Study setting
The study—a parallel-group randomized, double-blind (placebo/drug but not to exercise type) clinical trial—is approved by the local ethics committee (#5678/19) and it will be conducted at Instituto de Cardiologia do Rio Grande do Sul/Fundação Universitária de Cardiologia (study site). All eligible individuals must read and sign a free and informed consent (supplementary material) before entering the study. The study protocol is being developed following the principles of the Declaration of Helsinki. All study information is confidential; volunteers’ names will be kept confidential and all data obtained in the study will be used for academic purposes only. Research data will be stored for five years and then discarded according to the guidelines of CNS Resolution No. 466/12. Finaly, any adverse effect during the research will be reported to the local ethics committee that will have access to the adverse results and will make the decision in relation to terminating the study, if aplicable.
Trial Status
The study is registered on “www.clinicaltrials.gov” (ID: NCT04371757), as follows: Last update posted on May 1, 2020; Estimated study start on April 30, 2020; Estimated primary completation on December 20, 2020; Estimated study completation on December 23, 2021. At moment the recruitment status is “Recruiting”.
Eligibility criteria
Recruitment will be through on-site screening, media postings, and patient databases. Thus, hypertensive male and female individuals aged 35 to 55 years receiving health care at the study site, using anti-hypertensive drugs, who do not engage in regular physical exercise (≥2 sessions per week) are eligible to participate. Exclusion criteria are individuals with diabetes mellitus; chronic renal failure; body mass index (BMI) ≥35 kg/m2; coronary artery disease; heart failure; users of beta-blockers and/or alpha-blockers; smoking; lower limb injuries that prevent them from engaging in the study intervention; and females aged 35 to 55 with menopause.
Sample size
The sample size is calculated according to Atkinson et al. (26). In their study, they compared endothelium-dependent vasodilation (by FMD) after a single session of moderate-intensity AE for 30 minutes in healthy individuals receiving an α1- adrenergic blocker (prazosin 0.05 mg/kg-1) or placebo and reported a difference in means of 3.1% with standard deviations (SDs) of 1.13% for placebo and 1.91% for prazosin. For finding a similar difference in FMD (3.1%) between these two conditions (α1-adrenergic receptor block versus placebo) after our protocol’s exercise session (AE, RE and CE) considering α = 0.05 and 80% power, a sample of five volunteers per group (n = 15) is required.
For a comparison of FMD measurements among exercise types (AE, RE and CE), our sample calculation is based on pre-exercise SD (1.18%) and post-exercise SD (1.91%) for the prazosin group as described by Atkinson et al. Thus, to find a difference in FMD of 1.62% considering α = 0.05 and 80% power, a sample of 10 individuals per group (n = 30) is required. Assuming a 30% loss, a sample of 39 individuals would be required (n = 13 per group).
Randomization and blinding
Randomization of the study interventions (exercise groups) as well as assignment to intervention conditions (either an α1- adrenergic blocker or placebo) are carried out using computer program (www.randomization.com) with a coded numeric distribution. Allocation concealment is guaranteed; the random allocation of volunteers is kept in an inaccessible place and researchers will not have a priori knowledge of the intervention assignment to each volunteer. Randomization is carried out by an independent researcher not involved with the study.
Considering the volunteers’ age group (35 to 55 years) and that comparison analyses (primary and secondary outcomes) involve testing for baseline differences, we are conducting simple randomization divided into two steps: (1) by exercise type (AE, RE or CE) and (2) the order of condition (either placebo or α1-adrenergic blocker). All volunteers are blinded to the intervention allocation and will only have access to this information after a baseline evaluation. They are also blinded to the intervention condition (either placebo or α1-adrenergic blockade). The researchers responsible for the analyses of data are blinded to the intervention allocation and condition to minimize potential measurement bias.
Evaluation and interventions
Volunteers are selected based on information from their medical records. They are first contacted through a telephone call to ascertain whether they fit the inclusion criteria and do not meet any of the exclusion criteria. They are then pre-selected and invited to participate in the study. Those who agree to participate are scheduled their first visit (visit 1) at the study site.
Eligible individuals go through a four-step protocol as described below. Figure 1 shows the detailed flowchart of the study; Figure 2 summarizes the recruitment, study interventions and assessments; and Figure 3 describes the study intervention (exercise and condition).
