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Abstract
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 Ca2+ 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.
Nitroxyl (HNO), the 1-electron reduced and protonated form of nitric oxide (NO), exerts a bioactivity profile that differs markedly from NO1,2⇓ 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 myocardium4 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 Methods
In 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 Modification
AS increased myocyte shortening (?2-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 ?3-fold (Figure 1A). In the presence of DTT (2 mmol/L), HNO had no effect on SERCA activity (+3±11%; P=NS; n=3). Iodoacetamide binds preferentially to reactive thiolate anions at pH 6.59 and to cysteine 674 of SERCA, in particular.10,11⇓ Oxidative modification of SERCA thiols was assessed using biotinylated iodoacetamide (BIAM).7,9⇓ HNO (500 ?mol/L; 15 minutes) decreased BIAM binding to SERCA thiols by 27±3% (Figure 1B; P<0.0001; n=14). In some experiments, ARVMs exposed to AS for 15 minutes were washed for an additional 15 minutes. After washout, the amount of BIAM-labeled SERCA was similar in HNO-treated and control cells (Figure 1C and 1D; P<0.01; n=4), indicating that the HNO-mediated modification is reversible. This finding is consistent with our observation that HNO-stimulated SERCA activation is prevented by DTT and the prior observation by Tocchetti et al1 that the effects of HNO on cardiac myocyte function are reversed by DTT or removal of AS from the perfusion buffer.
Figure 1. HNO activation of SERCA involves reversible thiol modification. A, In ARVMs, AS (500 ?mol/L, 15 minutes) increased maximal SERCA activity (*P<0.001 vs vehicle control; n=4), measured as the initial rate of thapsigargin-sensitive 45Ca2+ uptake in a postnuclear supernatant fraction. This effect was prevented by DTT (online data supplement). B, AS (500 ?mol/L, 15 minutes) decreased the fraction of BIAM-labeled SERCA (†P<0.0001 vs vehicle control; n=14). C, This effect was reversed by subsequent washing (15 minutes) to remove AS (†P<0.01 vs vehicle control; n=4). D, Representative Western blot showing effects of AS and washout on BIAM-labeled and total SERCA. Data in B and D were normalized to the means of the control values.
Editor's Note: The authors have identified errors in this Figure. A revised figure and explanation can be found in the accompanying Correction Notice.
HNO Activates SERCA via S-Glutathiolation
We have shown that oxidative activation of SERCA7 and p21 Ras12,13⇓ is mediated via the formation of mixed disulfides leading to protein S-glutathiolation. S-Glutathiolation of SERCA was assessed by immunoprecipitation of SERCA followed by immunoblotting with an antibody directed against glutathione.13 HNO caused an 18±4% increase in SERCA S-glutathiolation (P<0.05; n=5), which was abolished by DTT (Figure 2A and 2B). To further examine the role of S-glutathiolation, glutaredoxin-1 (GRX) was overexpressed (?10-fold) via adenoviral infection (supplemental Figure II). In control ARVMs expressing ?-galactosidase, HNO decreased BIAM-labeled SERCA by 43±13% (P<0.05; n=4), whereas in GRX-expressing cells, the effect of HNO was abolished (Figure 2C and 2D; P=NS; n=4). In ?-galactosidase expressing cells, AS increased maximal SERCA activity by 42±5%, whereas in GRX-expressing cells HNO had no effect (Figure 2E; P=NS, n=4). Together with the demonstration that HNO increases S-glutathiolation of SERCA, the ability of GRX to prevent SERCA thiol oxidative modification and SERCA activation supports the conclusion that the major HNO-induced oxidative modification of SERCA is S-glutathiolation and is consistent with our prior demonstration that SERCA S-glutathiolation stimulates maximal enzyme activity in vascular smooth muscle cells and heart or purified SERCA in phospholipid vesicles.7
Figure 2. HNO activates SERCA via S-glutathiolation. A, AS (500 ?mol/L, 15 minutes) increases S-glutathiolation of SERCA (*P<0.05 vs control; n=5). B, Representative Western blot showing increased S-glutathiolation by addition of AS, and reversal by the addition of DTT (10 mmol/L) before BIAM labeling. C, GRX prevents the decrease in BIAM labeling caused by AS (*P<0.05 vs control; n=4). D, Representative Western blot. E, GRX prevents the AS-stimulated increase in maximal SERCA activity (*P<0.05 vs control; n=4). Data in A and C were normalized to the means of the control values.
