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[gamma]-Glutamyl transpeptidase ([gamma]-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH ([GSH.sub.o]). The objective of this study was to identify the role of [gamma]-GT in reversing pathogenic [K.sup.+] channel remodeling in the diseased heart. Chronic ventricular dysfunction was induced in rats by myocardial infarction (MI), and studies were done after 6-8 wk. Biochemical assays of tissue extracts from post-MI hearts revealed significant increases in [gamma]-GT activity in left ventricle (47%) and septum (28%) compared with sham hearts, which paralleled increases in protein abundance and mRNA. Voltage-clamp studies of isolated left ventricular myocytes from post-MI hearts showed that downregulation of transient outward [K.sup.+] current ([I.sub.to]) was reversed after 4-5 h by 10 mmol/l [GSH.sub.o] or N-acetylcysteine ([NAC.sub.o]), and that the effect of [GSH.sub.o] but not [NAC.sub.o] was blocked by the [gamma]-GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of [gamma]-glutamylcysteine synthetase by buthionine sulfoximine did not prevent upregulation of [I.sub.to] by [GSH.sub.o], suggesting that intracellular synthesis of GSH was not directly involved. However, pretreatment of post-MI myocytes with an SOD mimetic [manganese (III) tetrapyridylporphyrin] and catalase completely blocked recovery of [I.sub.to] by [GSH.sub.o]. Confocal microscopy using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate confirmed that [GSH.sub.o] increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, [GSH.sub.o]-mediated upregulation of [I.sub.to] was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, and AG1024) and thioredoxin reductase (auranofin and 13-cis-retinoic acid). These data suggest that [GSH.sub.o] elicits [gamma]-GT- and ROS-dependent transactivation of tyrosine kinase signaling that upregulates [K.sup.+] channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by [gamma]-GT catalysis of [GSH.sub.o] may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart. glutathione; voltage-dependent [K.sup.+] channel; thioredoxin; transient outward current

Redox imbalance elicited by oxidative stress contributes to pathogenic remodeling of ion channels that underlies arrhythmogenesis and contractile dysfunction in the failing heart. This study examined whether the expression of [K.sup.+] channels in the remodeled ventricle is controlled by the thioredoxin system, a principal oxidoreductase network regulating redox-sensitive proteins. Ventricular dysfunction was induced in rats by coronary artery ligation, and experiments were conducted 6-8 wk postinfarction. Biochemical assays of tissue extracts from infarcted hearts showed that thioredoxin reductase activity was decreased by 32% from sham-operated controls (P < 0.05), whereas thioredoxin activity was 51% higher postinfarction (P < 0.05). These differences in activities paralleled changes in protein abundance as determined by Western blot analysis. However, whereas real-time PCR showed thioredoxin reductase mRNA levels to be significantly decreased postinfarction, thioredoxin mRNA was not altered. In voltage-clamp studies of myocytes from infarcted hearts, the characteristic down-regulation of transient-outward [K.sup.+] current density was reversed by exogenous pyruvate (5 mmol/1), and this effect was blocked by the specific inhibitors of the thioredoxin system: auranofin or 13-cisretinoic acid. Real-time PCR and Western blot analyses of myocyte suspensions from infarcted hearts showed that pyruvate increased mRNA and protein abundance of Kv4.2 and Kv4.3 channel [alpha]-subunits as well as the accessory protein KChIP2 when compared with time-matched, untreated cells (P < 0.05). The pyruvate-induced increase in Kv4.x expression was blocked by auranofin, but the upregulation of KChIP2 expression was not affected. These data suggest that the expression of Kv4.x channels is redox-regulated by the thioredoxin system, which may be a novel therapeutic target to reverse or limit electrical remodeling of the failing heart. potassium channels; heart failure; electrical remodeling; transient-outward potassium current

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and [Ca.sup.2+] transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was ~36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and [gamma]-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and [gamma]-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged [Ca.sup.2+] transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of [Ca.sup.2+] handling proteins and contraction. redox; cardiomyopathy; pentose pathway

