Estrogen Receptor-β Activation Results in S-Nitrosylation of Proteins Involved in Cardioprotection
Background— It has been shown that the activation of estrogen receptor-β (ER-β) plays an important cardioprotective role against ischemia/reperfusion injury. However, the mechanism for this protection is not clear. We hypothesize that estrogen protects by ER-β activation, which leads to S-nitrosylation (SNO) of key cardioprotective proteins.
Methods and Results— We treated ovariectomized C57BL/6J mice with the ER-β selective agonist 2,2-bis(4-hydroxyphenyl)-proprionitrile (DPN), 17β-estradiol (E2), or vehicle using Alzet minipumps for 2 weeks. Isolated hearts were Langendorff perfused and subjected to ischemia and reperfusion. Compared with vehicle-treated hearts, DPN- and E2-treated hearts had significantly better postischemic functional recovery and decreased infarct size. To test the specificity of DPN, we treated ER-β–knockout mice with DPN. However, no cardioprotective effect of DPN was found in ER-β–knockout mice, indicating that the DPN-induced cardioprotection occurs through the activation of ER-β. Using DyLight-maleimide fluors and a modified biotin switch method, we used a 2-dimensional DyLight fluorescence difference gel electrophoresis proteomic method to quantify differences in SNO of proteins. DPN- and E2-treated hearts showed an increase in SNO of a number of proteins. Interestingly, many of these proteins also had been shown to have increased SNO in preconditioned hearts. In addition, the DPN-induced cardioprotection and increased SNO were abolished by treatment with a nitric oxide synthase inhibitor.
Conclusion— The activation of ER-β by DPN treatment leads to increased protein SNO and cardioprotection against ischemia/reperfusion injury, suggesting that long-term estrogen exposure protects hearts largely via activation of ER-β and nitric oxide/SNO signaling.
Received July 3, 2008; accepted April 27, 2009.
Premenopausal women have a reduced incidence of cardiovascular disease, including ischemic heart disease and heart failure. However, this incidence increases after menopause, suggesting a protective role for endogenous estrogen.1,2 Several animal studies have shown estrogen to be cardioprotective during ischemia/reperfusion (I/R). These include studies looking at short-term protective effects of estrogen treatment in perfused hearts and literature reporting decreased I/R injury in intact females exposed long term to estrogen.3–6 However, the lack of protection by estrogen in recent clinical trials such as the Women’s Health Initiative contrast with the protection seen in premenopausal women and the described animal studies.7 To understand this difference, we need to better understand the mechanism of estrogen-mediated protection in these animal models.
Editorial see p 190
Clinical Perspective on p 254
Traditionally, the effects of estrogen have been attributed to estrogen binding to the classic nuclear estrogen receptors (ERs), which act as ligand-gated transcription factors. Binding of estrogen to these receptors (both ER-α and ER-β) leads to altered protein expression. In addition to these genomic effects, estrogen has rapid effects in the heart, leading to activation of short-term signaling pathways. We have previously reported that female mice have increased activity of nitric oxide (NO) synthase (NOS) in the heart,6 whereas other studies have found that selective ER-β activation upregulates NO production in cardiomyocytes.8 Recent literature reports that estrogen binding to either ERs that are localized in the plasma membrane or an estrogen-activated G-protein–coupled receptor (called GPR30) can lead to short-term activation of signaling cascades such as phosphatidylinositol-3-OH kinase.9 Thus, estrogen can alter the level of proteins involved in cardioprotection and enhance protection through the activation of short-term signaling pathways.
We have previously found that intact female mice exhibit reduced I/R injury compared with males and ovariectomized females.10,11 This protection is lost in hearts from female mice that lack ER-β, suggesting a role for ER-β in cardioprotection.12 Additionally, we have found that this cardioprotection observed in females can be blocked by inhibition of NOS.10,13 Taken together, these data support a role for estrogen acting via ER-β through an NO-dependent mechanism to mediate cardioprotection.
