The Role of Muscle Protein Breakdown in the Regulation of Muscle Quality in Frail Elderly Individuals

Study Overview
=================
Brief Summary
-----------------
The purpose of this study is to investigate mechanisms underlying the reduction in muscle quality (the ratio between muscle strength and muscle size) with aging, and to investigate how these factors are affected by strength training and protein supplementation. It is already established that muscle quality defined as the ratio between the strength and the size of a muscle is improved with strength training, even in frail elderly individuals. However, the relative contribution of factors such as activation level, fat infiltration, muscle architecture and single fiber function is unknown. The main focus of this study is to investigate the relationship between muscle quality and muscle protein breakdown, as insufficient degradation of proteins is hypothesized to negatively affect muscle quality.

Detailed Description
-----------------
Aging is associated with impaired skeletal muscle function. This is evident not only by a reduced capacity to generate force and power at the whole muscle level, but also by a decline in individual muscle fiber contraction velocity and force generation. Combined with muscle atrophy, these changes lead to reduced muscle strength and quality and loss off physical function with age. Clinically, muscle quality may be a better indicator of overall functional capacity than absolute muscle strength. Thus, identifying the mechanisms underlying the age-related loss of muscle quality is of high relevance for the prevention of functional impairment with aging. The explanation for the loss of muscle quality with aging seems to be multifactorial, with alterations in voluntary muscle activation, muscle architecture, fat infiltration and impaired contractile properties of single muscle fibers being likely contributors. Single fiber specific force seems to be related to myosin heavy chain (MHC) content, which is thought to reflect the number of available cross-bridges. The reduction of single fiber specific force with aging may thus be a consequence of reduced synthesis of MHC and/or increased concentration of non-contractile tissue (e.g. intramyocellular lipids). Some studies in mice also indicate attenuated activity in some of the pathways responsible for degradation of muscle proteins with aging (especially autophagy). As a result, damaged proteins and organelles are not removed as effectively as they should, which could ultimately compromise the muscle's ability to produce force. In addition, reduced efficiency of mitophagy and lipophagy (two specific forms of autophagy), may indirectly affect single fiber specific force, through oxidative damage by reactive oxygen species (ROS) and increased levels of intramyocellular lipids, respectively. Although animal studies indicate attenuated autophagic function, exercise seems to restore the activity in this pathway. Whether this also is the case in humans is unknown. Thus, the purpose of this study is to investigate how the different factors contributing to reduced muscle quality in frail elderly individuals, with emphasis on the relationship between muscle quality and autophagy, may be counteracted by a specific strength training program targeting muscle quality and muscle mass. In this randomized controlled trial the investigators will aim to recruit frail elderly individuals, as muscle quality is shown to be low in this population. As a consequence, the potential for improved muscle quality is expected to be large. Subjects will be randomized to two groups; one group performing strength training twice a week for 10 weeks in addition to receiving daily protein supplementation. The other group will only receive the protein supplement. Several tests will be performed before and after the intervention period, including a test day where a biopsy is obtained both at rest, and 2.5 hours following strength training + protein supplementation or protein supplementation only. This will provide information about the regulation of muscle protein breakdown in a resting state, following protein intake and following strength training in combination with protein intake. As this will be done both before and after the training period, it will also provide information on how long-term strength training affects the activity in these systems.

Official Title
-----------------
The Role of Muscle Protein Breakdown in the Regulation of Muscle Quality in Frail Elderly Individuals

Conditions
-----------------
Sarcopenia

Intervention / Treatment
-----------------
* Other: Strength training
* Dietary Supplement: Protein supplementation


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Age > 65 Frail or pre-frail according to the Fried Frailty Criteria or Short Physical Performance Battery (SPPB) score <6. Mini Mental State Examination score > 18 Exclusion Criteria: Diseases or injuries contraindicating participation Lactose intolerance Allergy to milk Allergy towards local anesthetics (xylocain) Use of anticoagulants that cannot be discontinued prior to the muscle biopsy

