Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces I(Ca,L) and I(to) in rapid atrial pacing in rabbits.

OBJECTIVES: The purpose of the study was to characterize the ionic and molecular mechanisms in the very early phases of electrical remodeling in a rabbit model of rapid atrial pacing (RAP). BACKGROUND: Long-term atrial fibrillation reduces L-type Ca(2+) (I(Ca,L)) and transient outward K(+) (I(to)) currents by transcriptional downregulation of the underlying ionic channels. However, electrical remodeling starts early after the onset of rapid atrial rates. The time course of ion current and channel modulation in these early phases of remodeling is currently unknown. METHODS: Rapid (600 beats/min) right atrial pacing was performed in rabbits. Animals were divided into five groups with pacing durations between 0 and 96 h. Ionic currents were measured by patch clamp techniques; messenger ribonucleic acid (mRNA) and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS: L-type calcium current started to be reduced (by 47%) after 12 h of RAP and continued to decline as pacing continued. Current changes were preceded or paralleled by decreased mRNA expression of the Ca(2+) channel beta subunits CaB2a, CaB2b, and CaB3, whereas significant reductions in the alpha(1) subunit mRNA and protein expression began 24 h after pacing onset. Transient outward potassium current densities were not altered within the first 12 h, but after 24 h, currents were reduced by 48%. Longer pacing periods did not further decrease I(to). Current changes were paralleled by reduced Kv4.3 mRNA expression. Kv4.2, Kv1.4, and the auxiliary subunit KChIP2 were not affected. CONCLUSIONS: L-type calcium current and I(to) are reduced in early phases of electrical remodeling. A major mechanism appears to be transcriptional downregulation of underlying ion channels, which partially preceded ion current changes.

The antihistamine fexofenadine does not affect I(Kr) currents in a case report of drug-induced cardiac arrhythmia.

1. The human HERG gene encodes the cardiac repolarizing K(+) current I(Kr) and is genetically inactivated in inherited long QT syndrome 2 (LQTS2). The antihistamine terfenadine blocks HERG channels, and can cause QT prolongation and torsades de pointes, whereas its carboxylate fexofenadine lacks HERG blocking activity. 2. In the present study the ability of fexofenadine to block the K897T HERG channel variant was investigated. The underlying single nucleotide polymorphism (SNP) A2960C was identified in a patient reported to develop fexofenadine-associated LQTS. 3. K897T HERG channels produced wild-type-like currents in Xenopus oocytes. Even at a concentration of 100 micro M, fexofenadine did not inhibit wild-type or K897T HERG channels. Coexpression of wild-type and K897T HERG with the ss-subunit MiRP1, slightly changed current kinetics but did not change sensitivity to terfenadine and fexofenadine. 4. Western blot analysis and immunostaining of transiently transfected COS-7 cells demonstrated that overall expression level, glycosylation pattern and subcellular localization of K897T HERG is indistinguishable from wild-type HERG protein, and not altered in the presence of 1 micro M fexofenadine. 5. We provide the first functional characterization of the K897T HERG variant. We demonstrated that K897T HERG is similar to wild-type HERG, and is insensitive to fexofenadine. Although the polymorphism changes PKA and PKC phosphorylation sites, regulation of K897T HERG by these kinases is not altered. 6. Our results strongly indicate that QT lengthening and cardiac arrhythmia in the reported case of drug-induced LQT are not due to the K897T exchange or to an inhibitory effect of fexofenadine on cardiac I(Kr) currents. British Journal of

Comparative analysis of human and rat S1P(5) (edg8): differential expression profiles and sensitivities to antagonists.

Five guanine nucleotide-binding protein-coupled receptors (S1P(1-5)) for the lysophospholipid mediator sphingosine 1-phosphate (S1P) have thus far been described. Whereas tissue distribution and functional properties of the human S1P(1-4) genes are well characterized, only limited functional and expression data are available for S1P(5), todate. Northern blot analysis indicated that human S1P(5) (hS1P(5)) is an alternatively spliced gene, with a 5.4-kb transcript that is predominantly expressed in peripheral tissues, and a 2.4-kb transcript expressed in brain, spleen, and peripheral blood leucocytes. In contrast, rat S1P(5) (rS1P(5)) was exclusively detected in brain and skin. Expression of hS1P(5) and rS1P(5) in mammalian CHO-K1 or HEK293 cells conferred onto the cells the ability to mobilize intracellular calcium as determined by a functional Fluorometric Imaging Plate Reader assay, when challenged with S1P and dihydro S1P, respectively. Applying a lipid library with 200 bioactive lipids in a functional Fluorometric Imaging Plate Reader assay did not reveal additional agonists. However, both receptors exhibited differential sensitivity towards the S1P- and lysophosphatidic acid-receptor antagonist, suramin: rS1P(5)-mediated intracellular calcium mobilization was partly inhibited by suramin (IC(50): 5800 microM), whereas hS1P(5) was completely antagonized (IC(50): 130 microM). Both receptors were sensitive towards inhibition with the related drug (8,8'-(carbonylbis(imino-3,1-phenylene))bis(1,3,5-naphthalenetrisulfonic acid)) but IC(50) values differed significantly (340 microM for hS1P(5), 4000 microM for rS1P(5)). In addition, rS1P(5) displayed antiproliferative effects in transfected CHO-K1 and HEK293 cells in contrast to hS1P(5). Taken together, our data imply that differences between hS1P(5) and rS1P(5) will be an important point to be considered in the development of selective receptor antagonists.