Autophagy Flux: A Bottleneck in the Clearance of Damaged Organelles and Proteins after Skeletal Muscle Injury

Autophagy is a dynamic cellular recycling process that involves the formation of autophagosomes around damaged or dysfunctional organelles and proteins, the fusion of those autophagosomes with lysosomes, and the degradation of autophagolysosomes’ contents into their initial building block components (i.e. fatty acids, amino acids, and nucleotides) that can then be incorporated into the synthesis of new organelles or proteins. Current measures of autophagy generally involve quantifying autophagosome‐related protein content, however, this is a poor indicator of actual autophagy activity, or autophagy flux, because it fails to capture dynamic changes in autophagosome degradation. I have previously shown that autophagosome number increases exponentially with traumatic muscle injury, but the extent and mechanisms of increased autophagy flux after injury have not been fully elucidated. The purpose of this study was to investigate whether autophagy flux increases proportionally to meet the demand of numerous autophagosomes after injury and whether the autophagy initiating protein Unc‐51 like autophagy activating kinase (Ulk1) plays a role in regulating increases in autophagy flux. Autophagy flux was assessed through two different methods. First, by inhibiting lysosomal fusion through chloroquine (CQ) treatment. 4‐month‐old C57/BJ6 mice underwent unilateral traumatic freeze injury to the tibialis anterior (TA) muscle. 7 days post injury, mice were IP injected with 65mg/kg of CQ (n=6) or saline (n=8) and 2 hours later both injured and contralateral control limb TA muscles were harvested and prepped for immunoblots of LC3II (an autophagosome‐related protein). While injury did lead to a 7‐fold increase in LC3II expression overall (Main effect: Injury p<0.01), there was no significant interaction between CQ‐treated injured and saline‐treated injured limbs (p=0.19). Because the CQ results appeared to be somewhat inconclusive, a second more sensitive measure of autophagy flux was used involving a fluorescently labeled LC3 probe (GFP‐LC3‐RFP‐LC3ΔG plasmid) where the GFP portion is incorporated into the autophagosome and degraded with its contents while the RFP signal remains in the cytosol as an internal control. 3‐month‐old myogenin ‐Cre Ulk1 floxed mice (n=3) and their Cre negative littermates (n=3) were bilaterally transfected with GFP‐LC3‐RFP‐LC3ΔG plasmid. One month later, mice underwent unilateral freeze injuries and autophagy flux was assessed through the GFP:RFP ratio determined by in vitro two‐photon microscopy imaging of the TA muscle 7 days after injury. There was no difference observed between genotypes (p=0.48), however there was a 30% increase in the GFP:RFP ratio in the injured limbs (Main effect: Injury p=0.02) which suggest a reduction in autophagy flux. In conclusion, this data suggests that there is not a proportional increase in autophagy flux and there may even be a reduction in flux after injury. Therefore, inability to upregulate autophagy flux after injury may create a bottleneck leading to an accumulation of autophagosomes waiting to be degraded. Augmenting autophagy flux after muscle injury may provide a therapeutic target to enhance recovery especially in conditions where autophagy may be further reduced.