Identifying novel cardiovascular markers of exercise intolerance in adults living with atrial fibrillation.

This study will involve 2x 2-3 hour face-to face sessions at the Deakin's Institute for Physical Activity and Nutrition (IPAN) laboratory. Each session will be conducted at least greater than or equal to 48 hours apart with all measurements to be collected by study researchers. The first session will be a familiarization session provide the participant of an understanding and awareness of how the ultrasound imaging will be conducted, the set-up on the cycle ergometer and exercising on the ergometer and performing as well as conducting the strength based tests. In addition a fasting blood test (10 hour fast) at an Accredited Laboratory and dual-energy x-ray absorptiometry to assess body composition will be also be collected. This will approximately take 2.5 hours. The final visit will involve performing contrast-enhanced ultrasound imaging and a graded exercise test to determine the skeletal muscle microvascular blood flow response in participants. A 2 hour fasting period is required prior to testing on this visit and avoiding caffeine for greater than or equal to 6 hours prior to testing. Contrast-Enhanced Ultrasound Imaging (3 hours): To determine the skeletal muscle microvascular blood flow, contrast-enhanced ultrasound will be performed. The technique involves intravenous infusion of a commercially available contrast agent (Definity, Lantheus Medical Imaging) composed of haemodynamically inert, perflutren lipid microspheres sufficiently small in size to perfuse capillaries. Definity is indicated in diagnostic ultrasound imaging – both liver/kidney assessment (lesion characterisation) and echocardiography (chamber opacification, endocardial border definition and regional wall motion assessment) (Therapeutic Goods Administration approved). A/Prof Keske and her collaborators at the University of Virginia have used Definity for assessment of skeletal muscle perfusion in healthy, insulin resistant and type 2 diabetes humans to assess microvascular responses in skeletal muscle. A standard ultrasound machine will be used to image the vastus lateralis muscle (thigh muscle group) in cross-section. Depth, gain and focus will be optimised for each participant and maintained during repeated imaging sequences. A contrast agent suspension [one vial of Definity (1.5ml) will be added to 20-30 mL of saline] will be infused at 1.5–2.2mL/min (infused rate based on body weight) using a syringe pump. Images will be acquired using ultrasound and analysed using computer software. The acoustic signal generated from the contrast agent microspheres will be measured and is directly proportional to the number of capillaries open/active and volume of blood in the microvascular system. After a high energy pulse of ultrasound, all contrast agent microspheres within the ultrasound beam are destroyed. The rate of contrast agent microspheres reappearance within the ultrasound beam provides an indication of microvascular blood velocity which, combined with microvascular blood volume measurements, is used to determine total microvascular blood flow (i.e., microvascular function). Measurements will be collected before the graded exercise test, immediately following the graded exercise test and 60-mins post graded exercise test. Other ultrasound imaging (to be captured simultaneously as contrast enhanced imaging of the thigh): Femoral arterial diameter and blood velocity will be measured non-invasively using a high frequency L12-5 linear array transducer interfaced to the ultrasound system. Intra-cranial blood velocity recordings of the middle and posterior cerebral arteries will be measured using non-invasive transcranial Doppler. Extra-cranial blood flow measures of the internal carotid and vertebral arteries will be collected using a high frequency linear array transducer interfaced to the ultrasound system. Recordings of middle cerebral artery velocity and posterior cerebral artery velocity will be made at a frequency of 100Hz, at recommended depths, described in TCD guidelines. Diameter is assessed using 2D ultrasound and velocity assessed by Doppler ultrasound. Artery blood flow (ml/min) is calculated as pr2 x mean velocity x 60. Where radius (r) is cm and mean velocity is cm/s. Graded Exercise Test (within the 3 hours of contrast-enhanced ultrasound scanning): Participants will then be asked to sit on an upright cycle-ergometer and undergo one graded exercise test. Aerobic/exercise capacity (VO2 peak) will then be assessed during a symptom-limited cardiopulmonary exercise test. After a 5-minute rest period participants will begin cycling (approximately 60 revolutions per minute [RPM]) at a workload of 20-W which will increase stepwise at 10-20-W increments every 2 minutes thereafter until the participant is either exhausted and cannot maintain a cycling cadence of greater than or equal to 60 revolutions per minute, the participant wishes to stop, or if