Potent Inhibition of hERG Channels by the Over-the-Counter Antidiarrheal Agent Loperamide.

OBJECTIVES: The aim of this study was to determine the in vitro electrophysiological properties of loperamide. The authors' hypothesis was that loperamide is a potent blocker of the current carried by the human ether-à-go-go-related gene (hERG) potassium channel. BACKGROUND: Loperamide is a peripherally-acting µ-opioid agonist available worldwide as an over-the-counter treatment for diarrhea. Like most opioids, it is not currently known to be proarrhythmic. Recent cases of torsade de pointes in association with high-dose loperamide raise concern given its structural similarity to methadone, another synthetic opioid with an established arrhythmia risk. METHODS: Effects of loperamide on blockade of the hERG potassium channel ion current were assessed in Chinese Hamster Ovary (CHO) cells stably expressing hERG to elucidate current amplitude and kinetics. The concentration required to produce 50% inhibition of hERG current was assessed from the amplitude of tail currents and the impact on action potential duration was assessed in isolated swine ventricular cardiomyocytes. RESULTS: The 50% inhibitory concentration for loperamide inhibition of hERG ionic tail currents was approximately 40 nmol/l. In current-voltage measurements, loperamide reduced steady and tail currents and shifted the current activation to more negative potentials. Loperamide (10 nmol/l) also increased the action potential duration, assessed at 90% of repolarization, in ventricular myocytes by 16.4 ± 1.7% (n = 6; p < 0.004). The maximum rate of rise of phase 0 of the action potential, however, was not significantly altered at any tested concentration of loperamide. CONCLUSIONS: Loperamide is a potent hERG channel blocker. It significantly prolongs the action potential duration and suggests a causal association between loperamide and recent clinical cases of torsade de pointes.

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic ß-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.

Electrocardiographic studies of romidepsin demonstrate its safety and identify a potential role for K(ATP) channel.

PURPOSE: Romidepsin is a histone deacetylase inhibitor (HDI) approved for the treatment of both cutaneous and peripheral T-cell lymphoma (CTCL and PTCL). During development, a thorough assessment of cardiac toxicity was conducted. EXPERIMENTAL DESIGN: A phase II single-agent nonrandomized study of romidepsin was conducted in patients with CTCL or PTCL who had progressed after at least 1 prior systemic therapy. RESULTS: Results for the first 42 patients enrolled on the NCI 1312 phase II study of romidepsin in CTCL or PTCL showed no cardiac toxicity based on serial electrocardiograms (ECG), troponins, and MUGA scans/echocardiograms. The cardiac assessments reported herein confirm the safety of romidepsin among 131 enrolled patients, while supporting a role for electrolyte replacement. Heart rate increased an average 11 bpm following romidepsin infusion; there was no evidence of increased arrhythmia. Criteria for potassium/magnesium replacement were met before 55% of 1365 romidepsin doses; an association with hypoalbuminemia was confirmed. We propose a mechanism for ST segment flattening and depression, the most common ECG abnormalities observed: HDI-induced alteration of the activity or expression of KATP channels. In addition, examination of the variants of the active transporter of romidepsin, ABCB1, showed a trend toward smaller heart rate changes in the peri-infusion period among wild-type than variant diplotypes. CONCLUSIONS: We conclude that in the context of appropriate attention to electrolyte levels, the data support the cardiac safety of romidepsin.

Disruption of sarcolemmal ATP-sensitive potassium channel activity impairs the cardiac response to systolic overload.

Sarcolemmal ATP-sensitive potassium channels (K(ATP)) act as metabolic sensors that facilitate adaptation of the left ventricle to changes in energy requirements. This study examined the mechanism by which K(ATP) dysfunction impairs the left ventricular response to stress using transgenic mouse strains with cardiac-specific disruption of K(ATP) activity (SUR1-tg mice) or Kir6.2 gene deficiency (Kir6.2 KO). Both SUR1-tg and Kir6.2 KO mice had normal left ventricular mass and function under unstressed conditions. Following chronic transverse aortic constriction, both SUR1-tg and Kir6.2 KO mice developed more severe left ventricular hypertrophy and dysfunction as compared with their corresponding WT controls. Both SUR1-tg and Kir6.2 KO mice had significantly decreased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha and a group of energy metabolism related genes at both protein and mRNA levels. Furthermore, disruption of K(ATP) repressed expression and promoter activity of PGC-1alpha in cultured rat neonatal cardiac myocytes in response to hypoxia, indicating that K(ATP) activity is required to maintain PGC-1alpha expression under stress conditions. PGC-1alpha gene deficiency also exacerbated chronic transverse aortic constriction-induced ventricular hypertrophy and dysfunction, suggesting that depletion of PGC-1alpha can worsen systolic overload induced ventricular dysfunction. Both SUR1-tg and Kir6.2 KO mice had decreased FOXO1 after transverse aortic constriction, in agreement with the reports that a decrease of FOXO1 can repress PGC-1alpha expression. Furthermore, inhibition of K(ATP) caused a decrease of FOXO1 associated with PGC-1alpha promoter. These data indicate that K(ATP) channels facilitate the cardiac response to stress by regulating PGC-1alpha and its target genes, at least partially through the FOXO1 pathway.

Random assembly of SUR subunits in K(ATP) channel complexes.