NO is an important signaling molecule in cardioprotection.14 In addition to its effects via guanylyl cyclase, a number of recent studies have shown that NO can lead to reversible posttranslational protein modifications such as S-nitrosylation.10,15–17 S-nitrosylation is the covalent attachment of an NO moiety to protein sulfhydryl residues, resulting in the formation of an S-nitrosothiol group. It is a common posttranslational protein modification, analogous to phosphorylation,18,19 and has been shown to alter protein activity, leading to a cardioprotective state.10,15–17 In this study, we aim to test the hypothesis that ER-β, acting through NOS, leads to increased S-nitrosylation (SNO) of proteins involved in cardioprotection in females. We used DyLight-maleimide fluors in a modified biotin switch method and 2-dimensional (2D) fluorescence difference gel electrophoresis (DIGE) proteomic methods to identify proteins that were S-nitrosylated by estrogen and the ER-β–selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). We reasoned that if estrogen protection is mediated by ER-β activation of NOS, then estrogen and DPN should lead to a similar pattern of increased protein SNO.
C57BL/6J wild-type and ER-β knockout (βERKO) female mice were obtained from Taconic Laboratories (Washington, DC). Animals were bilaterally ovariectomized at 8 weeks of age and delivered to our laboratory at 9 weeks of age. Micro-osmotic Alzet pumps (model 1002, DURECT Corporation, Cupertino, Calif) were implanted subcutaneously into the mice. Each pump delivered a constant dose of vehicle (50% dimethyl sulfoxide in 85% saline), E2 (0.1 mg · kg−1 · d−1), or DPN (0.8 mg · kg−1 · d−1) for 2 weeks. These dosages were chosen on the basis of previous work by Nikolic et al.11 All animals were treated in accordance with National Institutes of Health guidelines.
I/R Protocol, Postischemic Functional Recovery, and Infarct Size Determination
The Langendorff heart perfusion, hemodynamic, and infarct size measurements were performed as previously described by Sun et al.16 Treatment protocols are shown in Figure 1. Two groups (with and without 10 μmol/L of N-nitro-l-arginine methyl ester [L-NAME], an NOS inhibitor) underwent a standard I/R protocol with adrenergic stimulation (Figure 1A). Hearts were equilibrated for 20 minutes, including treatment with 10 nmol/L isoproterenol 1 minute before 20 minutes of no-flow ischemia followed by 30 minutes of reperfusion with drugs and then an additional 90 minutes of reperfusion without drugs. When indicated, L-NAME was added 5 minutes before ischemia, and perfusion with L-NAME was continued for the first 30 minutes of reperfusion. Another 2 groups underwent a 30-minute no-flow ischemia I/R protocol without adrenergic stimulation (Figure 1B). Hearts were equilibrated for 20 minutes and then subjected to 30 minutes of no-flow global ischemia followed by 2 hours of reperfusion. When indicated, hearts were treated for 5 minutes with 10 μmol/L L-NAME before 30 minutes of ischemia followed by 30 minutes of reperfusion with L-NAME and then 90 minutes of reperfusion without L-NAME.
Identification of S-Nitrosylated Proteins by 2D DyLight DIGE Method
Total homogenates of each sample were prepared as previously described by Sun et al.16 In some studies, samples were pretreated with 1 mmol/L ascorbic acid before the initial thiol blockade as negative control. The modified biotin switch method20 using DyLight-maleimide sulfhydryl–reactive fluors (Pierce Biotechnology, Rockford, Ill; Figure 1C) and the 2D DyLight DIGE proteomic method for identification of S-nitrosylated proteins were previously described by Sun et al.16
Protein Expression Differences With the 2D CyDye DIGE Method
A 2D DIGE proteomic method with CyDye fluors (GE Healthcare/Amersham Biosciences, Piscataway, NJ) was used to determine protein expression differences between the DPN-treated group and the vehicle control group. An internal standard containing equal amounts of protein from the DPN and vehicle groups was labeled with Cy2 as an internal standard. The labeling reaction was performed on ice for 30 minutes in the dark and then quenched with 10 mmol/L lysine. Equal quantities of labeled protein from each group (Cy2, Cy3, and Cy5) were combined and separated with a 2D system involving isoelectric focusing (pH 3 to 10) on an Ettan IPGphor3 (GE Healthcare/Amersham Biosciences) and 5% to 20% SDS-PAGE (NextGen, Ann Arbor, Mich) on an Ettan DaltTwelve System (GE Healthcare/Amersham Biosciences). Each gel was scanned on a Typhoon 9400 variable mode imager (GE Healthcare/Amersham Biosciences) at a resolution of 100 μm. The gel was poststained overnight with SYPRO Ruby stain and scanned. Images from each gel were aligned and analyzed with Progenesis Discovery software (Nonlinear Dynamics, Newcastle Upon Tyne, UK). Protein spots were chosen for identification with mass spectrometry if they exhibited a >1.5-fold difference in fluorescence volume intensity between DPN and vehicle with a value of P<0.05. Automated protein extraction and protein identification are the same as in the 2D DyLight DIGE method described earlier by Sun et al.16
