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Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca2+ channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca2+ channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca2+ current (ICa,L) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α1C subunit of voltage-gated Ca2+ channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α1C were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased ICa,L in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized ICa,L stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca2+ channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca2+ channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca2+ channel remodeling of the failing heart. [ABSTRACT FROM AUTHOR]

γ-Glutamyl transpeptidase (γ-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH (GSHo). The objective of this study was to identify the role of -γ-GI in reversing pathogenic K+ 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 γ-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+ current (Ito) was reversed after 4-5 h by 10 mmol/l GSHo or N-acetylcysteine (NACO), and that the effect of GSHO but not NACO was blocked by the -γ-GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of -γ-glutamylcysteine synthetase by buthionine sulfoximine did not prevent upregulation of Ito by GSHO, 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 Ito, by GSHO. Confocal microscopy using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate confirmed that GSHO increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, GSH0mediated upregulation of Ito was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, and AG 1024) and thioredoxin reductase (auranofin and 13-cis-retinoic acid). These data suggest that GSHO elicits -γ-GT and ROS-dependent transactivation of tyrosine kinase signaling that upregulates K+ channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by -γ-GT catalysis of GSHO may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart. [ABSTRACT FROM AUTHOR]

In our previous studies, homocysteine (Hcys) has been reported to stimulate de novo ceramide synthesis and thereby induce NAD(P)H oxidase activation by increase in Rac GTPase activity in rat mesangial (RMG) cells. However, the mechanism by which Hcys-enhances Rac GTPase activity is still unknown. Given Vav1, a member of guanine exchange factors (GEFs), plays an important role in the activation of Rho/Rac GTPase activity, the present study tested the hypothesis that Rac1/NAD(P)H oxidase signaling induced by Hcys is associated with Vav1 in RMG cells. Real-time RT PCR and Western blot analysis confirmed that Vav1 was present in RMG cells. By Rac GTPase pull down assay, it was found that NSC23766, an inhibitor by interfering Rac1 interaction with the Rac-specific GEF, blocked the Hcys-induced Rac GTPase activity by 42%. In Vav1-siRNA transfected RMG cells, Hcys-induced Rac GTPase activity decreased by 52%, which was accompanied by 55% reduction of Hcys-induced superoxide (O2) production, as measured by ESR spectrometry. By immunofluorescence assay, it was found that Hcys stimulated Vav1 phosphorylated and then translocated to cell membrane. Similar results were obtained by C16-ceramide treatments of RMG cells. Based on these results, we conclude that Rac1/NADPH oxidase signaling induced by Hcys is Vav1-depentent in RMG cells, which involves its phosphorylation and subsequently translocation, an essential step for the activation of this GEF. [ABSTRACT FROM AUTHOR]

Chronic arsenic exposure in drinking water is associated with the abnormalities of cardiac tissue. Excessive generation of ROS induced by arsenic has a central role in arsenic-induced cardiotoxicity. (-)-Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, possesses a potent antioxidant capacity and exhibits extensive pharmacological activities. This study was aim to evaluate the effect of EGCG on arsenic-induced cardiotoxicity in vivo and in vitro. Treatment with NaAsO 2 seriously affected the morphology and ultrastructure of myocardium, and induced cardiac injuries, oxidative stress, intracellular calcium accumulation and apoptosis in rats. In consistent with in vivo study, the injuries, oxidative stress and apoptosis were also observed in NaAsO 2 -treated H9c2 cells. All of these effects induced by NaAsO 2 were attenuated by EGCG. These results suggest EGCG could attenuate NaAsO 2 -induced cardiotoxicity, and the mechanism may involve its potent antioxidant capacity. [ABSTRACT FROM AUTHOR]