Skeletal Muscle Mitochondria in Ageing (AGAMEMNON)

Exercise as a Countermeasure Against the Effects of Ageing on Muscle Mitochondria, Diffusive Oxygen Transport and Muscle Volume

Healthy ageing is associated with the loss of muscle mass and physical function. As a result, older people are limited in their independence. The aging of muscles typically begins around the age of 30. From this age onward, muscle strength, muscle mass, and the maximum oxygen uptake of muscles decrease. The reasons for this are not entirely clear, but it seems to be partly related to how oxygen moves from our blood vessels to the muscles and how muscles burn energy. The precise role of age and physical fitness, as well as whether exercise can counteract the effects of ageing, is still unknown. Therefore, in this study, we aim to investigate the muscle function of both physically active and inactive young and middle-aged individuals. We hypothesise that endurance training can mitigate some of the effects of ageing.

Study Overview

• Status: Recruiting
• Conditions: Aging
• Intervention / Treatment: Other: Maximal exercise test; Other: 3D Ultrasound; Other: Dynamometry; Other: Exercise test and occlusions; Other: Exercise test in MRI; Procedure: Muscle biopsy

Detailed Description

Healthy ageing is associated with a loss of muscle mass and physical function. This loss of physical function is underpinned by reductions in characteristics such as muscle strength, power, and maximal oxygen uptake (V̇O2max; reflecting exercise capacity). However, the causal contributors to these age-associated impairments, and the role of exercise training status in mitigating them, remain poorly defined. Skeletal muscle mitochondrial function has been proposed to be a key contributor to age-associated effects on physical function, however many conflicting results are present in the extant human literature. Moreover, diffusion of oxygen from capillaries to mitochondria is a key determinant of V̇O2max, however, whether the skeletal muscle diffusive capacity for oxygen (DmO2) declines with age is unknown. A new technique utilizing near-infrared spectroscopy (NIRS) will enable the non-invasive assessment of skeletal muscle diffusive capacity in young and elderly subjects for the first time to resolve this issue. The primary aims of this study are therefore to 1) compare DmO2 derived via NIRS between young sedentary, young endurance-trained, older sedentary, and older endurance-trained subjects; 2) to compare non-invasive (i.e. with NIRS and 31phosphorous magnetic resonance spectroscopy [31P-MRS]) and invasive (i.e. measures of mitochondrial morphology and respiration obtained by skeletal muscle biopsy) markers of mitochondrial function between the same groups, and 3) to assess the relationships between DmO2, mitochondrial measures and assessments of capillarization with functional measurements of muscle strength, power, and V̇O2max.

• Study Type: Observational
• Enrollment (Estimated): 60

Contacts and Locations

• Study Contact: Name: Ellen Breedveld, MSc; Phone Number: +31636318844; Email: e.a.breedveld@vu.nl

Study Locations

• Netherlands
  • Noord-Holland
    • Amsterdam, Noord-Holland, Netherlands, 1081HV
      • Recruiting
      • Vrije Universiteit Amsterdam
      • Contact: Ellen Breedveld, MSc; Phone Number: +31636318844; Email: e.a.breedveld@vu.nl

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

15 sedentary and 15 endurance-trained young and middle-aged individuals will be recruited per age group (i.e. a total of 60 individuals spanning 4 groups). An equal balance of the sexes will be aimed for in each group. All participants will be recruited from specific social media groups, through personal networks of friends, family and colleagues, flyers and posters at local sports clubs and on the VU Campus. All testing will be conducted at the VU or in the Department of Radiology and Nuclear Medicine at Amsterdam UMC location AMC. Therefore, participants should be currently residing in or willing to travel to Amsterdam.

