Wagoner et al, #529/R1
Expanded Methods
Patients: The study was approved by the Institutional Review Board of the University of Cincinnati College of Medicine. The disease group consisted of 232 unrelated sequential patients with ischemic (n = 84) or idiopathic dilated cardiomyopathy (n = 148) (age 49.5 ± 0.6 yrs, New York Heart Association functional class II-IV, left ventricular ejection fraction 26.7 ± 0.9%, 73% male, 80% Caucasian, 20% African-American) referred to the University of Cincinnati Heart Failure/Transplant Program. At the time of entry into the study, peripheral blood was drawn to determine b2AR genotype. Prior to exercise testing, patients in the study were aggressively treated with heart failure therapies, and all were ambulatory. Investigators managing the heart failure therapy, staff performing exercise testing, and patients were blinded as to the b2AR genotypes. Pertinent medications and frequency of use in the patient cohort included digoxin (93.9%), diuretics (92.1%), beta blockers (24.9%), angiotensin-converting enzyme inhibitors (ACEI) (90.0%), angiotensin II receptor blockers (9.6%), and amiodarone (17.5%).
A subset of twelve patients underwent detailed invasive exercise hemodynamics with respiratory gas exchange and cardiopulmonary exercise testing, Swan Ganz pulmonary artery catheterization, and systemic arterial monitoring. These twelve patients consisted of two paired groups: 6 patients with the Ile164 receptor and 6 patients with Thr164.
b2AR genotyping: Genomic DNA was isolated from peripheral blood using standard techniques. b2AR genotyping was carried out using the genetic bit analysis (GBA) method essentially as previously described in detail 1. Briefly, polymerase chain reactions were carried out using primers that provided products centered around nucleotides 47, 79, and 491 (encoding amino acids 16, 27. and 164, respectively) of the b2AR open reading frame. These products were then hybridized to primers that were immobilized to 96-well plates, and a primer extension reaction performed using fluorescein- and biotin-labeled ddNTPs. Incorporation of labeled nucleotide was detected by colorimetry at 405 and 620 nM.
Cardiopulmonary exercise testing (CPX): Patients undergoing CPX maintained all medications. Exercise was performed on a treadmill (Medgraphics) using a Modified Naughton protocol (1 mile per hour (mph) with 0% grade initially; workload was increased every 2 minutes to a symptomatic maximum (with the highest workload being 3 mph with 17.5% grade). Patients unable to exercise on a treadmill due to arthritis or other reasons (35 patients) were exercised on an upright bicycle ergometer (MedGraphics) (0 watts initially increasing by 10 watts per minute to symptomatic maximum). During exercise, respiratory gases were measured with a MedGraphics CPX metabolic cart equipped with O2 and CO2 analyzers and a turbine volume transducer calibrated within 1 hour of testing.
Minute oxygen consumption (VO2; ml/min), minute carbon dioxide production (VCO2, ml/min), and minute ventilation (VE; l/min) were measured with a breath-by-breath respiratory gas analyzer. The respiratory gas exchange ratio (RER) was calculated as VCO2/VO2. Minute VO2 was normalized for body mass by dividing by patient's weight in kg (VO2; ml/kg/min). Pertinent exercise parameters included peak VO2 (VO2 at peak exercise with RER > 1.0), VO2 at anaerobic threshold (AT) determined by the V-slope method, VO2 at RER = 1, VO2 ÷ predicted maximum based on Wasserman's tables x 100 to normalize for age and gender (%Max VO2), AT ÷ predicted maximum x 100 to assess effort level, metabolic equivalents (METS) at peak exercise, and exercise time 2.
