Abstract Body: Introduction: The severity of peripheral arterial disease (PAD) in women at advanced age compared to men indicates a possible role of circulating estrogen in skeletal muscle’s response to hypoxia. Hypoxia-inducible factor (HIF) 1α is a well-recognized contributor to myotubule stabilization and proliferation of satellite cells after hypoxia through transcriptional activation of angiogenic genes including VEGF. The effects of estrogen-mediated stimulation of GPER in this pathway have not been investigated in skeletal myocytes and prior work reveals conflicting effects in breast cancer cells and cardiomyocytes.
Hypothesis: We hypothesize that GPER plays a role in skeletal myocytic response to hypoxia through HIF1α stabilization in vivo and in vitro.
Methods: Hindlimb ischemia was induced through selective femoral artery ligation in homozygous male and female GPER knockout (-/-) and control C57BL/6 (WT) mice. Perfusion of the ischemic limb compared to the control limb was confirmed using laser speckle contrast imaging (LSCI). Calf muscle ischemic recovery was measured by myofibril diameter after 21 days. Human skeletal myoblasts were cultured and differentiated into skeletal myocytes. Cells were exposed to normoxic (21% O₂) or hypoxic (2% O₂) conditions for 18 hours. During exposure, cells were treated with GPER-specific agonist G1 or vehicle control. HIF1α and VEGFa were quantified by immunofluorescent staining.
Results: GPER^-/- and WT mice had similar response to femoral artery ligation by LSCI with 25% baseline perfusion initially and recovering to mean 70% baseline after 21 days. GPER^-/- mice had significantly reduced myofibril diameter compared to WT (23.3 vs 28.4 µm, P<0.0001). During in vitro studies, hypoxia induced HIF1α stabilization compared to normoxia, as expected. Upon treatment with G1, HIF1α stabilization was further significantly increased relative to control at all concentrations ranging from 1-1000 nM. VEGFa expression was noted to be significantly increased at all concentrations other than 100nM and 1000nM, with the most significant stabilization relative to control noted at 10 nM concentrations.
Conclusion: GPER^-/- animals had greater muscle ischemic injury despite normal perfusion recovery. GPER activation augments HIF1α in skeletal myocyte response to hypoxia. Further study is necessary to understand the biochemical pathways by which GPER stabilizes HIF1α and how this may inform future clinical treatments in PAD.