Nitroxyl (HNO) exerts inotropic and lusitropic effects in myocardium, in part via activation of SERCA (sarcoplasmic reticulum calcium ATPase). To elucidate the molecular mechanism, adult rat ventricular myocytes were exposed to HNO derived from Angeli's salt. HNO increased the maximal rate of thapsigargin-sensitive Ca 2؉ uptake mediated by SERCA in sarcoplasmic vesicles and caused reversible oxidative modification of SERCA thiols. HNO increased the S-glutathiolation of SERCA, and adenoviral overexpression of glutaredoxin-1 prevented both the HNO-stimulated oxidative modification of SERCA and its activation, as did overexpression of a mutated SERCA in which cysteine 674 was replaced with serine. Thus, HNO increases the maximal activation of SERCA via S-glutathiolation at cysteine 674. N itroxyl (HNO), the 1-electron reduced and protonated form of nitric oxide (NO), exerts a bioactivity profile that differs markedly from NO 1,2 and other reactive nitrogen species such as peroxynitrite. 3 In the cardiovascular system, HNO derived from Angeli's salt (AS) exerts inotropic and lusitropic effects in the myocardium 4 and causes relaxation of vascular smooth muscle. 5,6 These observations have raised the possibility that HNO is involved in cardiovascular regulation and/or may have therapeutic potential.In cardiac myocytes, HNO increases calcium cycling in association with increasing the activities of SERCA (sarcoplasmic reticulum ATPase) and the calcium release channel (CRC). 1 In vascular smooth muscle cells SERCA activity can be increased by NO-induced S-glutathiolation. 7 Accordingly, we hypothesized that in cardiac myocytes HNO can activate SERCA via S-glutathiolation. Materials and MethodsIn all experiments, adult rat ventricular myocytes (ARVMs) 8 were exposed for 15 minutes to 500 mol/L AS dissolved in 10 mmol/L NaOH. Detailed methods are provided in the online supplement at http://circres.ahajournals.org. Results and Discussion HNO Activation of SERCA Involves Reversible, Oxidative Thiol ModificationAS increased myocyte shortening (Ϸ2-fold) and accelerated relaxation ( Figure I in the online data supplement), confirming the findings of Tocchetti et al. 1 In the absence of dithiothreitol (DTT), AS (500 mol/L; 15 minutes) increased maximal SERCA activity Ϸ3-fold ( Figure 1A). In the pres-
Endothelial cell (EC) migration in response to VEGF is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well-understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca2+ regulation and migration. VEGF induced migration of human aortic EC into a scratch wound over 6 hours that was inhibited by overexpression of either catalase or SOD. EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca2+ influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knock down of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca2+ influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca2+ influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex Red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca2+ influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.
Nitric oxide is a gaseous signaling molecule that is well-known for the Nobel prize-winning research that defined nitric oxide as a physiological regulator of blood pressure in the cardiovascular system. Nitric oxide can signal via the classical pathway involving activation of guanylyl cyclase or by a post-translational modification, referred to as S-nitrosylation (SNO) that can occur on cysteine residues of proteins. As proteins with cysteine residues are common, this allows for amplification of the nitric oxide signaling. This review will focus on the possible mechanisms through which SNO can alter protein function in cardiac cells, and the role of SNO occupancy in these mechanisms. The specific mechanisms that regulate protein SNO, including redox-dependent processes, will also be discussed.
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