DPN and E2 Treatment Is Cardioprotective After I/R in Ovariectomized Hearts
Initial measurements of hemodynamic parameters before ischemia demonstrated no baseline differences in heart rate or left ventricular diastolic pressure among our treatment groups (see the Materials section in the online-only Data Supplement). Using a Langendorff-perfused heart model of I/R, we find that compared with the vehicle control, DPN or E2 treatment of ovariectomized female mice reduces I/R injury (Figure 2). The functional recovery, expressed as percent of the initial rate-pressure product, was significantly higher in the DPN and E2 groups compared with vehicle (36.9±3.2 and 31.4±4.0 versus 16.0±1.1, respectively; P<0.05). Consistent with higher postischemic recovery, infarct size, expressed as percent of the left ventricular area, was decreased in the DPN and E2 groups compared with vehicle (35.8±2.4 and 38.8±2.5 versus 60.1±2.1, respectively; P<0.05). These results show that DPN and E2 treatments are both cardioprotective in a female ovariectomized mouse model with no significant differences in the amount of cardioprotection provided by either treatment.
DPN and E2 Treatment Increases Protein SNO
We and others have reported that ER activation leads to increased NOS production and NO availability.6,8 Although NO can signal through different mechanisms in the cell, potentially leading to cardioprotection, one of its actions is protein SNO, a redox-based reversible posttranslational modification.21–23 We therefore examined whether treatment with DPN or E2 led to changes in protein SNO. We used a recently developed 2D DyLight DIGE method16 to determine SNO differences between various treatments. As seen in Figure 3A, S-nitrosylated proteins in each of 3 adrenergic-stimulated hearts (ie, DPN, E2, or vehicle) were labeled with a DyLight-maleimide fluor with a distinct excitation/emission wavelength. A representative 2D DyLight DIGE gel is scanned at each of these distinct wavelengths, showing the pattern of protein SNO for that particular treatment group. S-nitrosylated protein spots in the vehicle group (labeled by DyLight 488) fluoresce green; S-nitrosylated protein spots in the DPN (labeled by DyLight 649) and E2 (labeled by DyLight 549) groups fluoresce red and yellow, respectively. In Figure 3B, we show a representative overlay of the DPN/vehicle images (DyLight 649/488) over a Ruby image, which stains all protein spots in the 2D gel. Proteins with increased DyLight labeling will exhibit a slight shift to the positive/acidic (left) end of the 2D gel because each molecule of DyLight dye carries ≈3 to 4 negative charges. In this overlay, several S-nitrosylated proteins are shown as green spots in the vehicle group labeled by DyLight 488, which are slightly shifted compared with the original spot in the Ruby image, suggesting small amounts of protein SNO. The red spots, indicating increased levels of protein SNO in the DPN-treated sample labeled by DyLight 649, are further shifted to the positive/acidic (left) end of the 2D gel compared with the vehicle group. Figure 3C shows an overlay of E2-treated samples (labeled by DyLight 549; yellow spots in Figure 3A) with the DPN-treated samples (labeled red with DyLight 649). The orange (red+yellow=orange) on this overlay (Figure 3C) shows that E2 treatment leads to a pattern of protein SNO similar to that of DPN. These combined data provide evidence that DPN and E2 treatments increase the SNO of similar proteins in the mouse heart.
Selected protein spots found to be S-nitrosylated in our 2D DyLight DIGE studies were extracted from the 2D gels and identified by mass spectrometry. SNO levels were quantified and expressed as ratios of the 3 different treatment groups. As shown in Table 1, DPN- or E2-treated hearts showed an increase in SNO of a number of proteins. Interestingly, many of these proteins also have been shown to exhibit increased SNO in preconditioned hearts.16 Additionally, we identified 3 proteins that exhibit decreased SNO in DPN- and E2-treated groups compared with vehicle (Table 1).
