LAUSR

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that reverses glutathione (GSH) adducts of protein thiols generated through oxidative post-translational modifications. GSH-adducts regulate cellular signaling and transcription factors. We previously found that after femoral artery ligation Glrx knockout mice improved blood flow recovery in relation to activation of hypoxia-inducible factor (HIF)-1α via GSH adducts, indicating Glrx as an anti-angiogenic enzyme. Objective We hypothesized that local Glrx gene inhibition might improve poor blood flow recovery after ischemia in aging or diabetes. To inhibit Glrx gene expression in vitro and in vivo, we developed an improved protocol to produce adeno-associated viruses (AAVs) with a bicistronic expression system coding for Glrx shRNA (shGlrx) and mVenus, a yellow fluorescent protein. Methods Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation, aqueous two-phase partitioning, and an iodixanol gradient, which resulted in AAVs with adequate titer and purity for in vivo use. We tested the AAVs on C2C12 skeletal muscle cells and mouse gastrocnemius muscles. Results Quiescent confluent C2C12 cells transduced with a shGlrx-containing AAV exhibited a decrease of ~75% in the Glrx mRNA and protein levels after 4 days. Intramuscular injection (4.4 x 1012 viral genome/50 μl) of AAV-shGlrx transduced mVenus expression in skeletal muscle efficiently, but non-injected muscle, heart, or liver showed no mVenus expression. However, 4 weeks after intramuscular injection, Glrx protein levels remained unchanged compared to control-AAV injected or non-injected muscle, and inflammatory markers were increased. Six weeks after virus injection, Glrx expression in the muscle decreased 80% at the mRNA and 50% at the protein level. Conclusion Intramuscular injection of AAV-shGlrx resulted in muscle-specific attenuation of Glrx expression after at least 6 weeks. AAV intramuscular injection caused local inflammation in the early stage which may counteract the effects of shGlrx. (R01HL133013, R03AG051857, R01DK103750)