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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-

Myocardial failure is associated with increased oxidative stress and abnormal excitation-contraction coupling characterized by depletion of sarcoplasmic reticulum (SR) Ca 2+ -stores and a reduction in Ca 2+ -transient amplitude. Little is known about the mechanisms whereby oxidative stress affects Ca 2+ -handling and contractile function; however, reactive thiols may be involved. We used an in vitro cardiomyocyte system to test the hypothesis that short-term oxidative stress induces SR Ca 2+ -depletion via redox-mediated regulation of sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) and the sodium-Ca 2+ -exchanger (NCX) and that this is associated with thiol oxidation. Adult rat ventricular myocytes paced at 5 Hz were superfused with H 2 O 2 (100 μM, 15 min). H 2 O 2 caused a progressive decrease in cell shortening followed by diastolic arrest, which was associated with decreases in SR Ca 2+ -content, systolic [Ca 2+ ] i and Ca 2+ -transient amplitude, but no change in diastolic [Ca 2+ ] i . H 2 O 2 caused reciprocal effects on the activities of SERCA (decreased) and NCX (increased). Pretreatment with the NCX inhibitor KB-R7943 prior to H 2 O 2 increased diastolic [Ca 2+ ] i , and mimicked the effect of SERCA inhibition with thapsigargin. These functional effects were associated with oxidative modification of thiols on both SERCA and NCX. In conclusion, redox-mediated SR Ca 2+ -depletion involves reciprocal regulation of SERCA and NCX, possibly via direct oxidative modification of both proteins.