Description

Inclusion Criteria:

In order to be eligible to participate in this study, young sedentary participants must meet all of the following criteria:

• Aged between 18-30 years
• Male or female
• Not currently engaging in any formal exercise training or competitive sports
• No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise

In order to be eligible to participate in this study, young trained participants must meet all of the following criteria:

• Aged between 18-30 years
• Male or female
• Currently engaging in formal training (at least 3 times per week) in competitive endurance sports
• No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise

In order to be eligible to participate in this study, older sedentary participants must meet all of the following criteria:

• Aged between 50-65 years
• Male or female
• Not currently engaging in any formal exercise training or competitive sports
• No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise

In order to be eligible to participate in this study, older trained participants must meet all of the following criteria:

• Aged between 50-65 years
• Male or female
• Currently engaging in formal training (at least 3 times per week) in competitive endurance sports
• No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise

Exclusion Criteria:

• Age that falls outside of 18-30 years (young groups) or 50-65 years (middle-aged groups)
• Inability to provide informed consent
• History of claustrophobia
• Ineligibility to perform the exercise test described in this study protocol or follow instructions
• Taking any medications known to interfere with the physiological responses to exercise, e.g. e.g. systemic corticosteroids, statins, SGLT2 inhibitors, GLP1 receptor agonists
• Contraindication for MRI (e.g. pacemaker, claustrophobia)
• Being under investigation for non-diagnosed disease at the time of investigation
• Body Mass Index (BMI) >30 due to adiposity, since this is known to cause difficulties in obtaining muscle biopsies and NIRS measurements
• Pregnancy
• Are current smokers or have been a regular smoker within the last 12 months