Cysteine 674 Modulates HNO-Stimulated SERCA Activity
Of the 14 surface thiols of SERCA, iodoacetamide preferentially binds to cysteine 674.10,11⇓ To test the role of cysteine 674 in mediating the effect of HNO in ARVMs, we overexpressed (?5-fold; supplemental Figure III) wild-type SERCA or a mutated SERCA (C674S) in which cysteine 674 was replaced by serine. Of note, the amount of accessible SERCA thiols labeled by BIAM was reduced by 52±7% in cells expressing C674S (Figure 3A and 3B; P<0.01, n=3), indicating that cysteine 674 accounts for approximately half of the labeling of wild-type SERCA. In cells expressing wild-type SERCA, HNO decreased BIAM labeling by 65±8% (P<0.01, n=3) but, in contrast, caused no further decrease in the BIAM labeling of SERCA in cells expressing the C674S mutant (Figure 3A and 3B). In cells expressing wild-type SERCA, HNO stimulated SERCA activity by 59±12% but caused no stimulation in cells expressing the C674S mutant (Figure 3C). Likewise, HNO had no effect on myocyte contraction or relaxation in cells expressing the C674S mutant (supplemental Figure I). Thus, the cysteine 674 thiol is the molecular target that accounts for the HNO-mediated oxidative modification and activation of SERCA. Although it is possible that HNO causes oxidative modifications of other SERCA reactive cysteines14 and/or other amino acids, our data indicate that in cardiac myocytes S-glutathiolation of cysteine 674 is the most abundantly modified thiol and plays an essential role in HNO-mediated activation of SERCA.
Figure 3. Cysteine 674 modulates HNO-stimulated SERCA activation. A, AS decreases SERCA BIAM labeling in myocytes expressing wild-type SERCA (WT). In contrast, in myocytes expressing the C674S SERCA mutant, BIAM labeling is decreased as compared to wild type but is not decreased further by addition of AS (†P<0.01 vs control vehicle; n=3). B, Representative Western blot showing effect of C674S mutant on BIAM labeling. C, AS does not increase SERCA activity in myocytes overexpressing the C674S SERCA mutant (*P<0.05 vs vehicle control; †P<0.05 vs AS in wild type; n=3). Data in A were normalized to the mean of the wild-type control value.
Mechanism of Oxidative Modification
It remains to be determined how HNO causes S-glutathiolation of SERCA. It is possible that HNO leads to generation of NO and/or peroxynitrite. We previously found that peroxynitrite formed from NO can cause S-glutathiolation of SERCA in vascular smooth muscle cells7 and that this effect can be prevented by the peroxynitrite scavenger uric acid.7 Likewise, in ARVMs, we found that peroxynitrite decreases SERCA BIAM labeling and that this decrease is prevented by uric acid (100 ?mol/L; data not shown). However, uric acid had no effect on the decrease in SERCA BIAM labeling or SERCA activation caused by AS (supplemental Figure IV). This finding suggests that peroxynitrite does not mediate HNO-stimulated S-glutathiolation of SERCA. Although further studies will be required to determine the chemical mechanism, our data are consistent with a direct effect of HNO.15 In this regard, it has been proposed that HNO can mediate S-glutathiolation via the formation of a protein thiol N-hydroxysulfenamide intermediate followed by a reaction with GSH.15 Alternatively, HNO might cause GSH depletion, resulting in an oxidative environment that promotes protein S-glutathiolation.16
Role of Phospholamban
Recently, Froehlich et al17 showed that SERCA was not activated by HNO in the absence of phospholamban or with mutation of critical phospholamban cysteines, thus implicating oxidative cysteine modifications of phospholamban. Thus, the regulation of SERCA by HNO may involve oxidative modifications of both SERCA and phospholamban.17 For example, SERCA activation via oxidative modification of cysteine 674 may require and/or synergize with a conformational effect mediated via oxidative thiol modification of phospholamban.
Acknowledgments
Sources of Funding
Supported by NIH grants HL-061639 and HL-064750 (to W.S.C.); NIH grant HL31607 (to R.A.C.); and the National Heart, Lung, and Blood Institute-sponsored Boston University Cardiovascular Proteomics Center (contract no. N01-HV-28178 to R.A.C. and W.S.C.). S.L. was supported by La Fondation pour la Recherche Médicale grant SPE20051105207.
Disclosures
None.
Footnotes
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Original received December 19, 2008; revision received February 19, 2009; accepted February 23, 2009.
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