestation of atrial arrhythmias and SAN dysfunction.