Exercise Training & Statins for Cardiovascular Health

Sublingual Glyceryl Trinitrate (GTN): Following 10 minutes of supine rest, endothelium independent vasodilator function will be assessed simultaneously in the brachial and carotid artery. A 1-minute baseline recording of the brachial and carotid artery will be acquired on the same high-resolution ultrasound device before sublingual administration of glyceryl trinitrate (GTN; 400µg). GTN is a nitric oxide donor that will cause the arteries to vasodilate allowing a short-lived increase in blood flow. Both the brachial and carotid artery will be imaged for 10-minutes following the administration of GTN and peak diameter and blood flow will be recorded. GTN has been used for many decades in clinical and research work in this and other populations.

Cold Pressor Test: Following 15 minutes supine rest, carotid artery reactivity (CAR) to a sympathetic stimulation produced by the cold pressor test (CPT) will be measured. The same ultrasound probe will be repositioned onto the participant's neck and an image of the carotid artery will be recorded continuously for one minute before and then during 3 minutes of hand-to-wrist submersion in ice cold water (4 °C). After the 3 minute CPT, the participant will then remove their hand from the cold water and we will continue to image the carotid artery for an additional 3 minutes. Peak carotid artery diameter and blood flow velocity, and the time taken to reach these peaks during the CPT will be recorded. This procedure has been shown to be reliable and well tolerated in this population.

Carotid - femoral pulse wave velocity (CF-PWV) will be recorded following 15 minutes of supine rest via applanation tonometry. This measure is the accepted gold standard for non-invasive measurement of arterial stiffness and has been previously validated in this population. The arterial wave form will be recorded at the wrist, neck and groin using a high fidelity micromanometer tipped probe and gated to a 3-lead ECG for measurement of PWV via the SphygmoCor system (SphygmoCor, AtCor-Medical, Sydney, Australia). Pulse wave analysis (PWA) will also be used to measure aortic pressure using the SphygmoCor system.

Participants will then return to the assessment bed and lay supine for 10 minutes. Following this, participants will perform a carbon dioxide breathing challenge. The protocol will involve the participants breathing a fixed gas mixture of otherwise normal atmospheric air that has a mildly elevated fractional concentration of carbon dioxide (6%). This technique has been routinely used within this population and published guidelines will be followed. Normal atmospheric oxygen content is maintained throughout. Participants will be fitted with a face mask covering their nose and mouth with a one-way valve to prevent rebreathing. An elevation in the fractional concentration of carbon dioxide will be applied for 6 minutes and cerebrovascular blood flow will be measured continuously throughout this protocol, as per published guidelines. This procedure enables the maximal dilation capacity of the cerebrovascular blood vessels to be assessed.

Participants will perform a cyclical squat-to-stand protocol. Participants will start in a standing position and then adopt a squat position where the back of their legs are at a ~90◦ angle. This manoeuvre will be repeated and performed at frequency of 10 seconds of squatting, followed by 10 seconds of standing. The duration of the protocol will be 5 minutes. This technique has been used routinely in older populations and is well tolerated and validated.

After 15 minutes of rest, participants will perform a 10 - 15 minute bout of moderate intensity exercise at 50% predicted heart rate reserve (HRR) on a cycle ergometer.

Localised changes in cerebrovascular blood flow will be assessed during neuronal activation. The occipital lobe will be activated using a visual stimulus of a flashing checkerboard and participant's will then perform a series of short and repetitive sensory tasks. Sensory stimulation tasks will involve a simple cyclical eye opening and closing protocol, reading a short passage from an non-emotive text book and performing a simple visualisation cognitive task. This technique is frequently used in older populations and a standardised and validated protocol will be adopted.

Cognitive Function Assessment: Participants will be asked to complete the Montreal Cognitive Assessment. This paper-based assessment has been validated in this population and examines short term memory, visuospatial abilities, executive functions, attention, concentration and working memory, language and orientation to time and place. The test is scored based on a minimum score of 0 to a maximum score of 30, where a score of >26 is regarded as 'normal cognitive function' and a score of <26 indicative of mild cognitive impairment.

A 30ml blood sample will be taken from a vein in the arm and analysed for markers of cardiovascular risk. Blood samples will be analysed using the Randox Dayton+ analyser with standard proprietary reagents for total cholesterol, high-density lipoprotein, triglyceride (all lipid measures in mmol). Low-density lipoprotein will then be calculated using according to the Friedwald formula (mmol).

A 30ml blood sample will be taken from a vein in the arm and analysed for markers of cardiovascular risk. Blood samples will be analysed using the Randox Dayton+ analyser with standard proprietary reagents for glucose and insulin assayed using commercially available radioimmunoassay (both measures in mmol). Using fasting glucose and insulin concentrations, we then will calculate steady-state beta cell function and insulin resistance via the homeostasis model assessment.

A 30ml blood sample will be taken from a vein in the arm and simultaneously analysed for pro-inflammatory cytokines; Interleukin-1α, Interleukin-1β, Interleukin-6, Interleukin-8, Interleukin-10 Interferon-γ and Tumour Necrosis Factor-α,(all measured in pg/ml) using high sensitivity multiplex enzyme-linked immunosorbent assays (ELISA).