Since the study is designed to assess the acute effects of different types of exercise types under the condition of α1-adrenergic blockade or placebo, female volunteers are to undergo evaluations and exercise sessions during the luteal phase of the menstrual cycle.
Step 1: interview, anthropometric assessment and exercise test
Visit 1 takes place at the Laboratory of Clinical Investigation (LCI) at the study site. All inclusion and exclusion criteria are ascertained and the principal investigator (GW) will ask volunteers to sign a free and informed consent form. All research data collected will be stored in an appropriate safe space at the study site that is accessible to the principal investigator (GW) only.
They are then administered a medical questionnaire the International Physical Activity Questionnaire – long version (IPAQ, http://www.ipaq.ki.se). The volunteer is asked to remain in a seated position in a quiet room for 5 minutes before their blood pressure (BP) is measured (OMRON™ HEM-714INT device, Tokyo, Japan); three recordings are taken 1 minute apart on the arm with the highest values. Those who meet all inclusion criteria undergo an anthropometry assessment and a pre-scheduled exercise stress test.
The anthropometry assessment takes place at the study site. It includes measurements of total body mass (measured with appropriate one-piece [males] and two-piece clothing [females] in a standing position barefoot with feet together at the center of the scale); height (measured in a standing position with feet together, the head in the Frankfort plane with the stadiometer on the top of the head and readings taken while breathing in); waist circumference (measured in a relaxed standing position with arms folded across the chest with the measuring tape placed around the abdomen at narrowest point between the lower costal margin and the upper part of the iliac crest, the reading is made in the front perpendicular to the longitudinal axis of the body) (29).
The exercise stress test is performed on a treadmill (Inbramed, Porto Alegre, Brazil) using the Bruce Protocol and following the Brazilian Society of Cardiology guidelines (30). In a testing room at the study site a qualified technician and a cardiologist conduct the stress test followed with measurements of maximum oxygen consumption, respiratory parameters and maximum heart rate (HR) (ErgoPC 13, MICRIMED, Brasília, Brazil). Aerobic exercise is to be prescribed according to these measurements. The volunteers are then released by the cardiologist. They are contacted by phone to schedule visit 2.
Step 2: blood collection, assessment of arterial stiffness and maximum strength test
On visit 2 the volunteers are asked to come to the study site after a 12-hour overnight fast for blood collection and biochemical analysis (blood count, fasting blood glucose, HbA1c levels, triglycerides, total cholesterol, low-density lipoprotein cholesterol [LDL-c] and high-density lipoprotein cholesterol [HDL-c], creatinine and ultra-sensitive polymerase chain reaction [PCR] assay). LDL-c levels and glomerular filtration rate (GFR) are calculated as detailed below.
a) LDL-C = (total cholesterol – HDL-C) – (triglycerides/5);
b) GFR (mL/min/1.73 m2) = 175 x creatinine –1.154 x age –0.203 x 1.212 (for blacks) x 0.742 (for females) (31).
Volunteers are then offered a standard snack and asked to rest in a dark, quiet room at 23–24°C for 10 minutes. Arterial stiffness is assessed by carotid-femoral pulse wave velocity (PWV) (32) using the Complior Analyse (ALAM Medical, Paris, France). The straight distance between the two arterial sites is measured with an infantometer and data on BP, height, total body mass and distance between carotid and femoral artery sites are entered into the measuring device software for PWV calculation (32).
The volunteers then undergo the one-repetition maximum (1-RM) test to assess lower limb maximum strength (33) in a proper exercise room at the study site. They first learn the exercise techniques and are instructed on how to perform knee extension, knee flexion, leg pressure and plantar flexion exercises on a guided weight machine (Movement Perform W8; São Paulo, Brazil). Warm-up consists of 10–12 repetitions, 30% of 1-RM (2 concentric: 2 eccentric phases controlled by a metronome). After a rest of 3-5 minutes, they start the 1-RM test. It is interrupted to adjust the load when they are able to perform more than one repetition or when they are not able to lift the load. A 5-minute rest is allowed before a new attempt is made. The 1-RM is determined when the volunteer performs one complete movement (33). Their BP is measured 5, 10 and 15 minutes after the end of the test. The study interventions are scheduled afterwards.