Electrical remodeling of the diseased ventricle is characterized by downregulation of [K.sup.+] channels that control action potential repolarization. Recent studies suggest that this shift in electrophysiological phenotype involves oxidative stress and changes in intracellular glutathione (GSH), a key regulator of redox-sensitive cell functions. This study examined the role of GSH in regulating [K.sup.+] currents in ventricular myocytes from rat hearts 8 wk after myocardial infarction (MI). Colorimetric analysis of tissue extracts showed that endogenous GSH levels were significantly less in post-MI hearts compared with controls, which is indicative of oxidative stress. This change in GSH status correlated with significant decreases in activities of glutathione reductase and [gamma]-glutamylcysteine synthetase. Voltage-clamp studies of isolated myocytes from post-MI hearts demonstrated that downregulation of the transient outward [K.sup.+] current ([I.sub.to]) could be reversed by pretreatment with exogenous GSH or N-acetylcysteine, a precursor of GSH. Upregulation of [I.sub.to] was also elicited by dichloroacetate, which increases glycolytic flux through the GSH-related pentose pathway. This metabolic effect was blocked by inhibitors of glutathione reductase and the pentose pathway. These data indicate that oxidative stress-induced alteration in the GSH redox state plays an important, role in [I.sub.to] channel remodeling and that GSH homeostasis is influenced by pathways of glucose metabolism. redox; myocytes; failure; ion

A study was conducted to investigate the effects of two amphiphilic fatty acid metabolites on K+ currents of rat ventricular myocytes that are involve in the regulation of refractories and excitability of the heart. The metabolites are palmitoylcarnitine and palmitoyl-CoA. Results suggest that the fatty acid metabolites downregulate transient outward channels through a mechanism located in the cytoplasmic side to the membrane.

Research was conducted to examine the cellular mechanisms associated with changes in potassium channel function in failing hearts after myocardial infarction. A chronic, postinfarction model of heart failure was utilized in the study as Sprague-Dawley rats were intubated and ventilated with a respiratory under methohexital sodium. Results suggested that changes in myocyte channel function may be related to the impaired contractility and arrhythmogenesis related to failing hearts.

The mechanisms underlying decreased transient outward potassium (K+) current in cardiac myocytes isolated from diabetic rats were investigated by the whole cell patch-clamp technique. The results revealed that changes in transient outward K+ current were caused by diabetes-induced metabolic disturbances in glucose metabolism. These disturbances were normalized by an increase in glucose utilization.

The effect of excessive protons on ventricular myocytes during the early stages of diabetes mellitus was investigated by whole cell patch-clamp techniques on cardiac myocytes isolated from streptozoin-induced diabetic rats. Measurements of the calcium-dependent, transient outward potassium current and inward rectifier current in response to protons revealed the inhibition of the transient outward current. This inhibition was attributed to the different sensitivities of voltage-gated ion channels to protons.

Examination of the effect of human recombinant interleukin-1beta (IL-1) on the beta-adrenergic control of the cardiac L-type Ca2+ current (Ica) in adult guinea pig ventricular myocytes by whole cell voltage-clamp technique reveals that pretreatment with IL-1 inhibits the beta-responsiveness of ICa. The presence of extracellular L-arginine is necessary for the inhibition, and incubation with nitric oxide synthase (NOS) inhibitor cancels the inhibition by IL-1. A mechanistic study reveals the involvement of cellular mechanisms through L-Arg/NOS pathway in the inhibition by IL-1.

Analysis of the effect of interleukin-1 (IL-1) on the beta-adrenergic regulation of cardiac L-type calcium current (ICa) using whole cell voltage-clamp method indicates that IL-1 increases the beta-adrenergic responsiveness of ICa during acidosis, which inhibits ICa. Amiloride treatment that prevents Na+/H+ exchange or the use of protein kinase C inhibitors abolishes the increase in beta-responsiveness due to IL-1, indicating that IL-1 promotes beta-responsiveness by increasing Na+/H+ exchange. The cellular processes involving IL-1 are functional in modifying ventricular activities during myocardial ischemia-induced inflammation.