Sulfonylurea receptors (SURs) associate with Kir6.x subunits to form tetradimeric K(ATP) channel complexes. SUR1 and SUR2 confer differential channel sensitivities to nucleotides and pharmacological agents, and are expressed in specific, but overlapping, tissues. This raises the question of whether these different SUR subtypes can assemble in the same channel complex and generate channels with hybrid properties. To test this, we engineered dimeric constructs of wild type or N160D mutant Kir6.2 fused to SUR1 or SUR2A. Dimeric fusions formed functional, ATP-sensitive, channels. Coexpression of weakly rectifying SUR1-Kir6.2 (WTF-1) with strongly rectifying SUR1-Kir6.2[N160D] (NDF-1) in COSm6 cells results in mixed subunit complexes that exhibit unique rectification properties. Coexpression of NDF-1 and SUR2A-Kir6.2 (WTF-2) results in similar complex rectification, reflecting the presence of SUR1- and SUR2A-containing dimers in the same channel. The data demonstrate clearly that SUR1 and SUR2A subunits associate randomly, and suggest that heteromeric channels will occur in native tissues.

Role of the sarcolemmal adenosine triphosphate-sensitive potassium channel in hyperkalemic cardioplegia-induced myocyte swelling and reduced contractility.

BACKGROUND: Hyperkalemic cardioplegia (Plegisol) has been shown to result in myocyte swelling and reduced contractility. We have demonstrated the elimination of these detrimental effects by the addition of an adenosine triphosphate-sensitive K+ (KATP) channel opener. To examine whether the mitochondrial or sarcolemmal KATP channel might be involved, volume and contractility in isolated myocytes from wild-type mice and mice lacking the sarcolemmal KATP channel (Kir6.2-/-) were evaluated. METHODS: Myocytes were perfused for 20 minutes each with control 37 degrees C Tyrode's solution, test solution, and then control solution. Test solutions were (n = 10 per group) either 9 degrees C Plegisol or 9 degrees C Plegisol with 100 micromol/L of diazoxide, a putative mitochondrial-specific KATP channel opener. Cell volume and contractility were measured by digital video microscopy at baseline and during the test solution and reexposure periods. RESULTS: Myocytes from wild-type mice, perfused with 9 degrees C Plegisol, demonstrated significant cell swelling (11.2% +/- 0.4%; p < 0.01) and diminished contractility (32.5% +/- 9.6% reduction in percent shortening, 47.2% +/- 10.1% reduction in peak velocity of shortening, and 52.0% +/- 8.8% reduction in peak velocity of relengthening; p < 0.05) versus baseline. Cell swelling and diminished contractility were significantly reduced by the addition of diazoxide. In Kir6.2-/- myocytes, Plegisol caused a greatly reduced level of cell swelling (3.2% +/- 0.1%; p < 0.01), and this was unaffected by diazoxide. Contractility was unchanged in Kir6.2-/- myocytes after Plegisol. CONCLUSIONS: The sarcolemmal KATP channel appears necessary for exaggerated cell swelling and reduced contractility to occur after hyperkalemic cardioplegia in mouse myocytes.

Sarcolemmal K(ATP) channels: what do we really know?

K(ATP) channels are present at an extremely high density in the heart, and we know from in vitro studies that channel activation causes dramatic action potential shortening and contractile failure. But, if and when this happens in vivo is still a matter of debate. Twenty one years of intense study have led to a well-developed understanding of the molecular basis of K(ATP) channel activity. Structure-function studies, together with cellular experiments probing regulatory molecules have told us much about the way the K(ATP) channel can activate, and gene-targeting and proteomic tools have further elucidated determinants of in vivo function. However, the true physiological determinants of sarcolemmal K(ATP) activity remain elusive, we still await full illumination of the role of the channel in the intact heart.

Remodeling of excitation-contraction coupling in transgenic mice expressing ATP-insensitive sarcolemmal KATP channels.

Reducing the ATP sensitivity of the sarcolemmal ATP-sensitive K(+) (K(ATP)) channel is predicted to lead to active channels in normal metabolic conditions and hence cause shortened ventricular action potentials and reduced myocardial inotropy. We generated transgenic (TG) mice that express an ATP-insensitive K(ATP) channel mutant [Kir6.2(deltaN2-30,K185Q)] under transcriptional control of the alpha-myosin heavy chain promoter. Strikingly, myocyte contraction amplitude was increased in TG myocytes (15.68 +/- 1.15% vs. 10.96 +/- 1.49%), even though K(ATP) channels in TG myocytes are very insensitive to inhibitory ATP. Under normal metabolic conditions, steady-state outward K(+) currents measured under whole cell voltage clamp were elevated in TG myocytes, consistent with threshold K(ATP) activation, but neither the monophasic action potential measured in isolated hearts nor transmembrane action potential measured in right ventricular muscle preparations were shortened at physiological pacing cycles. Taken together, these results suggest that there is a compensatory remodeling of excitation-contraction coupling in TG myocytes. Whereas there were no obvious differences in other K(+) conductances, peak L-type Ca(2+) current (I(Ca)) density (-16.42 +/- 2.37 pA/pF) in the TG was increased compared with the wild type (-8.43 +/- 1.01 pA/pF). Isoproterenol approximately doubled both I(Ca) and contraction amplitude in wild-type myocytes but failed to induce a significant increase in TG myocytes. Baseline and isoproterenol-stimulated cAMP concentrations were not different in wild-type and TG hearts, suggesting that the enhancement of I(Ca) in the latter does not result from elevated cAMP. Collectively, the data demonstrate that a compensatory increase in I(Ca) counteracts a mild activation of ATP-insensitive K(ATP) channels to maintain the action potential duration and elevate the inotropic state of TG hearts.

Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions.

The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney. In the present study, the role of the PDZ binding motif in ROMK function is explored. Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel. Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR). As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK. Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK. Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR. Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR. Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR. These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney.