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

Knockdown of low-density lipoprotein receptors in skeletal muscle attenuates aging-related sarcopenia associated with mitochondrial fusion and ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Knockdown of low-density lipoprotein receptors in skeletal muscle attenuates aging-related sarcopenia associated with mitochondrial fusion and ferroptosis Ziyang Fang, Tao Feng, Xin Zhang, Xiaoyan Fang, Ying Li, Yinjun Luo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6891536/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract 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. Health sciences/Medical research Health sciences/Pathogenesis Sarcopenia Aging LDLR Skeletal muscle Mitochondrial dynamics Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Age-related sarcopenia, a progressive loss of skeletal muscle mass and strength occurring with aging, frequently results in disabilities, loss of independence and death[ 1 ]. The prevalence of sarcopenia in the elderly population will rise as the global population ages[ 2 , 3 ], and there is an urgent need to develop effective preventive and therapeutic strategies against this condition. Previous studies have found that abnormal lipid metabolism was closely related to muscle dysfunction and poor physical performance in older people with sarcopenia[ 4 , 5 ]. The low-density lipoprotein (LDL) receptor (LDLR) is a cell-surface glycoprotein that facilitates the internalization of LDL into cells via endocytosis. In general, the cholesterol carried by LDL is specifically transported into the mitochondria, where the biosynthesis of steroid hormones occurs[ 6 ]. However, increased cholesterol levels within mitochondria can impair mitochondrial function[ 7 ]. In order to maintain their normal morphology and functions, mitochondria constantly undergo the two essential processes of fusion and fission. The balance of mitochondrial fusion and fission is critical for preserving optimal muscle function, as increased mitochondrial fission has been associated with the progression of skeletal muscle atrophy[ 8 ]. Statins, a group of lipid-lowering drugs for treating hypercholesterolemia, inhibit cellular cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl (HMG) coenzyme-A reductase, and up-regulating the LDLRs in the liver and peripheral tissues, resulting in increased blood LDL cholesterol (LDL-C) clearance[ 9 , 10 ]. However, side effects of statins on muscle tissues are well-known and range from myalgia and cramping to an increase in creatine kinase (CK), myopathy, rhabdomyolysis and exacerbation of myasthenia gravis[ 11 , 12 ]. Marco and colleagues[ 13 ]reported that LDLR was increasingly expressed in the gastrocnemius muscle in adult male rats after being treated chronically with statins, which may provide the key link between LDLR and statins-induced myopathy. Although LDLR accumulation in the skeletal muscle s was found in male and female old rats[ 13 ], the underlying molecular mechanisms have not been clarified. Interestingly, a recent study reported that mitochondrial outer membrane protein phospholipase D6 (PLD6) hydrolyzes cardiolipin into phosphatidic acid facilitating the fusion of LDL-LDLR vesicles with mitochondria and accelerates the degradation of LDLR[ 14 ]. This revealed a novel link between mitochondrion and LDLR homeostasis. An interesting question arises as to whether LDLR accumulation in the aging skeletal muscle represents a potential mechanism that could lead to mitochondrial dysfunction and, in turn, to sarcopenia. However, the relationship between LDLR in the skeletal muscle and age-related sarcopenia has not been investigated. In this study, we speculated that a low expression of LDLR in skeletal muscles may attenuate aging-related skeletal muscle mass loss and muscle dysfunction. Therefore, the aim of this study was to investigate the effects of LDLR knockdown on skeletal muscle and to uncover the underlying mechanisms involved in this process. Materials and Methods Procedures on animals and AAV injections Tow-month-old C57BL/6J male mice were purchased from Viton Lihua Laboratory Animal Technology Company (Beijing, China), and sixteen-month-old C57BL/6J male mice were purchased from Ziyuan Laboratory Animal Technology Company (Hangzhou, China). 