Invasive Exercise Hemodynamic Testing: 12 patients (6 with Ile164 and 6 with Thr164) were enrolled in an invasive assessment of exercise hemodynamics. These patients held ACEI, ARB, and calcium channel blockers for 48 hours; none were being treated with beta blockers. Digoxin and diuretics were maintained; hydralazine was substituted for the 48 hour period. A Swan-Ganz catheter (Baxter CCO/sVO2) capable of continuous monitoring of the mixed venous (pulmonary artery) saturation was inserted through the right internal jugular vein and positioned in the pulmonary artery. A short polyethylene catheter was inserted into the radial artery. All patients were deemed to have compensated heart failure at the time of the study. Thirty min after instrumentation, the patient mounted a bicycle, the hemodynamics equilibrated for 5 min, and baseline hemodynamic measurements were made using a Gould Window-Graf 4 channel data acquisitions and analysis system: pulmonary arterial (PA) systolic, PA diastolic, and PA mean; pulmonary capillary wedge pressure (PCWP); central venous pressure (CVP); and systemic arterial (AP) systolic, AP diastolic, and AP mean. The arteriovenous oxygen difference was calculated as (arterial - venous O2 saturation) x (1.34 mL O2/g hemoglobin) x (hemoglobin concentration). Cardiac output (CO) was calculated by use of the Fick equation with arterial saturation from a pulse oximeter and PA saturation measured by the sVO2 catheter.
The patients exercised to a symptomatic maximum using a bicycle ergometry protocol (0 watts for the first minute, increasing the workload by 10 watts per minute). Respiratory gas and heart rate measurements were made continuously; hemodynamic measurements and arterial and PA saturations were recorded every 2 min of exercise, at peak exercise, and at 1, 3, and 5 min in recovery.
Statistical methods: The following exercise parameters were considered a priori to be potentially associated with b2AR genotype: peak VO2, % predicted VO2, the VO2 at AT, exercise time, and METS. Due to the relatively low frequency of the Ile164 allele in the normal and heart failure populations 3, we paired in a blinded fashion all 18 patients heterozygous for the Ile164 polymorphism with 18 randomly assigned patients with similar characteristics who were homozygous wild-type (Thr) at this position. The two groups were matched, except as noted, for NYHA functional class (NYHA FC), sex, age, race, and etiology of heart failure. (One patient was not matched for race, and one patient was not matched for etiology of heart failure.) We compared CPX parameters between these two groups using paired t-tests. Paired differences were adequately modeled by a normal distribution, so no transformation of the data was necessary. Potential confounders including the b2AR genotype at position 16 and 27 as well as left ventricular ejection fraction (LVEF), right ventricular ejection fraction (RVEF), and medication use, were examined using multiple regression models.
The frequent aminoterminal polymorphisms at positions 16 and 27 were analyzed in isolation as well as in combination. Thus, the results of CPX between all patients homozygous for Arg16 were compared to those homozygous for Gly16, without regard to polymorphisms at position 27. Similarly, results from individuals who were homozygous for Gln27 vs Glu27 were compared. The eighteen patients with Ile164 were excluded from this analysis. Multiple regression models were used for the analysis whereby potential confounders including age, sex, race, NYHA FC, etiology of heart failure, LVEF, RVEF, and medication use, were added to the model. We compared the results of CPX between the four combinations of homozygous genotypes at positions 16 and 27 also using multiple regression models with the same confounders.
The results of hemodynamics and exercise variables of the patients tested with invasive exercise testing were analyzed using paired t-tests. Pertinent hemodynamic and exercise variables were analyzed by paired t-tests using the percent change from baseline.
References
1. Liggett SB. Pharmacogenetics of relevant targets in asthma. Clin and Exp Allergy. 1998;28 (Suppl 1):77-9.
2. Weber KT, Janicki JS, McElroy PA, Reddy HK. Concepts and applications of cardiopulmonary exercise testing. Chest. 1988;93:843-7.
3. Liggett SB, Wagoner LE, Craft LL, Hornung RW, Hoit BD, McIntosh TC, Walsh RA. The Ile164 b2-adrenergic receptor polymorphisms adversely affects the outcome of congestive heart failure. J Clin Invest. 1998;102:1534-9.
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