Steps 3 and 4: α1-adrenergic receptor blockage, assessment of endothelial function, autonomic nervous system activation and exercise intervention
α1-adrenergic receptor blockage: α1-adrenergic blockage is used to assess the effects of sympathetic nervous system (SNS) activation on FMD in response to exercise as described in the literature (26). Since prazosin is no longer available in Brazil, its analogue doxazosin is used in this study (34). Briefly, 90 minutes before the start of the exercise intervention, the volunteers are asked to take a tablet containing either placebo (microcrystalline cellulose) or an α1-adrenergic blocker (doxazosin 0.05 mg.kg-1 of body weight) and to lie down and rest. This dose of an α1-adrenergic blocker has been reported as being able to block 80% of α1-adrenergic receptors in studies of sympathetic activation and vascular responses (35-37).
An independent researcher blinded to the study intervention gives a tablet to the volunteer on the day of the exercise session according to previous random assignment.
Assessment of endothelial function: endothelial function is assessed by FMD as described elsewhere (38, 39). In a quiet, dark room with controlled temperature (23–24°C) at the study site, a qualified evaluator blinded to the intervention condition simultaneously measures blood flow velocity and baseline brachial artery diameter on the volunteer's right arm. With the volunteer in supine position, arm extended and across the trunk at angle of ~40° after a 10-15-minute rest, measurements of baseline BP (left arm), resting HR, blood flow velocity and brachial artery diameter are taken. They are to be evaluated 10 minutes before and 10, 40 and 70 minutes after the exercise session.
For the assessment of FMD of the brachial artery, the volunteers are asked to come to the study site after 6-hour fasting except for anti-hypertensive medication. A rapid deflation cuff (Incoterm ™ EC500; Porto Alegre, Brazil) is placed around the forearm 5 cm distal to the antecubital fossa and B-mode artery images are obtained at the distal third of the arm with a linear multi-frequency transducer (12 MHz) connected to a high-resolution Doppler ultrasound system (Esaote MyLab™ 70 XVision; Genoa, Italy). The positions of the ultrasound probe and the cuff are marked on the skin so that they can be reproduced during the study. The position of the arm is photographed and the distance between the transducer and the antecubital fossa is measured so that the same can be reproduced in the second exercise session. The evaluator aligns the cursor of the sample volume to the width of the artery at an insonation angle of 60°. Baseline diameters are recorded for 1 minute and the forearm cuff is inflated to 50 mmHg above systolic blood pressure (SBP) for 5 minutes. Images are recorded 30 seconds before cuff deflation and for 3 minutes after cuff deflation. Real-time Doppler ultrasound images are recorded and stored into an USB video card (Dazzle; Taiwan) for offline data analysis. To minimize bias blood flow velocity and brachial artery diameter are evaluated via an edge-detection and wall-tracking software program (CardiovasculareSuit ™; Pisa, Italy). From this synchronized brachial artery diameter and blood flow velocity data, blood flow (the product of lumen cross-sectional area and Doppler velocity) is calculated at 30 Hz. Forearm peripheral vascular resistance is determined as mean BP divided by blood flow. FMD is calculated as the percentage change in peak diameter after cuff deflation from baseline. Time to peak diameter is calculated from the point of cuff deflation to peak artery diameter (39). The shear rate is calculated (4 x mean blood velocity / artery diameter) as the area under the curve between the point of deflation and maximal dilation (12).
Evaluation of autonomic nervous system activation: ANS activation is evaluated through heart rate variability (HRV) and blood pressure variability (BPV) as described by Atala et al. (40). Data are collected through continuous non-invasive measurements of pressure waves using digital photoplethysmography (Finometer®, Finapres Medical System BV, Netherland). A cuff device is worn on the middle finger of the right hand coupled to an analog-to-digital converter (AT / MCA-CODAS, DATAC Instruments Inc., Akron, Ohio, USA). Continuous signal sampling acquisition is obtained at 1,000 Hz after resting (lying down for 10 minutes) before FMD. The readings are made for 10 minutes before forearm cuff inflation for all volunteers either on the placebo or α1-adrenergic blocker groups.