3 to 4 mice were raised in each cage and maintained under the conditions of 24 ± 1°C and relative humidity of 50–60%, with a light/dark cycle of 12:12 hours, and they were fed with standard feed and water ad libitum. Adeno-associated viruses (AAVs) were administered via the tail vein after adaptation feeding until the adult C57BL/6 mice reached 3 months and the aged animals reached 19 months. All AAVs used in this study were of Serotype 9, which is a serotype with high levels of luciferase expression in the lower extremity skeletal muscles, and they exhibit a fast expression rate and maintain stable tissue expression[ 15 ]. The construction of a plasmid AAV-U6 > mLdlr-U6 > mLdlr backbone-based LDLR knockdown vector (LDLR KD ) was achieved by modifying the AAV-U6gRNA1-U6gRNA2-TnT-Cre adeno-associated viral vector (Addgene, #87682). The pAAV-tMCK-Cas9 vector was modified from the pAAV-nEFCas9 plasmid (Addgene, #87115) by inserting skeletal-muscle-specific muscle creatine kinase (MCK) promoter. The plasmids AAV-U6 > mLdlr-U6 > mLdlr and pAAV-tMCK-Cas9 were combined with autoclaved PBS as the solvent at a ratio of 1:1, with each vector having a titer of 1 x 10 12 . This mixture necessitates only a single injection to effectively knock down LDLR in the skeletal muscles of both adult and aged mice. Empty vectors AAV-U6-U6 without carrying LDLR gRNA sequences were prepared simultaneously as control. Adult mice were randomly divided into two subgroups: the A-LDLR KD (adult mice co-injected with AAV-U6 > mLdlr-U6 > mLdlr and pAAV-tMCK-Cas9 vectors to knockdown LDLR in the skeletal muscles, n = 11) group, and the A-LDLR vector (adult mice co-injected with empty vector AAV-U6-U6 and pAAV-tMCK-Cas9 as control, n = 9) group. Old mice were then randomly divided into two subgroups: the O-LDLR KD (old mice injected with AAV-U6 > mLdlr-U6 > mLdlr and pAAV-tMCK-Cas9 vectors to knockdown LDLR in the skeletal muscles, n = 8) group, and the O-LDLR vector (old mice injected with empty vector AAV-U6-U6 and pAAV-tMCK-Cas9 as control, n = 6) group. The mice that had completed their course of tail vein injections, were maintained and they were housed in an SPF environment with the same feed and drinking patterns. Particular attention was paid to observing whether the wound became infected, and the tail tip was ischemia. After 8 weeks of rearing, a muscle strength test was performed at 12 hours before death. All animal procedures were approved by the Institutional Animal Care and Use Committee of Youjiang Medical University for Nationalities (Approval number: 2022100801).The licensing committee approving the experiments, including any relevant details. The study was performed with regulations and by the ARRIVE guidelines. We confirm that all methods were carried out in accordance with relevant guidelines and regulations. Forelimb grip strength Prior to testing, mice were allowed to acclimate to the handgrip tester for 5 minutes. The forelimbs of the mouse were positioned on the sensing bar of the grip dynamometer. Once the forelimbs grasped the crossbar, the mouse's tail was pulled backwards horizontally until the forelimbs were released. The grasp force meter automatically recorded the maximum grasping force exerted by the mouse on the crossbar. The ratio of grip strength to body weight was used to calculate the relative grip strength, allowing for normalization of the grip strength measurement. Forelimb suspension test To assess the muscular endurance of mice, we conducted a forelimb suspension test. The mouse's tail was pinched to ensure that its forelimbs grasped the suspension wire securely. After confirming that the forelimbs were firmly gripping the rod, the mouse's tail was released. Immediately after releasing the mice, a timer was initiated to measure the duration from the