Identifying the link between bone and muscle in older-adults : effects of exercise

The study involves 2 phases. Phase 1 includes 2 visits: visit 1 and 2 (V1, 2) serves as baseline, screening and familiarisation prior to phase 2. Following phase 1, participants will be invited to phase 2. Phase 2 includes 3 separate testing conditions, blood sampling and optional muscle biopsy (none, 1 or 4). A portion of the muscle will be cultured, and stored for later analysis (described below). All visits will be performed by qualified and trained personnel. Phase 1, Cross sectional study: two separate visits (V1, V2) performed in any order and up to 14days apart. Participants will attend the laboratory following overnight fast (10hrs) and visits approx. 3-4 hours duration. V1; Following informed consent, participants will complete: Charlson comorbidity index for comorbidity burden, Nestle’ Mini-Nutritional Assessment and Falls Risk for Older People questionnaires. Height, weight will be measured and body mass index calculated. A 3-day dietary/physical activity log will be given, and returned (V2) to assess diet/physical activity, and to replicate similar diet for remainder visits. (a) Blood sampling; A singular blood draw (30mL) from the antecubital vein for assay of circulating osteoprogenitor (COP) cells, serum osteocalcin (OC), undercarboxylated osteocalcin (ucOC) and a variety of cardio-metabolic health markers. COP cells analysis will be performed as we previously described. Quantification of OC/ucOC; Total serum OC will be measured using an automated immunoassay (Elecsys 170; Roche Diagnostics). Serum ucOC will be measured by the same immunoassay after absorption of carboxylated OC on 5mg/mL hydroxyl-apatite slurry, as previously performed by our group. Insulin, b-isomerized C-terminal telopeptides (b-CTx) and procollagen 1 N-terminal propeptide (P1NP); will be analysed using a Roche Hitachi Cobas e602 immunoassay analyser, according to the manufacturer’s guidelines. Hormones (PTH), lipids, glucose and insulin, inflammation markers (C-reactive protein, serum interleukin-6 (IL-6) and potentially other cardiovascular/health markers will be analysed according to hospital standards. Genetic analysis; Genomic data will be extracted from the venous blood samples (EDTA tubes) using a standard kit. Genetic variants will be analysed using Taq man R-T PCR high throughout gene analysis and sequence analysis, to sequence target genes that may be associated with bone (i.e. OC) and muscle health and exercise intolerance. A genome-wide analysis approach will be performed on collected samples with either illumine OmniExpress array chips or Infinium CoreExome-24 Kit. Mitochondrial genome will be sequenced using a Next Generation Sequencing with the Ion Platform (Life Technologies, Thermo Scientific) library sequencing method. (b) Vascular/endothelial function; assessed by brachial artery flow-mediated dilatation (BAFMD) utilising ultrasound to capture images of brachial artery at baseline, during 5min occlusion and immediately after cuff release, for calculation of change in artery width post shear stress. Vascular stiffness assessed by measures of pulse wave velocity/reflection using applanation tonometry (SphygmoCor EXCEL system V1), a non-invasive diagnostic system for assessments of central blood pressure/pulse wave velocity (PWV). Pulse Wave Analysis (PWA): a cuff is placed on the upper arm (brachial artery) measuring pulsations (brachial) to produce central aortic pressure waveforms. (c) Exercise capacity; VO2Peak assessed on a cycle ergometer with 12-lead electrocardiogram (ECG; Mortara, X-Scribe II, Milwaukee, WI). Initial intensity will be 10-30 W and increase by 10-30W×min-1 according to participant ability. The test will be terminated according to participants’ self-reported fatigue perception (RPE of 17) or clinical signs or symptoms. (d) Familiarisation 1RM; Participants will perform a familiarisation session on leg press. Visit 2; (a) Body composition: a dual energy-X-ray absorptiometry (DXA) scan to assess total and regional lean body mass and BMD (total, neck of the femur and lumbar spine) used to define bone strength and fragility. (b) Fat infiltration, bone microarchitecture: will be assessed by Peripheral Quantified Computer Tomography (pQCT). Participants will undergo pQCT imaging to quantify muscle mass, density and adipose infiltration. pQCT and DXA performed by appropriate expert personnel. (c) Grip strength, Gait velocity; Grip strength measured using a hand dynamometer, less than 20kg for females and less than 30kgs for males will identify low muscle strength. A 4m gait velocity assessment will be performed using the instrumented walkway (GAITRite®) and/or by timing with a stop watch and less than 80cm/sec will determine reduced physical function. Grip strength and gait velocity thresholds are accepted as a measurement of sarcopenia. (d) Physical Performance Test (PPT); includes 4 functional tasks; gait velocity assessment (above), up-and-go test, stair climbing power (SCP) and stair descending. All will be scored in time (s). The up-and-go test is a simple performance based assessment, requiring minimal equipment including; standard arm chair (approx. 