24Autophagy plays an important role in mitochondrial maintenance, yet many details of skeletal 25 muscle autophagic activity are unresolved in the context of muscle stress and/or damage. 26Skeletal muscles from mice were stressed either by fatiguing contractions, eccentric contraction-27 induced injury (ECCI), or freeze injury (FI) to establish a timeline of mitochondrial function and 28 autophagy induction after different forms of muscle stress. Only FI was sufficient to elicit a 29 reduction in mitochondrial function (-88%, p=0.006), yet both ECCI and FI resulted in greater 30 autophagy-related protein content (28-fold, p0.008) suggesting a tunable autophagic response. 31Muscles from another cohort of mice were used to determine specific forms of autophagy, i.e., 32 flux and mitochondrial-specific, in response to muscle damage. Mitochondrial-specific 33 autophagy was evident by accumulation of autophagy-related proteins in mitochondrial-enriched 34 muscle fractions following FI (37-fold, p=0.017); however, autophagy flux, assessed by LC3II 35 accumulation with the lysosomal inhibitor chloroquine, was insignificant suggesting a 36 physiological bottleneck in the clearance of dysfunctional organelles following FI. Ulk1 muscle-37 specific knockout (Ulk1 MKO) mice were used to determine if autophagy is necessary for the 38 recovery of mitochondrial function after muscle damage. Ulk1 MKO mice were weaker (-12%, 39 p=0.012) and demonstrated altered satellite cell dynamics (e.g., proliferation) during muscle 40 regeneration after FI compared to littermate control mice, but determination of autophagy 41 necessity for the recovery of mitochondrial function was inconclusive. This study concludes that 42 autophagy is a tunable cellular response to muscle damaging stress and may influence muscle 43 fiber regeneration through interaction with satellite cells. 44 45 46 Key Points Summary 47 Muscle contractility dysfunction is well characterized after many different types of 48 muscle stress however, the timing and magnitude of mitochondrial dysfunction and 49 autophagy induction after different types of muscle stress is largely unknown. 50 In this study we found that only traumatic freeze injury causes mitochondria dysfunction 51 compared to fatigue contractions and eccentric contraction-induced injury, and that the 52 autophagic response to muscle stress scales to the magnitude of muscle damage, i.e., 53 freeze vs. eccentric contraction-induced injury. 54  We determined that total autophagy-related protein content has a greater response to 55 muscle fiber damage compared to autophagy flux likely reflecting a bottleneck of 56 autophagosomes awaiting degradation following muscle injury. 57  Using a skeletal muscle-specific autophagy knockout mouse (Ulk1), we found that 58 muscle contractility and satellite cell activity might be influenced by cellular events 59 within the adult muscle fiber following muscle damage. 60 61 62 63 64 157