Arrythmogenic reaction of phasic vagal stimuli with atrial ectopic pacemakers is modeled by a computer algorithm. Conducting atrial tissue is used to isolate the solitary ectopic pacemaker core, which, with a dominant sinus rode, forms the model. Timing and occurrence of atrial premature beats are hypothesized by the model. Induction of the vagal-stimulated entrance obstacle of the sinus impulse near the ectopic focus causes the beats. A sequence of experimentally obtained phase-reaction and rectified retrieval time curves are used to make the predictions. Vagal input and overdrive restraint are used to determine the regulation of ectopic pace-maker periodicity.

Phosphocreatine (PCr) is a compound used by the body to chemically store energy, and is particularly important in heart muscle cells. Cardiac levels of PCr decrease during conditions of oxygen starvation due to ischemia (poor blood flow) or other conditions. In turn, low levels of PCr and similar compounds are linked to irreversible cell damage. Resupply of PCr appears to be beneficial in preventing cell death during ischemia. In addition, PCr treatment has decreased cardiac arrhythmias (uncoordinated, ineffective heart beats) following heart attacks. It is not clear, though, whether the antiarrhythmic effects of PCr are linked to its function of energy storage. The effects of PCr and a related high-energy phosphate, phosphocreatinine (PCr-nine) on the electric activity of muscle cells under various conditions were investigated. PCr and PCr-nine had similar effects on muscle under normal conditions and had very little effect under ischemic-like conditions, suggesting that antiarrhythmic effects of PCr are not likely to be related to its ability to store energy. The experiments instead suggest that PCr can bind calcium, leading to a reduction of calcium levels inside muscle cells. During ischemia, intracellular calcium increases and can alter cellular electrical activity. Thus, the antiarrhythmic effects of PCr may be due to its ability to dampen calcium levels during ischemia, preventing changes in cellular electrical conductivity. Further research is needed to test this hypothesis. (Consumer Summary produced by Reliance Medical Information, Inc.)

The enhanced cardiac sympathetic afferent reflex (CSAR) contributes to the exaggerated sympathoexcitation in chronic heart failure (CHF). Increased sympathoexcitation is positively related to mortality in patients with CHF. However, the potential beneficial effects of chronic CSAR deletion on cardiac and autonomic function in CHF have not been previously explored. Here, we determined the effects of chronic CSAR deletion on cardiac remodeling and autonomic dysfunction in CHF. To delete the transient receptor potential vanilloid 1 receptor-expressing CSAR afferents selectively, epicardial application of resiniferatoxin (50 µg/mL), an ultrapotent analog of capsaicin, was performed during myocardium infarction surgery in rats. This procedure largely abolished the enhanced CSAR, prevented the exaggerated renal and cardiac sympathetic nerve activity and improved baroreflex sensitivity in CHF rats. Most importantly, we found that epicardial application of resiniferatoxin largely prevented the elevated left ventricle end-diastolic pressure, lung edema, and cardiac hypertrophy, partially reduced left ventricular dimensions in the failing heart, and increased cardiac contractile reserve in response to β-adrenergic receptor stimulation with isoproterenol in CHF rats. Molecular evidence showed that resiniferatoxin attenuated cardiac fibrosis and apoptosis and reduced expression of fibrotic markers and transforming growth factor-β receptor I in CHF rats. Pressure-volume loop analysis showed that resiniferatoxin reduced the end-diastolic pressure volume relationships in CHF rats, indicating improved cardiac compliance. In summary, cardiac sympathetic afferent deletion exhibits protective effects against deleterious cardiac remodeling and autonomic dysfunction in CHF. These data suggest a potential new paradigm and therapeutic potential in the management of CHF. [ABSTRACT FROM AUTHOR]