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Young, sedentary participants; Aged between 18-30 years; Male or female; Not currently engaging in any formal exercise training or competitive sports; No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise	Other: Maximal exercise test; Participants will undertake an incremental ramp test on a cycle ergometer to determine maximal oxygen uptake (V̇O2max) and the gas exchange threshold (GET). Throughout the exercise test, muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: 3D Ultrasound; Muscle volume and morphological characteristics will be assessed via 3D ultrasound imaging.; Other: Dynamometry; To determine the contractile properties of the knee extensors, participants will perform maximal isometric and isoinertial contractions of the knee extensors on a dynamometer.; Other: Exercise test and occlusions; Participants will perform a series of moderate-intensity constant power output exercise bouts on a cycle ergometer following which the recovery rates of muscle V̇O2 will be determined via a series of intermittent arterial occlusions. Throughout all tests, pulmonary gas exchange and ventilation will be determined and muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: Exercise test in MRI; Exercise will be performed on a custom-built magnetic resonance-compatible cycle ergometer in supine position for determination of muscle phosphocreatine recovery kinetics using 31phosphorous magnetic resonance spectroscopy [31P-MRS].; Procedure: Muscle biopsy; A muscle biopsy will be obtained from the vastus lateralis using a modified Bergström needle technique with suction.
Young endurance-trained participants; Aged between 18-30 years; Male or female; Currently engaging in formal training (at least 3 times per week) in competitive endurance sports (i.e. running, cycling, swimming, triathlon, etc.); No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise	Other: Maximal exercise test; Participants will undertake an incremental ramp test on a cycle ergometer to determine maximal oxygen uptake (V̇O2max) and the gas exchange threshold (GET). Throughout the exercise test, muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: 3D Ultrasound; Muscle volume and morphological characteristics will be assessed via 3D ultrasound imaging.; Other: Dynamometry; To determine the contractile properties of the knee extensors, participants will perform maximal isometric and isoinertial contractions of the knee extensors on a dynamometer.; Other: Exercise test and occlusions; Participants will perform a series of moderate-intensity constant power output exercise bouts on a cycle ergometer following which the recovery rates of muscle V̇O2 will be determined via a series of intermittent arterial occlusions. Throughout all tests, pulmonary gas exchange and ventilation will be determined and muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: Exercise test in MRI; Exercise will be performed on a custom-built magnetic resonance-compatible cycle ergometer in supine position for determination of muscle phosphocreatine recovery kinetics using 31phosphorous magnetic resonance spectroscopy [31P-MRS].; Procedure: Muscle biopsy; A muscle biopsy will be obtained from the vastus lateralis using a modified Bergström needle technique with suction.
Middle-aged, sedentary participants; Aged between 50-65 years; Male or female; Not currently engaging in any formal exercise training or competitive sports; No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise	Other: Maximal exercise test; Participants will undertake an incremental ramp test on a cycle ergometer to determine maximal oxygen uptake (V̇O2max) and the gas exchange threshold (GET). Throughout the exercise test, muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: 3D Ultrasound; Muscle volume and morphological characteristics will be assessed via 3D ultrasound imaging.; Other: Dynamometry; To determine the contractile properties of the knee extensors, participants will perform maximal isometric and isoinertial contractions of the knee extensors on a dynamometer.; Other: Exercise test and occlusions; Participants will perform a series of moderate-intensity constant power output exercise bouts on a cycle ergometer following which the recovery rates of muscle V̇O2 will be determined via a series of intermittent arterial occlusions. Throughout all tests, pulmonary gas exchange and ventilation will be determined and muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: Exercise test in MRI; Exercise will be performed on a custom-built magnetic resonance-compatible cycle ergometer in supine position for determination of muscle phosphocreatine recovery kinetics using 31phosphorous magnetic resonance spectroscopy [31P-MRS].; Procedure: Muscle biopsy; A muscle biopsy will be obtained from the vastus lateralis using a modified Bergström needle technique with suction.
Middle-aged endurance-trained participants; Aged between 50-65 years; Male or female; Currently engaging in formal training (at least 3 times per week) in competitive endurance sports (i.e. running, cycling, swimming, triathlon, etc.); No chronic health conditions likely to affect exercise tolerance or the physiological responses to exercise	Other: Maximal exercise test; Participants will undertake an incremental ramp test on a cycle ergometer to determine maximal oxygen uptake (V̇O2max) and the gas exchange threshold (GET). Throughout the exercise test, muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: 3D Ultrasound; Muscle volume and morphological characteristics will be assessed via 3D ultrasound imaging.; Other: Dynamometry; To determine the contractile properties of the knee extensors, participants will perform maximal isometric and isoinertial contractions of the knee extensors on a dynamometer.; Other: Exercise test and occlusions; Participants will perform a series of moderate-intensity constant power output exercise bouts on a cycle ergometer following which the recovery rates of muscle V̇O2 will be determined via a series of intermittent arterial occlusions. Throughout all tests, pulmonary gas exchange and ventilation will be determined and muscle oxygenation and deoxygenation will be monitored by NIRS.; Other: Exercise test in MRI; Exercise will be performed on a custom-built magnetic resonance-compatible cycle ergometer in supine position for determination of muscle phosphocreatine recovery kinetics using 31phosphorous magnetic resonance spectroscopy [31P-MRS].; Procedure: Muscle biopsy; A muscle biopsy will be obtained from the vastus lateralis using a modified Bergström needle technique with suction.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Maximal oxygen uptake (V̇O2max)	ml/min/kg	Baseline (visit 1)
Muscle volume	cm^3	Baseline (visit 1)
Muscle strength	Newton-metre (Nm)	Baseline (visit 1)
Muscle power	Watt (W)	Baseline (visit 1)
Muscle diffusing capacity for oxygen (DmO2)	Differences in recovery constant k (min-1) obtained under conditions of high, medium or low O2 availability	Baseline (visit 1) and visit 2-4. In total 4 weeks.
Muscle mitochondrial fragmentation index (A.U.)	Degree of fragmentation of the mitochondrial pool.	Visit 6 muscle biopsy (+/- after 4 weeks).