46cm), 3-meter walkway, floor mark and stopwatch and performed as time (s) taken to rise from a seated position, walk 3 meters, turn, walk back to chair and sit. The SCP will consist of a rapid ascent of 10 stairs, where; Stair Climbing Power equals: body weight (kg) x 9.8ms-2) x step height (m) x number of steps x time -1 (s-1) The stair descent will be time to descend safely 10 stairs. Rest between ascent and descent will be 45s. 3 attempts will be given on each task (40-s rest intervals) and best time recorded. PPT score equals sum of the fastest times recorded for each. (e) maximal strength; 1 repetition maximum (IRM) test on leg press will be performed, defined as the heaviest weight lifted once, with proper technique and without compensatory movements. Results will guide prescription for the acute resistance exercise (RE). Leg Muscle Quality (LMQ); shown to decrease with age is described as the amount of force a muscle group can produce per unit of muscle mass. (Leg strength (1RM), leg lean mass (DXA) Calculated as; LMQ equals: leg strength (kg) / L leg lean mass (kg) + R leg lean mass (kg) Phase 2: Acute exercise; randomised, controlled, crossover (V3, 4, 5); examining effects of acute aerobic (AER) and RE on bone/muscle metabolism and metabolic/CV risk (glucose, lipids, inflammation etc.). Phase two will be performed up to 14days following baseline testing. Participants will be randomised using sealed envelope (block allocation) to determine order of interventions; AER, RE and control (CTRL) (to prevent carry forward effects), performed by external personnel. Participants will perform each condition but in any order (randomised), and thus will serve as their own control. Each intervention is approx. 30mins duration and 3hrs total duration, visits performed 7 days apart. Participants will be under the direct supervision of the study coordinator, who is an AEP, and for all invasive measures under close supervision of the appointed medical doctor. In between visit, during the wash out period, participants will be asked to refrain from vigorous activity in preceding min. 24hours, and may or may not be requested to wash out of particular medications as advised by the medical doctor if electing for muscle biopsy. All of this information is managed and communicated to participants by study coordinater. (a) Blood sampling; On arrival, a cannula will be inserted (as described above) and blood sampling at immediately post exercise (0mins), 30, 60 and 120mins to assess changes post exercise. 25mL sample will be taken immediately post exercise at 0mins, 30, 60 and 120min to observe changes in COP, OC and ucOC measures. (b) Muscle biopsies; Participants elect to have none, 1 (at rest) or 4 (1 at rest a baseline for all interventions and 1 following each condition) muscle biopsies. from vastus lateralis under local anesthesia (xylocaine 1%) by percutaneous needle biopsy technique, modified to include suction. Excised tissue immediately snap frozen with liquid nitrogen and stored at -80degrees for later analysis. All muscle samples will be analysed for signalling proteins involved in muscle degradation/hypertrophy. (c) interventions, randomised (V3, 4 and 5) I. Acute CTRL: 30 mins rest. II. Acute AER: 30 mins cycle ergometer 70-75% of HRPeak (based on GXT data). III. Acute RE: 30mins of power exercises at 70-75% (pre-determined from 1RM) includes: leg press, 5 x 10 rapidly concentric (“as fast as possible”) and slow eccentric (4 second) reps and jumping sequences; 5 x 10 jumps with 2mins recovery between sets. Power training is effective to increase muscle strength, bone density and is safe for older-adults. Baseline biopsy: In-vitro study: A portion of muscle sample obtained in V3 will be used for Cell culture: examining effect of different treatments (including, but not limited, ucOC) on human skeletal muscle myotubes. Markers of muscle degradation/ hypertrophy, see list below, and glucose uptake will be assessed. Analysis of muscle sample: Muscle will be analysed for protein degradation pathways; ubiquitin-proteasome, autophagy-lysosome and caspase-3-mediated proteolytic pathways and protein synthesis; mammalian target of rapamyacin, mTOR. Other factors implicated in aging/pathways i.e. Smad3-signalling, TGF-beta, myostatin and growth and differentiation factor II will be assessed. We will investigate these pathways following 3 separate conditions. Protein extraction, Western blotting; specialised technique to detect specific proteins via gel electrophoresis to separate proteins by length of polypeptides or by 3D structure. Proteins are transferred to a membrane where using antibodies, specific proteins can be targeted.