Data are recorded into BeatsScope® (Smart Medical, Gloucestershire, United Kingdom) and LabChart® (ADInstruments, Sydney, Australia) software programs for data analysis of histograms showing BPV and pulse interval series (RR interval) for power spectrum analysis (HRV). All heartbeats are identified through a specialized algorithm using MAT-LAB MT (MATLAB 6.0, Mathworks, USA) with automatic detection of systolic and diastolic pressure wave events. Pulse intervals (PIs) are calculated as the difference between start and end points of the cycle (PI = time point 1 – time point 0). For the time-domain analysis of HRV and BPV data, such as spectrum composition (fast Fourier transform), the CardioSeries® software program is used (www.danielpenteado.com/cardioseries). This analysis requires the removal of transient and discrepant elements and interpolation of consecutive fragments of a time series. The following spectral frequencies are evaluated: very low frequency (VLF, 0.007-0.04 Hz); low frequency (LF, 0.04-0.15 Hz); high frequency (HF, 0.15-0.4 Hz); and total power (TP, VLF + LF + HF). Spontaneous baroreflex sensitivity (SBE) is inferred by the square root of (LF / HRV m2) / (LF / BPV m2) (40).
Reproducibility of FMD: the reproducibility of FMD measurements by the study evaluator (GW) (who has performed more than 150 assessments) was assessed according to current recommendations (38, 39). Eleven healthy individuals (6 males and 5 females aged 38.4 ± 13.9 years) were tested in the morning (~9 h) after a 15-minute rest in supine position (38) from October 23 to November 29, 2019. They were evaluated at two time points one hour apart. Reproducibility for each time interval between pairs of values was expressed as the coefficient of variation (CV) of a single measurement, defined as the standard deviation of the difference between paired values divided by the mean and divided by √2) (38, 41). The mean CV for baseline diameter of healthy volunteers was 1.4 mm and mean CV for FMD was 11.0%. The recommended CV values for diameter (mm) are <2% and for FMD are <15% (38).
The exercise sessions are performed in two afternoon visits (1 pm) in random order (placebo or α1-adrenergic blocker). The volunteers are asked to fast over 6 hours before the session. Sessions are held 5 to 7 days apart and include AE on a cycle ergometer; RE; and CE (AE + RE) as described elsewhere (33).
Aerobic exercise protocol: AE session is performed on a horizontal cycle ergometer (Movement BM4500 Pro, São Paulo, Brazil) and consists of warm-up (5 minutes) followed by moderate-intensity AE (60% HR reserve) for 40 minutes. The volunteers are monitored using a heart rate monitor (POLARTM RS800CX RUN, Helsinki, Finland) and a subjective effort scale (Borg scores 6 to 20). BP and HR measures and Borg scores are recorded at the start, every 5 minutes during the session and up to 15 minutes after exercise.
Resistance exercise protocol: RE consists of lower limb exercise (knee extension, knee flexion, leg pressure and plantar flexion exercises) on a guided weight machine (Movement Perform W8; São Paulo, Brazil), 4 sets with 12 repetitions, 60% of 1-RM (2 concentric: 2 eccentric phases controlled by a metronome). There is a 90-second rest between sets and exercises and the session lasts 40 minutes. BP, HR and Borg scale are recorded at the start and end of set 4 of each exercise as well as up to 15 minutes after exercise. While performing lower limb strength exercises, the volunteers are not be allowed to hold the hand rests, and an adjustable seat belt is used to stabilize the hip during knee extension and knee flexion (33).
Combined exercise protocol: CE consists of AE for 20 minutes + RE for 20 minutes (two sets of each exercise) as described elsewhere. Similarly, BP, HR and Borg scores are recorded at the start and end of RE as well as at the start, every 5 minutes and up to 15 minutes after AE.
Additional information: the stress exercise test, 1-RM test or exercise session are not started when resting BP is greater than 160 mmHg (SBP) and/or 105 mmHg (DBP) (42). If BP remains above the recommended levels at a second check the volunteers are referred to the study cardiologist for evaluation. They will be excluded from the study if the cardiologist determines that they cannot participate.
Data management: to ensure data security and data integrity, all research data will be entered and stored in the REDCap platform at the study site overseen by one of the contributors (LFCB). Only the authors of this study will have access to it. Each participant has the right of access to their own research results.
Statistical analysis
The Shapiro-Wilk is used to test the normality of data. Values are presented as means ± standard deviations or medians and 95% confidence intervals (95% CIs) according to the distribution of variables. A multivariate analysis of data is performed using the generalized estimation equations (GEE) for repeated measures with Bonferroni's post-hoc. Pearson’s and Spearman’s tests are used to assess potential correlations between variables according to their distribution. Statistical analyses are carried out using the Statistical Package for Social Sciences (SPSS) 24.0. A level of significance of <0.05 is set for all analyses.