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Gas exchange and ventilatory variables (gas exchange threshold, respiratory compensation point, maximal ventilation)	L/min	Baseline (visit 1)
(Peak) power output	Watt (W)	Baseline (visit 1)
Mean response time of the V̇O2 slope during ramp exercise	sec	Baseline (visit 1)
(Maximum) heart rate (HR)	beats per minute (bpm)	Baseline (visit 1) and during visit 2-4 (max 4 weeks in total)
Maximal O2 pulse	ml/beat	Baseline (visit 1)
Slope of ventilation (VE) versus carbon dioxide (VCO2) output during ramp exercise (i.e. ventilatory efficiency)	V̇E/V̇CO2 slope	Baseline (visit 1)
V̇O2/HR slope during ramp exercise	beats/L/min	Baseline (visit 1)
Maximal respiratory exchange ratio (RER)	RER = VCO2/VO2	Baseline (visit 1)
Maximal ventilatory equivalents	VE/VCO2 and VE/VO2	Baseline (visit 1)
Maximal end-tidal pressures for oxygen (O2) and carbon dioxide (CO2)	mmHg	Baseline (visit 1)
Capillary lactate concentration	mmol/L	Baseline (visit 1)
Maximal respiratory frequency	breaths/min	Baseline (visit 1)
Pulmonary oxygen uptake - baseline and steady state V̇O2	L/min	Baseline (visit 1) and visit 2-4 (max 4 weeks in total)
Pulmonary oxygen uptake kinetics - Phase II V̇O2 time constant and time delay	sec	Visit 2-4 (max 4 weeks in total)
Pulmonary oxygen uptake kinetics - Phase II V̇O2 amplitude	L/min	Visit 2-4 (max 4 weeks in total)
Concentrations of NIRS derived muscle oxy- and deoxygenated [haemoglobin + myoglobin] (HbO2, Hbb) and tissue saturation index (TSI).	HbO2 and Hbb: % maximal value, TSI (%) = HbO2/(HbO2+Hbb) For all variables resting concentration, baseline cycling concentration, (sub)maximal exercise concentration will be reported.	Baseline (visit 1) and visit 2-4 (max 4 weeks in total)
NIRS derived muscle oxy- and deoxygenated [haemoglobin + myoglobin] (HbO2, Hbb) and tissue saturation index (TSI) versus relative and absolute work rate.	HbO2 and Hbb: % maximal value, TSI (%) = HbO2/(HbO2+Hbb) versus relative (%max) and absolute power output (W) Relative and absolute work rates comparisons will be reported for all variables: resting concentration, baseline cycling concentration, (sub)maximal exercise concentration.	Baseline (visit 1)
Initial and secondary slope of increase during incremental exercise will be reported for NIRS derived muscle oxy- and deoxygenated [haemoglobin + myoglobin] (HbO2, Hbb) and tissue saturation index (TSI) versus relative work rate.	concentration[Hbb/HbO2/TSI]%/delta%peak power(W)	Baseline (visit 1)
Initial and secondary slope during incremental exercise will be reported for NIRS derived muscle oxy- and deoxygenated [haemoglobin + myoglobin] (HbO2, Hbb) and tissue saturation index (TSI) versus absolute work rate.	concentration[Hbb/HbO2/TSI]%/deltaW	Baseline (visit 1)
Muscle (de)oxygenation breakpoint during incremental exercise	Power output (W) and maximal oxygen uptake (L/min)	Baseline (visit 1)
Rate constant of mV̇O2 recovery kinetics under conditions of high, medium and low O2 availability	sec	Baseline (visit 1)
Muscle morphology - Fascicle length	cm	Baseline (visit 1)
Muscle morphology - pennation angle	degrees	Baseline (visit 1)
Muscle morphology - (effective) physiological cross-sectional area (PCSA)	cm2	Baseline (visit 1)
Muscle morphology - vastus lateralis specific force	N/cm2	Baseline (visit 1)
Muscle morphology - estimated muscle fiber number	PCSA/muscle fiber cross-sectional area	Baseline (visit 1)
Adipose tissue thickness at the site of NIRS measurement	mm	Baseline (visit 1)
Mitochondrial respiratory function (background, LEAK, N-linked respiration, OXPHOS, ETS, succinate (S) + rotenone (ROT)-linked uncoupled respiration, )	pmol/s/mg	Visit 6 muscle biopsy (+/- after 4 weeks).
Respiratory control ratios	OXPHOS/ETS, LEAK/ETS, LEAK/OXPHOS, LEAK/NADH-linked, ROT+S/ETS, (OXPHOS-LEAK)/ETS, (OXPHOS-LEAK)/OXPHOS, (ETS-LEAK)/ETS, (ETS-OXPHOS)/ETS	Visit 6 muscle biopsy (+/- after 4 weeks).
Intrinsic mitochondrial respiration (each respiratory state outlined below will be normalised to mitochondrial volume density)	(pmol/s/mg)/mitochondrial area density (%) For: background, LEAK, N-linked respiration, OXPHOS, ETS, succinate + rotenone-linked uncoupled respiration	Visit 6 muscle biopsy (+/- after 4 weeks).