DPN and E2 Treatment Alters Protein Levels
As described earlier, estrogen activation of the classic ERs can alter the expression of many genes.11 If estrogen and DPN treatments reduce protein expression, the decrease in SNO might be due to decreased levels of available protein for SNO. As a result, the actual ratio of protein SNO per level of total protein might be unchanged. To evaluate whether DPN alters protein levels in the ovariectomized female mouse hearts, we used a 2D CyDye DIGE method to measure differences in protein expression. Figure 4 shows a representative 2D CyDye DIGE gel comparing vehicle- and DPN-treated hearts. As listed in Table 2, DPN treatment increased protein expression of aconitase and myosin light chain 1 while decreasing protein expression of α-actin-2, myosin regulatory light chain 2, tropomyosin-2, and ATP synthase subunit e. Interestingly, as shown in Table 1, α-actin-2 exhibits decreased levels of protein SNO in the DPN- and E2-treated groups. This suggests that the decreased protein SNO of α-actin-2 may result from decreased protein expression of α-actin-2. On the other hand, the increased protein expression of aconitase might contribute to its significant increase in protein SNO. Finally, the increase in protein levels of myosin light chain 1, coupled with decreased SNO, suggests that this protein has reduced SNO under conditions of DPN treatment.
DPN-Induced Cardioprotection Is Dependent on the Activation of NO/SNO Signaling
Because we have shown that female mice have increased NOS activity6 and others have demonstrated that estrogen increases NOS expression,8 the NOS inhibitor L-NAME was used to test whether the blockade of NOS would abolish the DPN-induced cardioprotection. As shown in Figure 5, 10 μmol/L L-NAME abolished the DPN-induced cardioprotection in these I/R hearts under adrenergic stimulation; ie, the postischemic functional recovery (Figure 5A) and infarct size in hearts treated with L-NAME (Figure 5B) were comparable to vehicle-treated control. Because the increased intracellular Ca2+ associated with adrenergic stimulation might also lead to the activation of Ca2+-dependent NOS and NO/SNO formation, it is important to test whether DPN can activate SNO and protect the hearts against I/R injury in the absence of isoproterenol. With 20 minutes of no-flow global ischemia, the protection observed in females occurs only under conditions of increased contractility.5,6,10–13 Because others have reported protection in females in the absence of increased contractility but with longer periods of ischemia, we considered whether a more severe ischemic injury might reveal protection in females in the absence of isoproterenol. We therefore increased the time of global ischemia from 20 to 30 minutes. As shown in Figure 5, with 30 minutes of ischemia in the absence of isoproterenol, DPN treatment significantly increased postischemic functional recovery (Figure 5C) and decreased infarct size (Figure 5D), which could also be blocked by L-NAME pretreatment.
On the basis of the data in Figure 5, we were interested in determining the levels of SNO that occurred with DPN treatment in the absence of isoproterenol. The representative 2D DyLight DIGE gel shown in Figure 6 clearly demonstrates that DPN treatment alone without isoproterenol resulted in a significantly increased SNO level (ie, only a few yellow spots in vehicle without isoproterenol [labeled with DyLight 549] versus multiple spots and increased fluorescence intensity in DPN without isoproterenol [labeled with DyLight 488]). However, adrenergic stimulation did not further increase the SNO level in these DPN-treated hearts, which was indicated by the yellow overlay images of DPN without isoproterenol (green) compared with DPN plus isoproterenol (labeled with DyLight 649, red). Thus, isoproterenol treatment is apparently not necessary for the DPN-induced increase in SNO.
Because L-NAME pretreatment abolished the DPN-induced cardioprotection (Figure 5), we were interested in determining whether L-NAME also would block the DPN-mediated increase in SNO. As shown in Figure 7A, the SNO level in L-NAME–pretreated isoproterenol-stimulated hearts was significantly decreased to low basal levels. In addition, the pattern of SNO modifications in DPN-treated hearts without isoproterenol stimulation (Figure 7B) was very similar to that found in DPN plus isoproterenol samples, and the increase in SNO levels induced by DPN was significantly attenuated by L-NAME pretreatment. To confirm the specificity of the DyLight/biotin switch method for labeling SNO, we performed a control in which the extract was pretreated with ascorbic acid before blocking free thiols in the modified DyLight Switch. Ascorbic acid has been used as a specific reducing agent to decompose SNO.20 This sample, labeled with DyLight 649 and shown on right side of Figure 7, shows little or no fluorescence, thus demonstrating the specificity of the method for labeling SNO.
