The purpose of this original research is to characterise the effect of exercise training on angina status, anxiety, depression and the peripheral blood transcriptome, in patients with stable angina and coronary artery disease. Gene expression outcomes aim to provide an insight into the mechanism of the beneficial effects of exercise training in coronary artery disease and on collateral blood vessel development which might in turn provide evidence as to the impact upon cardiac morbidity in these patients.

This is a prospective randomised control comparison study. After completion of a baseline assessment, participants will be randomly assigned to either a structured exercise or a usual care control group. Patients will be randomised by an independent researcher via code numbers using nQuery software (nQuery Advisor 6.01, Statistical Solutions, USA). The randomisation sequence will not be not disclosed to the researcher responsible for the day-to-day running of the trial until patients have completed baseline assessments. It is not possible to blind participants or investigators to treatment allocation, but data collected will be blinded prior to analysis to eliminate bias. This research will be conducted in the Clinical Research Facility (CRF) at the Northern General Hospital, Sheffield, UK and the Centre for Sport and Exercise Science at Sheffield Hallam University, UK. The researchers involved are experienced clinicians and scientists. Ethical approval was obtained for this study through Bradford Research Ethics Committee.

Patients with stable angina will be identified from cardiology clinics at Sheffield Teaching Hospitals NHS Foundation Trust, UK. Written, informed consent will be obtained from each potential volunteer before baseline assessment.

1) Class I to III angina pectoris (classified according to the Canadian Cardiovascular Society [CCS]) with documented myocardial ischemia or coronary artery disease on angiography.

2) Ability to read and speak English to a level allowing satisfactory completion of written questionnaires and to understand instruction during the exercise programme.

3) Age 30-80 years of either sex.

1) Acute coronary syndromes or recent myocardial infarction (< 2 months).

2) Left main coronary artery stenosis > 25% or high-grade proximal left anterior descending artery stenosis.

3) Known reduced left ventricular function (ejection fraction < 40%).

4) Significant valvular heart disease.

5) Insulin-dependent diabetes mellitus.

6) Occupational, orthopedic, and other conditions that preclude regular exercise.

7) Patients who's ECG prevents interpretation of an exercise test (LBBB, RBBB, pacemaker implantation).

8) Patients already performing greater than 30 min continuous exercise three times weekly (self-reported).

Patients randomised to exercise training will attend an Exercise Science facility based at Sheffield Hallam University. This facility has a range of aerobic exercise equipment, including cycle ergometers, treadmills and cross-trainers. The intervention will comprise sessions of aerobic exercise training for 12 weeks total, up to three times weekly. An interval training regimen will be used, incorporating three minutes of exercise followed by one minute active recovery for 30 minutes total. Heart rate will be monitored throughout; ensuring patients do not exceed 85% of predicted maximum heart rate or 10 b.m^-1 below the angina threshold [¹]. Individuals who were taking medication that alters maximum heart rate (e.g. beta blockers) will have their exercise intensity monitored using the Borg Ratings of Perceived Exertion [RPE] scale [¹⁸]. Exercise intensity in this instance will be set at that point just below the onset of angina. Each exercise session will be preceded by 10-15 minutes of light intensity/gentle mobility exercise and followed by a 10-15 minute period of cool-down activities. If angina is provoked during an interval, exercise will halt until angina subsides, either with rest or GTN treatment. If angina continues longer than 5 min the principal investigator or another clinically qualified investigator will be contacted to assess the patient. Once angina has subsided, blood pressure will be checked, and the intervals will restart at a heart rate 10 b.m^-1 below the level which caused the angina symptoms. Subsequent sessions may exceed this level of intensity if the angina threshold is found to have increased (due to exercise training adaptation or alteration of medication).

Hence, each supervised exercise session will last 50-60 minutes in total and patients will be encouraged to attend as many sessions as possible up to three times weekly over the 13 week period. Exercise frequency, intensity, duration and modality, as well as rate of progression, will be logged. After completing the first six weeks, participants will have a week break in training (during which visit 3 takes place), so that acute gene expression changes in response to exercise do not confound the analysis.

Outcome measures will be performed at CVBRU at baseline, midpoint (week 7), endpoint (week 14), and 6 month follow up. At assessment visits patients will have fasted for at least 12 h. On arrival, the following blood tests will be obtained; full blood count (FBC), fasting glucose (FG), fasting lipids (Lip), and peripheral blood RNA (RNA). The patients will then be offered a light meal. They will complete two questionnaires: The Seattle Angina questionnaire (SAQ), and the Beck Depression/Anxiety Inventory (BDI). The questionnaires are well established, widely used and well validated tools for assessing these outcomes [^19-21]. In addition a full drug history will be obtained (DH). Height and weight will also be recorded (H&W). Following completion of these questionnaires, an exercise tolerance test will be performed (ETT) to give an indication or aerobic exercise tolerance and assess angina threshold. RNA samples (stored in a HTA licensed Biorepository) will be extracted and used to bind to Affymetrix Human Genome HU133 microarrays to assess whole genome peripheral blood gene expression. See Figure 1 for study flow diagram.

As our primary outcome measure is gene expression in whole blood, a priori sample size calculations are not possible. Based on the pilot study guidelines of [²²], we will recruit 30 volunteers for each group, making a total sample size of 60. The results of this pilot will provide information on recruitment, compliance and attrition, and will identify logistical constraints that might limit the number of potential recruits. Variability data for the outcome measures and estimates of the treatment effect could be used to inform design a subsequent larger-scale randomised controlled trial, if post hoc analysis reveals this initial study to be underpowered.

Outcomes will be compared at each assessment point using a mixed-design factorial ANOVA. Where any significant difference existed at baseline between groups, ANCOVA procedures will be implemented, with baseline values used as the covariate [²³]. Statistical significance will be set at p < 0.05. Bivariate relationships between exercise tolerance and other outcome variables will be evaluated using Pearson's correlation analysis. Data will be analysed using SPSS (SPSS U.K. Ltd, Woking U.K.). The data will be analysed on an intention to treat basis with all analyses including missing data by imputing change across time to be zero. Parametric data will be presented as mean differences of change scores between time points, with 95% confidence intervals and effect sizes (calculated as Cohen's d i.e. mean of the treatment effect divided by the standard deviation of the controls). Gene expression data will be analysed using our previously published probabilistic method for analysis of microarray data that improves probe level sensitivity [^24-26].