31P-MRS-derived mitochondrial bioenergetic function - resting and steady-state exercising concentrations and amplitude of exercise-induced changes of skeletal muscle Phosphocreatine [PCr], [inorganic phosphate] and pH	mM pH = unitless	Visit 5 MRI
31P-MRS-derived mitochondrial bioenergetic function rate constant of PCr	Rate constant of Phosphocreatine [PCr] on- and off-kinetics (sec)	Visit 5 MRI
31P-MRS-derived mitochondrial bioenergetic function - maximal rate of oxidative ATP synthesis	mM/s	Visit 5 MRI
Muscle fiber type distribution	Type I, IIa, IIx and hybrid fiber-type proportions (%)	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
(mean) fiber cross-sectional area, also fiber type specific	um	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Weighted fiber cross-sectional area	um2	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Mean and fiber type specific succinate dehydrogenase activity	A660/um/s	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Integrated fiber succinate dehydrogenase activity	A660.um/s	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Mean and fiber type specific myoglobin concentrations	mM	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - capillary density	number of capillaries/mm2	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - capillary-to-fiber ratio	capillary-to-fiber ratio	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - mean number of capillaries surrounding a fiber (CAF)	number of capillaries	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - mean number of capillaries surrounding a fiber in relation to fiber area (CAFA)	number of capillaries/um2	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - capillary-to fiber-perimeter exchange index (CFPE)	CFPE (unitless)	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - length of capillaries relative to fiber perimeter (LC/PF)	percentage (%)	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Muscle capillarization - sarcomere length	um	Visit 6 muscle biopsy (+/- after 4 weeks) - staining within time window of 2 years.
Mitochondrial dynamics proteins (Mfn1, Mfn2, OPA1, Drp1, Parkin, PINK1, Fis1, MTFP1, NRF1&2, PGC1a, TFAM, OXPHOS protein content (complexes I-V and total protein content)	ug/mg loaded sample	Visit 6 muscle biopsy (+/- after 4 weeks) - western blot within time window of 2 years.
Physical activity status - pedometer	Number of steps per day	Baseline 7 days
Mitochondrial area density	Percentage (%)	Visit 6 muscle biopsy (+/- after 4 weeks) - electron microscopy (EM) within time window of 2 years.
Mitochondrial volume density	um3.um3.10^2	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial number	number/um2	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial area	um2	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial height, width, perimeter and maximal+minimal Feret's diameter	um	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial surface area-to-volume ratio	um/um^2	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial circularity	Circularity (AU)	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial roundness	Roundness (AU)	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial aspect ratio	Aspect ratio (AU)	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.
Mitochondrial cristae area density	Percentage (%)	Visit 6 muscle biopsy (+/- after 4 weeks) - EM within time window of 2 years.

Collaborators and Investigators

• Sponsor: VU University of Amsterdam
• Investigators: Principal Investigator: Richie Goulding, PhD, VU University of Amsterdam

Study record dates

Study Major Dates

• Study Start (Actual): March 25, 2024
• Primary Completion (Estimated): December 1, 2025
• Study Completion (Estimated): December 1, 2026

Study Registration Dates

• First Submitted: July 18, 2024
• First Submitted That Met QC Criteria: August 2, 2024
• First Posted (Actual): August 6, 2024

Study Record Updates

• Last Update Posted (Actual): August 6, 2024
• Last Update Submitted That Met QC Criteria: August 2, 2024
• Last Verified: August 1, 2024

More Information

Terms related to this study

• Keywords: Exercise; Mitochondria; Skeletal muscle
• Other Study ID Numbers: 2023.0746

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No