Involvement of Muscle Mitochondrial Dysfunction in Frailty in Older Adults. Role of Exercise

Background and Rationale:

Frailty is a multifactorial geriatric syndrome characterized by a decline in physiological reserves across multiple systems, leading to reduced resilience, weakness, and increased vulnerability to stressors such as acute illness, injury, or surgery. Clinically, frailty manifests as diminished strength, slowed mobility, exhaustion, and unintentional weight loss, often culminating in disability, hospitalization, and premature mortality. Among its core biological features, skeletal muscle deterioration, encompassing losses in muscle mass, contractile function, and regenerative capacity, plays a central role in the onset and progression of functional impairment and dependence in older adults.

At the cellular level, mitochondrial dysfunction has emerged as a key pathophysiological driver of frailty and sarcopenia. Mitochondria are essential for energy production, redox balance, and regulation of calcium homeostasis, apoptosis and inflammation. With aging, mitochondrial content, dynamics, and efficiency decline, leading to reduced ATP generation, increased reactive oxygen species (ROS) production, and accumulation of damaged mitochondrial DNA (mtDNA). These alterations compromise muscle bioenergetics and promote catabolic pathways that accelerate muscle atrophy. Moreover, defective mitophagy, an essential quality control process, leads to the persistence of dysfunctional organelles, perpetuating oxidative stress and inflammation.

Systemic metabolic disturbances further exacerbate these mitochondrial deficits. A chronic condition such as type 2 diabetes mellitus (T2DM) is frequently associated with frailty and share common mechanisms. Increased levels of pro-inflammatory cytokines (e.g., IL-6, TNF-α) and mitochondrial stress markers signal ongoing tissue damage and maladaptive stress responses. These interactions between metabolic dysregulation, inflammation, and mitochondrial dysfunction form a self-reinforcing cycle that underlies the molecular pathogenesis of frailty.

Exercise training, and particularly resistance exercise, is among the most potent non-pharmacological interventions to counteract frailty-related declines. Regular exercise improves muscle mass and strength, enhances glucose and lipid metabolism, and promotes mitochondrial biogenesis and function through activation of key molecular pathways. Furthermore, exercise induces the release of myokines that mediate intercellular communication between muscle and distant organs, influencing systemic metabolism, inflammation, and repair processes. However, despite well-established clinical benefits, the molecular and cellular mechanisms by which exercise remodels mitochondrial networks and restores metabolic homeostasis in frail or metabolically compromised individuals remain incompletely understood.

Elucidating how exercise modulates mitochondrial quality control, energy metabolism, and signaling pathways in the context of frailty could reveal new therapeutic targets to delay or reverse physiological decline. Integrating metabolic markers with molecular indicators of mitochondrial stress and muscle-derived factors offers a multidimensional approach to understanding the bioenergetic and inflammatory signatures of frailty and their reversibility through targeted interventions.

Study Objectives:

This project aims to:

• Determine the contribution of skeletal muscle mitochondrial dysfunction to frailty in older adults, including those with T2DM.
• Evaluate the effects of a 6-week supervised resistance training program on functional, molecular, and metabolic outcomes.
• Characterize adaptations within the perimuscular interstitial microenvironment (fibroblasts, immune cells, vascular, neural and muscle stem cells as well as extracellular matrix components) that may mediate exercise-induced improvements in muscle health.

Through these objectives, the study seeks to integrate clinical, physiological, and molecular analyses to uncover mechanisms linking mitochondrial remodelling, aging, and exercise adaptation

Study Design:

This is a longitudinal, interventional study including 120 participants divided into three groups:

• Older adults with T2DM (≥65 years) - 40 participants
• Healthy older adults (≥65 years) - 40 participants
• Young healthy adults (19-35 years) - 40 participants (controls) All participants will complete baseline assessments, a 6-week supervised resistance training program, and post-intervention evaluations. Optional muscle biopsies will be obtained from participants who consent before and after training.

Intervention:

Participants will undergo a 6-week progressive resistance training program targeting the quadriceps muscles, consisting of two sessions per week (a total of 12 sessions), each lasting 30-50 minutes.

• Exercises include leg press and knee extension, progressing from submaximal to near-maximal effort as tolerated.
• Training intensity and progression will be individualised according to functional capacity.
• All sessions will be supervised by qualified exercise specialists to ensure safety and adherence.

This short, structured, and closely monitored training protocol was designed to be both safe and feasible for older adults with varying levels of frailty or diabetes.

Assessments and Procedures:

Baseline and Post-Intervention Evaluations:

Participants will undergo comprehensive testing before and after the exercise program, including:

• Blood sampling for metabolic, inflammatory, and aging biomarkers.
• Body composition analysis using DXA, ultrasound, and bioimpedance.
• Functional assessments of strength, balance, gait speed, and mobility.
• Stool sampling for gut microbiota composition.
• Optional muscle biopsies (quadriceps) under local anesthesia for mitochondrial and tissue-level analyses.

Muscle Biopsy Analyses

Muscle tissue samples will be used to examine:

• Mitochondrial function, oxidative stress, and biogenesis markers.
• Cellular and molecular characteristics of the perimuscular niche via single-nuclei and histological analyses.
• Changes in fibrosis, immune infiltration, vascular density, and extracellular matrix remodelling following exercise.

