Atorvastatin Impairs Pathways Associated With Skeletal Muscle Function and Health in Myoblasts From Older Adults

Background: Statins are widely prescribed to reduce low‐density lipoprotein (LDL) cholesterol to decrease the risk of cardiovascular disease. However, there are ongoing concerns surrounding the frequently reported skeletal muscle side effects. These include muscle pain, weakness and reduced function and are defined as statin‐associated muscle symptoms (SAMS). This study aimed to characterise the biological processes, which underlie SAMS through analysing in vitro muscle cell phenotypic and transcriptomic effects of atorvastatin, the most prescribed statin, using human myoblasts from older adults. Methods: Human myoblasts were isolated from vastus lateralis biopsies of 11 female older adult participants (average age 78.4 years) who were not on statin therapy from the Hertfordshire Sarcopenia Study extension (HSSe). Myoblasts were treated with 1, 5, or 10 μM atorvastatin for 4 days in proliferating or differentiating cultures. In proliferating cells, cytotoxicity, senescence and proliferation were measured using LDH cytotoxicity, β‐galactosidase (β‐gal) and 5‐ethynyl‐2′‐deoxyuridine (EdU) assays. To understand the influence of atorvastatin treatment across myoblast differentiation, immunocytochemistry (ICC) was undertaken analysing Myogenic Differentiation 1 (MyoD), Myogenin (MyoG) and Myosin Heavy Chain (MyHC). RNA sequencing (RNA‐seq) was performed on a subset of 10 differentiating myoblast cultures treated with 10 μM atorvastatin followed by gene ontology and protein–protein interaction (PPI) pathway analysis (Metascape). Results: Atorvastatin treatment was not significantly toxic to myoblasts at any of the concentrations tested (1 μM p = 0.32, 5 μM p = 0.21 or 10 μM p = 0.76). Senescence increased with atorvastatin at 5 μM (p = 1.95 × 10−4) and 10 μM (p = 9.77 × 10−4). Myoblast proliferation decreased at all of 1 μM (p = 1.86 × 10−2), 5 μM and 10 μM (p = 9.77 × 10−4) concentrations. In differentiating cells, ICC identified MyoD significantly decreased with 10 μM atorvastatin; decreased MyoG at 1, 5, and 10 μM atorvastatin (p = 9.77 × 10−4); and decreased MyHC at 1 μM (p = 3.2 × 10−2), 5 μM (p = 2.93 × 10−3) and 10 μM atorvastatin (p = 1.37 × 10−2). RNA‐Seq analysis following 10 μM atorvastatin treatment in differentiating myoblast cultures revealed 822 genes upregulated and 888 genes downregulated in expression (false discovery rate [FDR] < 0.05). Pathway and MCODE analysis identified key networks downregulated, including muscle contraction and cell cycle process, and upregulated pathways implicated in cholesterol and fatty acid synthesis. Conclusions: These findings show that atorvastatin treatment negatively impacts skeletal muscle at the cellular level by disrupting many key gene regulatory pathways involved in muscle maintenance, function and health. Identification of such disruption, which likely underpins SAMS, provides novel molecular mechanisms, which could be targeted through pharmaceutical/nutraceutical interventions to reduce the negative effects of statins on skeletal muscle health.