Skeletal muscle mass and function depend on a balanced coordination of protein synthesis and degradation. The autophagy‑initiating kinases ULK1 and ULK2 were investigated as candidate regulators of broad protein metabolism in skeletal myofibers. The study tested whether ULK1/2 jointly modulate autophagy flux, anabolic signaling, and rates of protein synthesis and degradation to influence muscle size and quality.
The authors used mouse models with skeletal muscle‑specific knockout of Ulk1 and Ulk2 (Ulk1/2skmDKO) and wild‑type (WT) littermates. Experiments included both lifelong deficiency and short‑term knockdown in adult muscle achieved by electroporating plasmids encoding microRNAs targeting Ulk1 and Ulk2 into 4‑month‑old wild‑type mice.
Protein metabolic fluxes were quantified using deuterium oxide (D2O) labeling to estimate synthetic (Ksyn) and degradative (Kdeg) rates in subcellular protein pools (myofibrillar, mitochondrial, sarcoplasmic). Autophagy flux was assessed biochemically via LC3‑II accumulation in response to colchicine. Anabolic signaling responses were probed after insulin and leucine administration to assess mTORC1 and AKT pathway activities. Physiological outcomes included fiber histology (central nucleation, MyHC fiber types and diameters) and contractile force of dorsiflexor and plantar flexor muscles.
Lifelong Ulk1/2 deficiency produced a marked impairment in autophagy flux: LC3‑II accumulated with colchicine only in WT mice (P<0.001), demonstrating defective flux in Ulk1/2skmDKO animals.
Histologically, lifelong Ulk1/2 loss increased centrally nucleated fibers, reflecting compromised muscle quality. Central nucleation rose from 0.1% to 4.5% in females and from 0.8% to 22.7% in males (P<0.001), with changes primarily involving MyHC type 2b fibers. Functionally, lifelong Ulk1/2 deficiency decreased contractile force: male dorsiflexors and plantar flexors showed ~20% lower force (P<0.01), and female plantar flexors showed ~24% lower force (P<0.01).
Despite these deficits in quality and strength, Ulk1/2 deficiency drove substantial hypertrophy: diameters of all major MyHC fiber types increased in the tibialis anterior and soleus by approximately 10–15% in males and 14–20% in females (P<0.05).
Short‑term Ulk1/2 knockdown in adult muscle (up to 4 weeks) also produced myofiber hypertrophy (≈13%, P<0.05) but did not produce increased central nucleation or measurable impairments in contractile force, suggesting an early phase in which hypertrophy occurs without immediate loss of muscle quality.
Using D2O labeling, the investigators identified altered protein turnover that favors net accretion in Ulk1/2‑deficient muscle. Myofibrillar protein synthesis was higher (Ksyn increased by 23%, P<0.05). Concurrently, degradation rates were reduced in non‑myofibrillar pools: mitochondrial Kdeg was ~16% lower (P = 0.09) and sarcoplasmic Kdeg was ~14% lower (P<0.05). These combined changes—enhanced myofibrillar synthesis with lowered mitochondrial and sarcoplasmic degradation—provide a mechanistic basis for increased fiber size despite impaired autophagy flux.
Hypertrophy observed with Ulk1/2 deficiency was accompanied by increased activity of mTORC1, a central anabolic regulator, occurring independently of AKT activation. The data indicate that ULK1 and ULK2 normally act to limit mTORC1‑driven protein synthesis while sustaining autophagy; loss of ULK1/2 removes that restraint, permitting elevated mTORC1 signaling and myofibrillar protein synthesis.
The temporal comparison in the study highlights a tradeoff. Short‑term Ulk1/2 deficiency in adult muscle produced hypertrophy without immediate evidence of impaired muscle quality or force, suggesting a window in which growth occurs without functional cost. In contrast, lifelong deficiency compromised autophagy flux, increased markers of poor muscle quality (central nucleation), and reduced force, indicating that chronic loss of ULK1/2 leads to accumulation of structural/functional deficits despite larger fiber size.
Collectively, the results support a model in which ULK1 and ULK2 jointly sustain autophagy and restrain mTORC1‑driven protein synthesis to coordinate protein turnover in skeletal muscle. Lifelong loss increases muscle size at the expense of quality and contractile function, while short‑term loss allows hypertrophy without immediate functional decline. The authors propose ULK1/2 as a regulatory node that could be investigated for therapeutic strategies targeting atrophy and weakness.
The source reports no competing interests. Funding sources reported in the preprint include the National Institutes of Health, a Fraternal Order of Eagles Diabetes Research Center award, the University of Iowa Office of the Vice‑President for Research, and the U.S. Department of Veterans Affairs. Details of experimental protocols, sample sizes, and additional data are those provided in the preprint source.