Ages Eligible for Study
-----------------
Minimum Age: 65 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Strength training + protein supplement<br>Two sessions of strength training each week in addition to daily protein supplementation for 10 weeks. | Other: Strength training<br>* Heavy load strength training performed twice a week for 10 weeks.<br>* Other names: Resistance training;Dietary Supplement: Protein supplementation<br>* Dietary protein supplement (protein-enriched milk with 0,2 % fat). 0,33 l each day for 10 weeks.<br>|
| Experimental: Protein supplement<br>Daily protein supplementation for 10 weeks. | Dietary Supplement: Protein supplementation<br>* Dietary protein supplement (protein-enriched milk with 0,2 % fat). 0,33 l each day for 10 weeks.<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Single fiber specific force | A measure of muscle quality at the single fiber level. Biopsies obtained from m. Vastus Lateralis | Change from baseline at 10 weeks | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Lean mass | Measured by a Dual-energy X-ray absorptiometry (DXA) scan | Change from baseline at 10 weeks | 
| Fat mass | Measured by a Dual-energy X-ray absorptiometry (DXA) scan | Change from baseline at 10 weeks | 
| Bone mineral density | Measured by a Dual-energy X-ray absorptiometry (DXA) scan | Change from baseline at 10 weeks | 
| Muscle strength of m. quadriceps | Maximal isometric and dynamic muscle strength of m. quadriceps | Change from baseline at 10 weeks | 
| Muscle size of m. quadriceps | Cross-sectional area of m. quadriceps measured by a Computed Tomography scan | Change from baseline at 10 weeks | 
| Fat infiltration of m. quadriceps | Fat infiltration of m. quadriceps measured by a Computed Tomography scan | Change from baseline at 10 weeks | 
| Muscle activation | Voluntary activation level during a maximal isometric knee extension using the interpolated twitch technique | Change from baseline at 10 weeks | 
| Fractional Breakdown Rate | Measurement of fractional breakdown rate by the use of orally provided Deuterium Oxide, biopsies and blood samples | Measured over the last 14 days of the intervention period | 
| m. Vastus Lateralis thickness | Measured by ultrasound | Change from baseline at 10 weeks | 
| Chair stand performance | Time (sec) to stand up from a chair five times | Change from baseline at 10 weeks | 
| Habitual gait velocity | Time (sec) to walk 6 meters at habitual gait velocity | Change from baseline at 10 weeks | 
| Maximal gait velocity | Time (sec) to walk 6 meters as fast as possible | Change from baseline at 10 weeks | 
| Level/cellular location of Microtubule-associated protein 1A/1B-light chain 3 (LC3) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Level/cellular location of p62/Sequestosome-1 | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Level/cellular location of Lysosome-associated membrane glycoprotein 2 (LAMP2) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Level/cellular location of forkhead box O3 (FOXO3a) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Phosphorylation status and total level of ribosomal protein S6 kinase beta-1(P70S6K) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Phosphorylation status and total level of eukaryotic elongation factor 2 (eEF-2) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Phosphorylation status and total level of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP-1) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Level/cellular location of muscle RING-finger protein-1 (Murf-1) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Level/cellular location of ubiquitin (Ub) | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Blood serum glucose | Fasted | Change from baseline at 10 weeks | 
| Blood serum insulin | Fasted | Change from baseline at 10 weeks | 
| Blood plasma Hemoglobin A1c (HbA1c) | Fasted | Change from baseline at 10 weeks | 
| Blood serum Triglycerides | Fasted | Change from baseline at 10 weeks | 
| Blood serum High-density lipoproteins (HDL) | Fasted | Change from baseline at 10 weeks | 
| Blood serum Low-density lipoproteins (LDL) | Fasted | Change from baseline at 10 weeks | 
| Blood serum C-reactive protein (CRP) | Fasted | Change from baseline at 10 weeks | 
| forkhead box protein O3 (FOXO3A) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| forkhead box protein O1 (FOXO1) mRNA mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| hepatocyte growth factor (HGF) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| insulin-like growth factor I (IGF1) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| myostatin (MSTN) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| E3 ubiquitin-protein ligase TRIM63 (TRIM63) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| p62/Sequestosome-1 mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| muscle RING-finger protein-1 (Murf-1) protein 1 (4EBP-1) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Atrogin1 mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Microtubule-associated protein 1A/1B-light chain 3 (LC3) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| BCL2/adenovirus E1B interacting protein 3 (BNIP3) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| PTEN-induced putative kinase 1 (PINK1) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| TNF receptor associated factor 6 (TRAF6) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| transcription factor EB (Tfeb) mRNA | Biopsies from m. Vastus Lateralis analyzed by western blot | Before and 2.5 hours after acute training session both at baseline and after 10 weeks | 
| Intramyocellular lipids | Oil-Red-O staining of muscle sections. Biopsy from m. Vastus Lateralis analyzed by immunohistochemistry | Change from baseline at 10 weeks | 
| Muscle fiber type distribution | Biopsy from m. Vastus Lateralis analyzed by immunohistochemistry | Change from baseline at 10 weeks | 
| Muscle fiber cross-sectional area | Biopsy from m. Vastus Lateralis analyzed by immunohistochemistry | Change from baseline at 10 weeks | 
| Muscle satellite cells | Biopsy from m. Vastus Lateralis analyzed by immunohistochemistry | Change from baseline at 10 weeks | 
| Myonuclei | Biopsy from m. Vastus Lateralis analyzed by immunohistochemistry | Change from baseline at 10 weeks | 
| Myonuclei number | Biopsy from m. Vastus Lateralis analyzed by confocal microscopy | Change from baseline at 10 weeks | 
| Myonuclei location | Biopsy from m. Vastus Lateralis analyzed by confocal microscopy | Change from baseline at 10 weeks | 
| Amount of mitochondria | Biopsy from m. Vastus Lateralis analyzed by confocal microscopy | Change from baseline at 10 weeks | 
| Location of mitochondria | Biopsy from m. Vastus Lateralis analyzed by confocal microscopy | Change from baseline at 10 weeks | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
Sarcopenia, Frailty, Strength training, Autophagy