Role of HIF-1α in Skeletal Muscle Aging

Study Overview
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Brief Summary
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Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Sarcopenia is the age-related loss of skeletal muscle mass and function. It is now recognised as a major clinical problem for older people and research in the area is expanding exponentially. This interest stems from the fact that sarcopenia is both common and associated with serious health consequences in terms of frailty, disability, morbidity and mortality. The age-related loss of human skeletal muscle mass is due to a decrease in myofibre size and number with the loss of both fast and slow type myofibres, although the loss of fast myofibres tends to start earlier, at ∼70 years. Many factors influence the decrease in muscle mass. A significant contributor is an anabolic resistance of older skeletal muscle to protein nutrition as seen during immobilisation which can be ameliorated at least in part by resistance exercise and dietary supplementation. Other intensive areas of research are related to the loss of innervation and oxidative damage. Moreover, ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. It is now widely accepted that satellite cells, and generally adult stem cells, are normally quiescent and tend to reside in hypoxic areas of the tissue to preserve their undifferentiated state. To govern these processes, cells have developed a very complex machinery that is mainly regulated by a group of transcription factors known as hypoxia-inducible factors (HIFs). In particular, several observations support the idea that oxygen deprivation and HIF-1a may play a key role during ischemia to activate the regeneration process, which, after an initial hypoxic insult, needs to proceed under normoxia. On these bases, in this study we will investigate the role of HIF-1a in skeletal atrophy during aging.

Detailed Description
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Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Sarcopenia is the age-related loss of skeletal muscle mass and function. It is now recognised as a major clinical problem for older people and research in the area is expanding exponentially. This interest stems from the fact that sarcopenia is both common and associated with serious health consequences in terms of frailty, disability, morbidity and mortality. The age-related loss of human skeletal muscle mass is due to a decrease in myofibre size and number with the loss of both fast and slow type myofibres, although the loss of fast myofibres tends to start earlier, at ∼70 years. Many factors influence the decrease in muscle mass. A significant contributor is an anabolic resistance of older skeletal muscle to protein nutrition as seen during immobilisation which can be ameliorated at least in part by resistance exercise and dietary supplementation. Other intensive areas of research are related to the loss of innervation and oxidative damage. Moreover, ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. It is now widely accepted that satellite cells, and generally adult stem cells, are normally quiescent and tend to reside in hypoxic areas of the tissue to preserve their undifferentiated state. To govern these processes, cells have developed a very complex machinery that is mainly regulated by a group of transcription factors known as hypoxia-inducible factors (HIFs). In particular, several observations support the idea that oxygen deprivation and HIF-1a may play a key role during ischemia to activate the regeneration process, which, after an initial hypoxic insult, needs to proceed under normoxia. On these bases, in this study we will investigate the role of HIF-1a in skeletal atrophy during aging. In particular, we will isolate satellite cells from muscular biopsies harvested from old sarcopenic patients and from young patients. The, we will measure HIF-1a levels and we will compare them.

Official Title
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Role of Hypoxia Inducible Factor-1α (HIF-1α) in Skeletal Muscle Aging

Conditions
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Sarcopenia

Intervention / Treatment
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* Biological: Harvesting of muscular biopsies


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Sarcopenic patients (measured by DXA) affected by hip osteoarthritis, developmental dysplasia of the hip or hip fracture and undergoing hip replacement surgery (for the sarcopenic group) Patients affected by traumatic ACL tears and undergoing ACL reconstruction surgery (for the control group) Patients between 18 and 35 years old (for the control group) Patients between 65 and 90 years old (for the sarcopenic group) 18 ≤ Body Mass Index (BMI) ≤ 30 kg/m2 Exclusion Criteria: Diseases that can affect bone or muscle metabolism Pharmacological therapies that can interact with bone or muscle metabolism Bone metastases Bone infections HIV, hepatitis B virus, hepatitis C virus or Treponema pallidum positivity BMI ≥30kg/m2

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 90 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Sarcopenic group<br>Harvesting of muscular biopsies Muscular biopsies will be harvested from old sarcopenic patients undergoing hip replacement surgery | Biological: Harvesting of muscular biopsies<br>* Muscular biopsies will be harvested during surgery; them satellite cells will be isolated and characterized in vitro.<br>|
| Control group<br>Harvesting of muscular biopsies Muscular biopsies will be harvested from young patients undergoing Anterior Cruciate Ligament (ACL) reconstruction surgery | Biological: Harvesting of muscular biopsies<br>* Muscular biopsies will be harvested during surgery; them satellite cells will be isolated and characterized in vitro.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Evaluation of HIF-1a expression levels in satellite cells isolated from sarcopenic and young | Western blot analysis of HIF-1a levels and of its downstream targets (Vascular Endothelial Growth Factor: VEGF, phosphoglycerate kinase: PGK, Prolylhydroxilases: PHD2) | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Evaluation of sarcopenic profile in satellite cells harvested from old sarcopenic patients | Real Time polymerase chain reaction: PCR analysis of MURF1 and atrogin1 (markers of muscular aging) | 6 months | 
| Correlation of HIF-1a levels with the degree of sarcopenia in the satellite cells of old sarcopenic patients | chromatin immunoprecipitation: ChiP seq analysis of HIF-1a downstream targets | 1 year | 
| Evaluation of differences in satellite cells number between young and old patients | To evaluate differences in cell number two different immunohistochemical analyses will be used in order to obtain a ratio: 1. Immunofluorescence for PAX7 (satellite cell markers) and 2. BrdU will be performed and the ration between PAX7 positive cells and bromodeoxyuridine: BrdU positive cells will be calculated. | 6 months | 
| Evaluation of HIF-1a stabilization on satellite cells treated with PHDs inhibitors | Prolylhydroxilases: PHDs inhibitors (FG-4592 e PLG) will be tested in vitro to analyze HIF-1a downstream targets (Vascular Endothelial Growth Factor: VEGF, phosphoglycerate kinase: PGK, Prolylhydroxilases: PHD2) by real time PCR | 1 year | 
| Evaluation of satellite cell proliferation in cells treated with PHDs inhibitors | Prolylhydroxilases: PHDs inhibitors (FG-4592 e PLG) will be tested in vitro to analyze cell proliferation (RealTime Glow and CellTox kits) | 1 year | 
| Evaluation of satellite cell differentiation in cells treated with PHDs inhibitors | Prolylhydroxilases: PHDs inhibitors (FG-4592 e PLG) will be tested in vitro to analyze cell differentiation (Immunofluorescence and Realt Time PCR analysis for differentiation markers: MyoD, Myogenin and Myosin Heavy Chain) | 1 year |