moment the mouse's forelimbs were suspended from the wire until they fell. Three trials were conducted for each mouse, with a 5-minute interval between trials. The relative suspension time was calculated as the ratio of forelimb suspension time to body weight. Biochemical measurements Blood samples were collected via the retro-orbital sinus in animals under terminal anesthesia. The isolated plasma was centrifuged at 1500rpm for 20 minutes at 4°C and kept at 80°C until the assays were performed. LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triacylglycerol (TG) levels were determined by the colorimetric method (Elabscience) following the manufacturer's protocols. Tissue Preparation and Collection Under baseline conditions, the muscle tissues were extracted from adult (5-month-old) and old (21-month-old) mice. The quadriceps muscles were immediately dissected and cleaned to remove blood and fur before being snap-frozen in liquid nitrogen and stored at -80 ℃ before the examination. Separation of mitochondria Mitochondrial separation was carried out following the manufacturer's instructions (Beyotime). Briefly, 120 mg of the quadriceps muscle was minced and homogenized in an EP tube containing a cold isolation buffer together with pancreatic extracts. After rotating the mitochondrial suspension at 600g for 10 minutes, the supernatants were centrifuged at 11000g for 10 minutes. The mitochondria obtained were frozen at -80 ℃ for future analysis. Mitochondria ROS measurement The Reactive Oxygen Species Assay Kit was used to determine mitochondrial ROS levels (Beyotime, China). Measurement of mitochondrial ROS was performed as described previously[ 16 ]. Total Mitochondrial ROS was measured using dichlorofluorescein diacetate from mitochondria (DCFH-DA, Beyotime). Briefly, the mitochondrial storage solution containing mitochondria was incubated at 37°C for 20 minutes together with 10 µM DCFH-DA and the fluorescence was measured on a microplate reader. Electron microscopy The isolated mitochondria were collected and fixed in 2.5% glutaraldehyde (H7650, Japan, Hitachi). Subsequently, the mitochondrial pellets were treated with 1% osmic acid for 2 hours at ambient temperature. The pellets then underwent dehydration through a series of increasing ethanol concentrations. Following dehydration, the mitochondria were embedded in Epon-Araldite resin. Finally, ultrathin sections were mounted onto copper grids, stained with uranyl acetate and lead citrate, and examined via transmission electron microscopy (H-7650, Hitachi, Japan). RNA extraction and quantitative real-time PCR The RNA was extracted from skeletal muscles by using TIANGEN reagent and following the manufacturer's instructions. The concentration and purity of RNA were assessed by measuring the absorption at 260/280 nm. To synthesize cDNA, 1g of total RNA was reverse transcribed with HiScript® III RT Super Mix for qPCR (+ gDNA wiper) cDNA reverse transcription reagent (R323-01, Vazyme, China). Real-time PCR was carried out using Light Cycler96 (Roche Diagnostics) and Brilliant III Ultra-Fast SYBR QPCR master mix amplification kit (B21202, Bimake, China). The primers utilized are listed in Supplementary Table 1 . Each reaction contained 0.5µL of both forward and reverse primers, 2µL of cDNA, with a final volume of 10µL. All primers exhibited optimal amplification efficiencies (within the range of 90–110%). A control PCR for the housekeeping gene, alpha-actin, was performed. The thermocycling conditions included initial denaturation at 95˚C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 60 s, extension at 72°C for 15 s, and a final extension at 95°C, 60°C for 1 min, and 95°C. The expression data were normalized to alpha-actin and presented as 2 −ΔΔCt units. The PCR products were verified by using a melting curve analysis. Western blotting Skeletal tissues were homogenized in a lysate buffer that contained a high concentration of RIPA buffer (R0010, Solarbio), 1mM PMSF (R0100, Solarbio), and protein phosphatase inhibitors (P1260, Solarbio). To remove insoluble material, the tissue lysates were centrifuged at 1,000 rpm for 10 minutes at 4°C. The concentration of protein was determined using the Bicinchoninic Acid (BCA) Assay (P0010, Beyotime). 