DPN-Induced Cardioprotection Does Not Occur in Hearts Lacking ER-β
We initially measured the ratio of uterine weight to body weight in each of our treatment groups (see the Materials section of the online-only Data Supplement) to confirm that DPN, a selective ER-β agonist, was acting only through ER-β and not ER-α.11 To further confirm that the DPN-induced cardioprotection was due to the specific activation of ER-β, DPN- or vehicle-containing minipumps were implanted into ovariectomized βERKO mice, and after 2 weeks of treatment, the hearts were Langendorff perfused and subjected to 30 minutes of ischemia without adrenergic stimulation. No cardioprotective effect of DPN was found in ovariectomized βERKO mice (Figure 8A and 8B), indicating that DPN mediates cardioprotection through activation of ER-β. In addition, the similar pattern and fluorescence intensity of DyLight labeling (Figure 8C) in DPN- and vehicle-treated ovariectomized βERKO mice indicated that DPN did not lead to increased NO/SNO signaling in βERKO mice, suggesting that the DPN-induced activation of SNO also is dependent on activation of ER-β.
Estrogen has been shown to be cardioprotective in a number of different animal studies.3–6 Previous reports suggest that this cardioprotection is mediated by ER-β12 in an NO-dependent manner.10 In this study, we find that long-term treatment with DPN is cardioprotective in ovariectomized female mice to an extent similar to E2 and that DPN- and E2-treated hearts have similar protein SNO patterns. Additionally, the DPN-mediated cardioprotection is abolished in βERKO mice. Taken together, these results provide evidence that ER-β is the primary receptor responsible for long-term cellular changes leading to cardioprotection in female mice.
We find that ovariectomized female mice treated for 2 weeks with either E2 or DPN have reduced postischemic contractile dysfunction and smaller infarcts than vehicle-treated mice after I/R. However, this protection is abolished in DPN-treated mice that are pretreated with the NOS inhibitor L-NAME. Additionally, our 2D DIGE studies show that E2 and DPN result in increased SNO of an overlapping set of cardiac proteins. These observations lend strong support to the hypothesis that estrogen leads to cardioprotection via ER-β specifically through an increase in NO signaling. Many of the proteins found to be S-nitrosylated by DPN and estrogen such as aconitase, heat shock protein, mitochondrial F1-ATPase, creatine kinase, and malate dehydrogenase also have been shown to be S-nitrosylated in other models of cardioprotection.16
As shown in Table 2, we identified 3 proteins that exhibit decreased SNO in the DPN- and E2-treated groups compared with vehicle. SNO levels are determined by SNO and denitrosylation pathways.24,25 Estrogen clearly leads to activation of SNO pathways, as illustrated by the decrease in SNO in the presence of L-NAME. It is also possible that estrogen might selectively activate or target denitrosylation mechanisms, which might contribute to reduced SNO of these proteins. Another possibility for this decreased SNO may be that estrogen alters protein levels, leading to decreased availability of protein for SNO. To address this question, we performed a 2D CyDye DIGE to determine whether DPN altered protein expression levels compared with vehicle. Of interest, we noted that DPN causes a decrease in protein expression of α-actin-2, which was found to have decreased SNO in our E2- and DPN-treated groups. This finding would suggest that the decreased levels of α-actin-2 reduce the availability of this protein for SNO. As a result, the overall level of protein SNO decreases. However, a decrease in protein level does not account for the decrease in SNO of myosin light chain 1 and pyruvate dehydrogenase E1 β subunit.
The effects of estrogen have typically been attributed to estrogen-ER–mediated changes in gene expression. We were therefore surprised by the small number of proteins that were substantially increased or decreased with DPN treatment; only 2 proteins were significantly increased and 4 proteins were decreased with DPN treatment. This small number of changes at the protein level contrasts with the large number of changes (≈145) at the mRNA level identified by microarray.11 There are several possible reasons for this discrepancy. Many of the changes at the mRNA level were in transcription factors and relatively low-abundance signaling proteins that are not readily observed in the 2D CyDye DIGE method, which is biased toward high-abundance proteins. It is also likely that some of the changes in gene expression do not result in significant changes in protein levels. Despite these differences, it is interesting that most of the protein changes observed with the 2D CyDye DIGE method were contractile proteins. In our previous microarray study, we also observed a large number of changes in contractile proteins.11 This alteration in contractile proteins is intriguing in light of the study by Petre et al26 reporting sex differences in contractile reserve.