clinical signs or symptoms of metabolic or cardiorespiratory abnormalities appear. Expired respiratory gases will be collected through a breath-by-breath pneumotach system connected to gas analysers. The breath-by-breath data will be averaged for each 15 sec interval, and the mean values for oxygen (VO2), carbon dioxide (VCO2), ventilation (VE), and respiration exchange ratio (RER) for each interval will be calculated. Each graded exercise test will take between 8-12 minutes. Electrocardiography (ECG) will be recorded continuously, and blood pressure measured during, throughout, and after exercise. An ultrasound system will be used before exercise, immediately after exercise, and 30 minutes after exercise, to determine cardiac function, thigh arterial blood flow, and muscle microvascular blood flow responses to maximal exercise. A venous blood sample will be taken before exercise, immediately after exercise, and 15 and 30 minutes post-exercise. Samples will be analysed for lactate, oxygen saturation and blood glucose levels. 1 repetition maximum testing (15 minutes): The participant will first complete progressive warm-up sets at a light to moderate-intensity load (rating of perceived exertion [RPE]: 10-12/20) of 5 repetitions, then 3 repetitions. The load selection will be completed by the supervising Accredited Exercise Physiologist which will be progressed based on the participant’s RPE. Following the warm-up, the weight will then be increased for an attempt to determine the participant’s 1RM. If the participant can successfully complete the lift with safe technique and reporting they could complete another repetition, the weight will then be increased again by the Accredited Exercise Physiologist based off the RPE and the repetitions in reserve (ie. how many more repetitions they think they could complete at that load). A 3-5 minutes rest will be provided between attempts, with the aim to obtain the 1RM within 3 attempts. The maximum weight attempted with safe and correct technique without being able to be repeated will be recorded as the 1RM for the chest press and leg press, respectively. Hand Grip Strength (5 minutes): To determine if a participant is at risk of sarcopenia, hand grip strength will be evaluated using the American Society of Hand Therapists (ASHT) protocol. Participants will sit in an armless chair with the elbow bent to 90 degrees and will be instructed to grip the handle of a hand grip dynamometer with the thumb facing up towards the sky. A researcher will instruct the participant to grip the handle as hard as they possibly can for three seconds using their right hand. The researcher will encourage the participant throughout the hand grip assessment and record the hand grip strength from the dynamometer in kilograms. After the right hand has been assessed, a hand grip assessment of the left hand will be completed. Three trials will be taken on each hand and will be averaged to obtain the hand grip score for the right and left hand, respectively. Venous Blood Sampling: Research staff qualified to perform cannulation and venepuncture will collect blood samples via intravenous catheter and venepuncture. 10mL of venous blood will be taken at each timepoint. Additionally, an accredited commercial pathology laboratory - Dorevitch Pathology (located on campus) will be used to collect fasting blood samples and for processing. These will be collected by one of the certified phlebotomists at Dorevitch Pathology. Participants may be provided with a Dorevitch Pathology request form where the participant can elect to go to the Dorevitch Pathology centre at Deakin University Burwood or one of their accredited laboratories of their choice. All venous blood samples processed by Dorovitch Pathology will have a deidentified code and results will be emailed to PI Way. Samples collected at Deakin University will be stored in a -80 degree freezer and labelled with a deidentified code. Dual-energy x-ray absorptiometry (DXA): will be used to assess total body fat and lean body mass (LBM). In addition, the DXA and Hologic software will be used to assess fat mass in the abdominal region as well as bone mineral density to exclude osteoporosis. The participants will be asked to lie-down on their back on the DXA table in light closing (no metal) or a gown for approximately 10-15 minutes while the machine scans their body. All DXA measurements will be conducted by trained a and accredited staff at Deakin University. Physical Activity Levels and Sedentary Time (minutes/week): These measures will be collected using the ActiGraph wGT3X-BT accelerometer (ActiGraph, Pensacola, Florida) worn over the right hip for 7 days (5 weekdays and 2 weekend days), excluding water-related activities (e.g. bathing or swimming). The widely used ActiGraph wGT3X-BT accelerometer is a lightweight, compact accelerometer that captures movement across three axes (y-, x- and z-axis), and is valid and reliable across a range of physical activity intensities and modes.