All biopsy procedures will be performed by trained clinicians following hospital safety protocols, with local anaesthesia and post-procedure monitoring.

Outcomes and Data Integration:

The study will generate a comprehensive dataset encompassing:

• Clinical and functional outcomes: muscle strength, mobility, balance, and composite frailty scores.
• Molecular and biochemical measures: mitochondrial respiration, oxidative stress and inflammatory markers, and pathways related to muscle regeneration.
• Circulating biomarkers: metabolic, inflammatory, and aging-related factors.
• Microbiome analyses: gut bacterial diversity and composition.
• Cross-sectional comparisons: differences across age and diabetes status.
• Longitudinal effects: pre- vs. post-intervention changes in functional and molecular endpoints.

This integrated approach will allow exploration of mechanistic links between exercise-induced mitochondrial remodelling, systemic inflammation, and functional improvements.

Safety Considerations:

The exercise program carries minimal risk and will be supervised at all times. Potential risks include mild muscle soreness or fatigue. Blood sampling may cause minor bruising or lightheadedness. Muscle biopsy, when performed, carries a small risk of pain, bleeding, or infection; all procedures will be conducted by trained clinical personnel with appropriate monitoring and follow-up.

Participants' safety will be prioritised throughout the study, with adherence logs, post-biopsy monitoring, and regular communication with healthcare providers.

Potential Benefits:

Participants may experience improvements in strength, balance, and mobility, as well as greater awareness of their health and functional capacity.

Beyond individual benefits, this project is expected to provide key insights into:

• The role of mitochondrial dysfunction and diabetes in age-related muscle decline.
• The cellular mechanisms underlying exercise-induced rejuvenation of skeletal muscle.
• Evidence-based strategies for designing personalised exercise programs to delay or prevent frailty.

Significance and Expected Impact:

By comparing diabetic and non-diabetic older adults with younger controls, this study will clarify the contribution of mitochondrial dysfunction and systemic metabolic stress to muscle aging and functional decline. It will also identify how short-term, supervised resistance training promotes beneficial adaptations within muscle fibers and their surrounding microenvironment.

The results will contribute to developing practical, safe, and effective interventions to maintain independence, improve quality of life, and reduce healthcare burden in aging populations. Ultimately, this project aims to bridge the gap between basic mitochondrial biology and translational geroscience, providing new insights into the prevention and management of frailty in older adults.

Inclusion criteria

• Inclusion criteria for young controls

• Healthy young adults (18-35 years old)
• Without any pathology or treatment
• Inclusion criteria for older controls

• Healthy older adults (65 years or older)
• Not currently enrolled in a resistance training program
• Inclusion criteria for cases

• Age over 65 years
• Diagnosis of frailty or robustness
• Diagnosis of type 2 diabetes mellitus
• Glycosylated hemoglobin (HbA1c) less than 9%
• Treatment with metformin
• Not currently enrolled in a resistance training program

Exclusion criteria

• Exclusion criteria for young controls

• Pregnancy or suspected pregnancy
• Body mass index greater than 35 kg/m2
• Treatment within the last 30 days with oral corticosteroids
• Edema or severe fluid regulation disorders that could alter bioimpedance results
• Allergy to the local anesthetic mepivacaine
• Prostheses, metal implants, or surgical staples lodinated or barium contrast for other imaging tests within the last 7 days
• Exclusion criteria for older controls

• Decompensated and uncontrolled chronic diagnoses
• Body mass index greater than 35 kg/m2
• Treatment within the last 30 days with oral corticosteroids
• Systemic diseases, active oncological disease, liver cirrhosis, untreated hypothyroidism, or severe chronic obstructive pulmonary disease (COPD).
• Severe edema or fluid regulation disorders that could alter bioimpedance results
• Allergy to the local anesthetic mepivacaine
• Prostheses, metal implants, or surgical staples Iodinated or barium contrast for other imaging tests in the last 7 days
• Exclusion criteria for cases

• Diagnosis of pre-frailty
• Age over 80 years
• Body mass index greater than 35 kg/m2
• Time since onset of diabetes mellitus type 2 less than 10 years
• Treatment with insulin
• Treatment with allopurinol
• Treatment with anticoagulant or antiplatelet drugs
• Treatment within the last 30 days with oral corticosteroids
• Advanced chronic complications of diabetes mellitus type 2 :

Renal failure (clearance <30 ml/min/m2) Macroalbuminuria greater than 200 mg/g Ischemic vascular diseases (acute myocardial infarction) Myocardial infarction, angina, stroke) Proliferative retinopathy or laser therapy Ulcerated diabetic foot or lower limb amputations, except for digital amputations

• Systemic diseases, active oncological disease, liver cirrhosis, untreated hypothyroidism, or severe chronic obstructive pulmonary disease
• Physical limitations in the lower limbs (acute or acute-on-chronic inflammatory processes, limited joint range of motion, chronic pain (visual analogue scale for pain > or = 4, etc.)
• Senile dementia
• Edema or severe fluid regulation disorders that may alter bioimpedance results
• Allergy to the local anesthetic mepivacaine
• Pacemaker, metallic prostheses or implants, surgical staples, and radiopaque tubes or catheters Iodinated or barium contrast for other imaging tests within the last 7 days