M.A. Moore1, S. Hayes1, B. Akpinar1, Y. Li1 1Western Michigan University Homer Stryker MD School of Medicine, Kalamazoo, MI, USA
Introduction: Muscle cell hypoxic preconditioning (HP) is a strategy that uses transient exposure to low oxygen conditions to improve cell survival in subsequent states of stress. HP involves a balance between the processes of autophagy, a catabolic process that recycles intracellular components, and apoptosis, a programmed cell death pathway that eliminates damaged cells. Autophagy and apoptosis have similar but conflicting roles in muscle cells. Both pathways can be upregulated in response to nutrient deprivation to remove damaged cellular components and the protein X-linked inhibitor of apoptosis (XIAP) is involved in both pathways. Apoptosis can recycle damaged cellular components under sub-lethal states of stress or nutrient deprivation but cells may be overwhelmed under extended or severe states of stress, resulting in the accumulation of damaged proteins and triggering apoptotic cell death. Understanding the interplay of autophagy and apoptosis is important for designing HP procedures to optimize the beneficial adaptations of muscle cells without reaching the point of inducing cell death. This has applications in improving muscle cell resilience to ischemic conditions. This study investigates the molecular pathways involved in HP of muscle cells, aiming to understand the interplay of autophagy and apoptosis in cells under hypoxic stress.
Methods: C2C12 cells were cultured for 48 hours in either hypoxia (1% or 5% oxygen) or in normoxia (21% oxygen) and with or without an autophagy inhibitor chemical 3-methyladenine (3-MA). Expression of Bcl-2, Bax, Caspase 9, Beclin-1, and XIAP were measured via qPCR. Protein expression was confirmed via Western Blot.
Results: C2C12 cells showed increased expression of XIAP, Bcl-2, and Beclin-1 under hypoxia, indicating an upregulation in autophagy. Cells cultured with 3-MA in hypoxia showed similar expression of autophagy-related Beclin-1 as cells cultured in normoxia. Cells cultured in hypoxia showed decreased expression of apoptosis-related Bax and Caspase 9, indicating less flux through autophagic pathways.
Conclusion: Hypoxic exposure induces higher expression of autophagy-related markers in C2C12 cells and decreased expression of apoptosis-related markers after 48 hours of hypoxic exposure. These findings support the idea that hypoxia can enhance flux through autophagic pathways to recycle cellular components and act as a protective mechanism against states of stress. Conversely, the decreased expression of apoptosis-related markers suggests that apoptotic cell death may be suppressed under transient hypoxic conditions.