We have shown that long-term E2 and DPN treatments mediate cardioprotection in female mice and alter the SNO of several cardiac proteins. How does altered protein SNO potentially contribute to cardioprotection? SNO can have 2 primary effects on a protein. It can alter the activity of enzymes through changes in protein structure or function, and it can protect the modified cysteine from further oxidation. These 2 principles have been suggested to contribute to SNO-mediated cardioprotection.16,22,23 One protein of interest is creatine kinase, which was found to have increased SNO in DPN- and E2-treated and in preconditioned hearts.16 The SNO of creatine kinase might prevent the target cysteine residue(s) from further irreversible oxidative damage on reperfusion.
We also found increased SNO of the F1-ATPase α1 subunit in E2- and DPN-treated hearts. Sun et al16 recently reported that preconditioning and S-nitrosoglutathione treatment lead to the SNO of mitochondrial F1-ATPase, reversibly decreasing its activity. As much as 50% of the ATP generated during ischemia by glycolysis is consumed by the F1-ATPase functioning in reverse mode. The decrease in F1-ATPase activity, conferred by increased SNO, may therefore reduce the ischemic consumption of ATP.16 Several other important mitochondrial proteins were found to have increased SNO in our E2- and DPN-treated groups compared with the vehicle group. Future investigation will focus on whether SNO of these mitochondrial proteins alters their activities, leading to potential cardioprotection.
There are some interesting clinical implications from these findings. Although estrogen is cardioprotective in women, it has a number of detrimental effects, including an increased risk for breast and uterine cancer, both of which are largely mediated by ER-α activation. The finding that DPN, an ER-β selective agonist, is cardioprotective and increases SNO of the same set of cardiac proteins as E2 offers a potentially unique therapy in terms of a selective ER modulator that confers cardioprotection without the increased risk for cancer. Additionally, estrogen-mediated protein SNO may provide a partial explanation for the failure of the Women’s Health Initiative to show cardioprotection with hormone replacement therapy. It has been suggested that NO signaling might decrease in aging because of decreased levels of tetrahydrobiopterin27 or an increase in arginase activity.28
We thank the Proteomics Core Facility of the National Heart, Lung, and Blood Institute for help with 2D-DIGE analysis and peptide mass fingerprinting.
Sources of Funding
Dr Lin was supported by the Sarnoff Foundation and Harvard Medical School. Dr Steenbergen was supported in part by National Institutes of Health grant HL–39752. Drs Murphy and Sun were supported by the National Institutes of Health Intramural Program.
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Premenopausal women have reduced cardiovascular disease, and this protection is lost as women enter menopause. A number of studies in animal models have shown that estrogen treatment is cardioprotective. In contrast, the Women’s Health Initiative showed no protection with hormone replacement therapy, although some questions have been raised about the age of the women at treatment. This difference emphasizes the need for a better understanding of the mechanism by which estrogen protects in animal studies. This study shows that estrogen acts via the β-estrogen receptor in the heart to increase the S-nitrosylation of many proteins that have been shown to be cardioprotective. S-nitrosylation is a common posttranslational protein modification with covalent attachment of a nitric oxide moiety to protein sulfhydryl residues. S-nitrosylation not only leads to changes in the structure and function of target proteins but also prevents the modified cysteine residue(s) from further oxidative modification. These data might prompt future researchers to investigate whether age-related alterations in nitric oxide signaling contribute to the lack of protection by estrogen treatment in older women. It has been suggested that NO signaling might decrease with age because of a decrease in the levels of tetrahydrobiopterin or an increase in arginase activity. Additionally, the finding that a β-estrogen–specific agonist is protective provides a potential novel treatment therapy that might promote the beneficial effects of estrogen without specific adverse effects on other tissues such as the uterus.
Guest Editor for this article was Aruni Bhatnagar, PhD.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.868729/DC1.