Metformin Attenuates Statin Associated Myopathy by Restoring Mitochondrial Function and Oxidative Stress in C2C12

다국어 초록 (Multilingual Abstract)

Statin associated muscle symptoms (SAMS) affect 10-25% of statin users and represent the primary reason for treatment discontinuation, significantly increasing cardiovascular risk. Atorvastatin, an HMG-CoA reductase inhibitor, is the most prescribed medication in the United States, while metformin ranks second, making their potential interaction clinically significant. However, no experimental study has examined combined treatment effects in skeletal muscle cells. This study investigated whether metformin co-treatment attenuates statin induced myopathy in differentiated C2C12 myotubes. To establish a statin associated myopathy model, C2C12 myotubes were treated with 20 µM atorvastatin for 24 hours, which significantly increased atrogin-1 expression, an established SAM marker, confirming myopathy induction. Atorvastatin upregulated atrogin-1, disrupted myotube morphology and elevated cellular and mitochondrial reactive oxygen species levels. Co-treatment with 75 µM metformin significantly restored these alterations. To elucidate the molecular mechanisms, next-generation sequencing (NGS) analysis was performed. The analysis identified Mss51, an inhibitor of mitochondrial respiration, and Gdf11, a muscle atrophy promoter, as key differentially expressed genes. Quantitative PCR confirmed statin induced upregulation of Mss51, which was reduced by metformin. To investigate the functional implications of these differentially expressed genes, gene Ontology pathway analysis revealed that atorvastatin upregulated genes related to neuromuscular signaling and mitochondrial transport while downregulating genes involved in immune response, cell cycle, and insulin metabolism; metformin partially normalized these changes. To investigate the protective mechanism, comprehensive mitochondrial functional assays were performed. Atorvastatin impaired mitochondrial respiratory capacity and decreased mitochondrial membrane potential while elevating mitochondrial ROS production. Metformin co-treatment restored mitochondrial respiration and normalized mitochondrial membrane potential while reducing mitochondrial ROS production.
Our findings demonstrate that metformin protects against atorvastatin-induced myopathy by restoring mitochondrial function, suppressing oxidative stress, and preventing muscle atrophy. Metformin's protective effects are primarily mediated through preservation of mitochondrial integrity and function, highlighting its potential utility in preventing SAMS in patients requiring statin therapy.

목차 (Table of Contents)

• TABLES OF CONTENTS
• ABSTRACT ⅳ
• TABLES OF CONTENTS ⅶ
• LIST OF FIGURES ⅺ
• LIST OF TABLES ⅻ
• I. Introduction 1
• 1. Statins and their mechanism of action 1
• 1.1 Pharmacological mechanisms of statins 1
• 2. Statin associated myopathy (SAM) 3
• 2.1 Pathophysiological features and mechanisms of myopathy 3
• 2.2 Mitochondrial dysfunction and oxidative stress in SAM 6
• 3. Protective effects of metformin on skeletal muscle 8
• 3.1 Mechanism of action of metformin 8
• 3.2 Regulatory effects of metformin on SAM 10
• 4. Research purpose 12
• II. Materials and Methods 13
• 1. Cell culture and treatments 13
• 2. Measurement of intracellular ROS production using DCFDA. 14
• 3. Immunofluorescent staining of C2C12 myotubes 15
• 4. Myotube morphology assessment 16
• 5. Western blot 17
• 6. Assessment of cell viability 18
• 7. Total RNA preparation and quantitative reverse transcription PCR 19
• 8. Seahorse XF-based analysis of mitochondrial respiration 21
• 9. Next generation sequencing (NGS) analysis 22
• 10. Mitochondrial membrane potential assay 23
• 11. Measurement of ROS production in mitochondrial using MitoSOX Red 24
• 12. Statistical analysis 25
• III. Results and Discussion 26
• 1. Determination of optimal atorvastatin and metformin concentration in C2C12 Cells 26
• 2. Metformin alleviates atorvastatin-induced muscle atrophy and improves myotube morphology 28
• 3. Metformin reduces oxidative stress induced by atorvastatin in C2C12 cells 33
• 4. NGS-based gene expression profiling of atorvastatin and metformin-treated C2C12 cells 37
• 5. Metformin restores mitochondrial respiratory capacity impaired by atorvastatin 45
• 6. Metformin improves mitochondrial function by restoring membrane potential and reducing mitochondrial ROS 49
• IV. Conclusion 55
• V. References 58
• 국문 초록 62