Knockdown of low-density lipoprotein receptors in skeletal muscle attenuates aging-related sarcopenia associated with mitochondrial fusion and ferroptosis

Background: Age-related sarcopenia is defined by a gradual decline in skeletal muscle mass and strength, typically involving a reduction in muscle fibers and an increase in intramuscular fat. Lipid accumulation is suggested to be a potential mechanism that may contribute to mitochondrial dysfunction and subsequently lead to sarcopenia. While previous studies have shown the accumulation of low-density lipoprotein receptor (LDLR) in the skeletal muscles of aged rats, a specific connection between LDLR and age-related sarcopenia has not been investigated. This study aimed to investigate the effects of LDLR knockdown on skeletal muscle. Methods: Wild-type and LDLR skeletal muscle-specific knockdown mice were randomly divided into adult and old groups. The control group consisted of adult and old mice that were injected with AAV-gRNA empty vector virus. The grip strength was measured before sacrifice. Following scarification, skeletal muscles were collected for atrophy assessment using histopathological and immunofluorescent methods. Mitochondria were isolated from skeletal muscle and their morphology and ROS levels were assessed. LDLR expression, atrophy-related proteins, mitochondrial fission, and fusion-related proteins, and ferroptosis pathway were measured by western blotting. Results: In aged mice, there was a significant decrease in muscle mass normalized to body weight (1.3±0.04 vs 1.5±0.05 %, p < 0.05) and forelimb grip strength (2.01±0.13 vs 2.38±0.08 g/g, p < 0.05) as well as increased levels of lipofuscin, mitochondrial ROS (3924±369 vs 2527±326 a.u., p < 0.01) and the ferroptosis-related protein, ACSL4, in the quadriceps muscle, when compared to adult mice. Following LDLR knockdown, there was an increase in muscle mass normalized to body weight (1.50±0.02 vs 1.36±0.03%, p < 0.01), particularly in fast-twitch muscle fibers, as well as an increase in forelimb grip strength (2.34±0.05 vs 1.97±0.11 g/g, p < 0.05) in LDLR knockdown aged mice (O-LDLR ^KD group), when compared to the old mice injected with empty vector (O-LDLR ^vector group). Additionally, lipofuscin levels and the atrophy-related protein, MuRF, were decreased in the O-LDLR ^KD group compared to the control ^{group. Mitochondrial ROS and the Drp1 mitochondrial fission protein ( p < 0.01) levels were significantly decreased, while the Mfn2 mitochondrial fusion protein levels increased ( p < 0.05). Among the ferroptosis-related markers, ACSL4 showed a marked decrease ( p ＜0.01), while SLC7A11 increased ( p ＜0.05) in the O-LDLR KD group compared to the O-LDLR vector group. Conclusions: Our results suggest that LDLR-specific knockdown in skeletal muscle can attenuate muscle atrophy and loss of strength in aged mice, potentially associated with enhanced mitochondrial fusion and suppressing ferroptosis.}