2.1 Participants and Study Design
The Brazilian Heart Study (BHS), an observational prospective cohort registered at ClinicalTrials.gov (NCT02062554) (detailed elsewhere ) enrolled 354 participants. Briefly, consecutive STEMI patients admitted to Coronary Care Unit at the Hospital de Base do Distrito Federal (Brasilia, Brazil) who met to these inclusion criteria were enrolled: (i) £ 24 hours from the onset of MI symptoms, (ii) ST-segment elevation ³ 1 mm (frontal plane) or 2 mm (horizontal) in contiguous leads, and (iii) myocardial necrosis evidenced by scores ³ the 99th percentile of the reference limit for CK-MB (25 U/L) and troponin I (0.04 ng/mL), followed by a decline of both.
A complete medical evaluation was performed upon admission (D1) with patients followed in-hospital until the fifth day (D5) after MI. Attending physicians decided on each medical treatment, including the choice of reperfusion therapy, without influence from the investigation team. Functional endothelial vascular reactivity was assessed 30 days after STEMI.
2.2 Clinical Evaluation
In the first 24 hours after the onset of symptoms, a standardized interview was performed to assess medical history, all drugs currently used, and lifestyle factors. To assist these assessments, blood samples were taken. Hypertension was defined either by use of antihypertensive drugs prior hospital admission or by repeatedly elevated blood pressure exceeding 140 over 90 mmHg during in-hospital stay. Diabetes was defined by use of hypoglycemic drugs and/or insulin, fasting blood glucose ≥ 126 mg/dL or glycated hemoglobin (Hb1Ac) ≥ 6.5%. Current smoking was defined by using 1 or more cigarette/day for more than 1 year before the coronary event. Former smoking was defined by smoking cessation for at least 6 months prior to hospitalization. Sedentary lifestyle was defined by not practicing physical activities for more than 30 minutes over at least 4 days a week. Drugs with vasodilation effects were defined by in-hospital use of calcium channel blockers (CCB), angiotensin receptor blockers (ARB) and/or angiotensin converting enzyme inhibitors (ACEi). Nitrates were not included by ruling out from analyses participants exposed to these drugs during hospitalization. Medical and biochemical assessments were repeated as clinically needed during hospital stay, with glycemic scores being necessarily reassessed at D5.
2.3 Biochemical Analyses
Blood samples were drawn at admission and a second round of clinical biochemistry assays was also done after a 12-h overnight fast at D5. Samples in EDTA were centrifuged at 4500 rpm for 15 minutes at 5°C, and plasma was used in an automatic chemical analyzer to performe the following assessments, in duplicate: glucose, gycated hemoglobin A1c (HbA1c), insulin, C-peptide, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), creatinine, ultra-sensitive C-reactive protein (us-CRP), troponin and creatine kinase-myoglobin binding (CK-MB), with reagents from Roche Diagnostics (Mannheim, Germany), Bio-Rad Laboratories (Hercules, USA) or Dade Behring (Marburg, Germany). LDL cholesterol (LDL-C) was calculated by the Friedewald formula. Glomerular filtration rate (GFR) was estimated by the Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation: eGFR (mL/min/1.73 m2) = 141 x min (SCr x 0.0113/k, 1)a x max (SCr x 0.0113/k, 1)-1.209 x 0.993Age x 1.018 (if female) x 1.159 (if black), where SCr is serum creatinine, k is 0.7 for females and 0.9 for males, a is -0.329 form females and -0.411 for males.
2.4 Glucose Homeostasis Model Assessment
The Homeostasis Model Assessment (HOMA) Calculator, version 2.2.2, was used to assess insulin sensitivity (HOMA2S) based on plasma insulin and b-cell function (HOMA2B) based on plasma C-peptide  in all enrolled participants. The Disposition Index (DI) was calculated as (HOMA2B x HOMA2S)/100.
2.5 DNA Extraction and Analysis
Genomic DNA was obtained from peripheral blood leukocytes. The rs41279104 NOS1 SNP was genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method originally published elsewhere . Briefly, the GenBank was search for the NOS1 gene (NG_011991.2) and a pair of primers was designed to amplify a 150 bp-fragment with the SNP of interest (-84 G/A) (forward 5’-CTGACTGCCCTTGTCTCTCC-3’ and reverse 5’-GCGACTGGGGTTTAATTGAC-3’). Each reaction (25 µL) was composed of 100 ng of DNA, 25 mM Tris-HCl pH 8.3, 75 mM KCl, 1.5 mM MgCl2, 0.2 µM of each primer, 0.2 µM of each dNTP, 0.25 mg/mL of ovalbumin and 0.5 unit of Taq Polymerase (Phoneutria, Minas Gerais, Brazil). Amplification had an initial denaturation at 94ºC for 2 min, followed by 36 cycles of denaturation at 94ºC for 40 s, annealing at 63ºC for 45 s and extension at 72ºC for 50 s, followed by a final extension at 72ºC for 5 min. Enzymatic digestion was carried out at 37ºC overnight in 15 µL, using 0.1 U of Fnu4HI (New England BioLabs, USA) to yield two fragments (92 and 58 bp) visible under electrophoresis in 2.2% agarose gel.
2.6 Measurement of total NO/nitrite/nitrate
For quantification of total serum levels of nitric oxide (NO), the Griess diazotization reaction based on conversion of nitrate to nitrite (NO2−) by nitrate reductase was employed, performed following the manufacturer's instructions (R&D Systems Inc., MN, USA), with readings done by colorimetric detection in a Biotek ELX 800 (DeMorellis, SP, Brazil) device, set at a 540 nm wavelength.
2.7 Brachial artery reactivity
Endothelial-dependent vasodilation was assessed by ischemia-induced reactive hyperemia on the 30th day post-STEMI, after a 12-hour overnight fasting and 24 hours after withdrawal of vasoactive drugs (described elsewhere ). Briefly, after 10 minutes of rest, longitudinal images of the brachial artery were taken by an experienced physician blinded to the patient’s history, using ultrasound with a high-resolution linear transducer (model IE 33, 3–9 MHz transducer, Philips Medical Systems, MA, USA) synchronized with EKG monitoring, and participants in supine position. A blood pressure cuff placed on the forearm was inflated to 50 mmHg above the systolic pressure for 5 min, and then deflated. The flow-mediated dilation (FMD) was scanned for 2 min, and the change in diameter was calculated respective to the resting state.
2.8 Statistical Analysis
Assumptions for parametric models (linearity, normality of distribution and equal variance) were checked using histograms, normal probability plots and residual scatter plots by means of the Kolmogorov-Smirnov test. Normally distributed and skewed data were tested using ANOVA or Kruskal-Wallis, respectively, with a Tukey post-hoc test if necessary. ANCOVA was used to adjust the effect of the SNP rs41279104 on glycemic scores, NO levels and brachial artery reactivity for potential confounders. Changes between D1 and D5 were additionally adjusted for baseline levels to attenuate the effect of regression toward the mean. Categorical data were tested using the chi-square test. A two-sided p-value of < 0.05 was considered statistically significant, with analyses performed using SPSS for Mac version 25.0.