20µg of proteins were loaded onto 10–12% gels, separated via SDS-PAGE, and transferred onto polyvinyl fluoride membranes (LABSELECT TM-PVDF-S-45, PVDF membrane, 0.45µm, China). The membranes were incubated for 2 hours at room temperature in a blocking solution composed of Tris-buffered saline with 5% non-fat dried milk, 1% BSA and 0.1% Tween 20. Following this, the membranes were subjected to overnight incubation at 4°C with primary antibodies. Anti-LDL receptor antibody (1:500; ab52818; Abcam), alpha-actin polyclonal antibody (1:2000; 23660-1-AP; Proteintech), MuRF1 (C-11) (1:500; sc-398608; Santa Cruz), MyHC-2X/D (1:1000; M4276; Sigma-Aldrich), Drp1 (1:1000; ab184247; Abcam), OPA1 (D-9) (1:500; sc-393296-I; Santa Cruz), Mfn2 (XX-1) (1:400; sc-100560; Santa Cruz), Fis1(B-5) (1:300; sc-376447-i; Santa Cruz), D73D12(VDAC) (1:1000; 4661T; CST), SLC7A11(1:1000; PA1-16893; Invitrogen), GPX4 (1:1000; 14432-1-AP; Proteintech), ACSL4 (1:500; sc-365230; Santa Cruz), TFRC (1:500; BA0462-2; Boster), MyHC II (1:1000; M4276, clone MY-32; Sigma-Aldrich) were used as primary antibodies in this study. Membranes were washed (3×10min) in TBS-T and subsequently incubated with the appropriate HRP-conjugated secondary antibodies. These included: HRP goat-anti-rabbit IgG (H + L) (1:2000; AS014; ABCLONAL) and goat anti-mouse IgG secondary antibody HRP-conjugated (1:1000; #L3032; SAB). Both antibodies were diluted in secondary antibody diluent (P0258, Beyotime) and incubated with the membranes for 1 hour at room temperature. Membranes were then washed (3×5min) in TBST. Protein bands were visualized using an enhanced chemiluminescence substrate (PW30701S, Monad) and imaged with the automatic chemiluminescence image analysis system (Tanon-5200Multi). Protein content was normalized with alpha-actin. Image J (NIH image) was used to analyze the gray value and the ratio between the target protein and the standard protein was calculated. GraphPad Prism (version 8.0) was used to analyze the statistical difference. Hematoxylin and Eosin (H&E) staining The muscle tissues were fixed in 10% neutral formaldehyde and then they were dehydrated by a fully automatic dehydrator, followed by embedding in paraffin wax, and sectioned. These were stained with hematoxylin for 15 minutes, washed with running water for 3minutes, graded with 3% saline for 5 seconds, and blue washed again for 15 minutes. After the sections underwent staining with eosin for 3 minutes, the sections were immersed in 70, 80, 90, 95 and 100% ethyl alcohol, respectively, each for 5 minutes, and subsequently in xylene twice, each time for 10 minutes. The sections were sealed with neutral balsam and then imaged using a microscope. Each section was magnified under 40x microscopy to observe the gross lesions of all tissues, and then 3 magnified microscopic images wer e collected by selecting the area to be observed. Muscle fiber area and distribution To determine the fiber area distribution, we utilized Image J software to trace the outlines of approximately 200 muscle fibers in the skeletal muscle from each HE-stained sample. Additionally, we traced approximately 80–100 muscle fibers from each immunofluorescence sample, which included both type I (slow-twitch) and type II (fast-twitch) fibers. The cross-sectional area (CSA) of these fibers was measured. The total fiber area was divided by the total number of fibers to calculate the average fiber CSA and analyzed the fiber type area distribution. The results were expressed in square micrometers (µm²). Immunofluorescence Immunofluorescence assays were conducted on the quadriceps muscle by following standard protocols. The muscle tissue s were sliced at a thickness of 3µm for immunofluorescence staining. After being fixed in 95% alcohol for 20 minutes, the tissue sections underwent three washes with phosphate-buffered saline (PBS) and were then subjected to a 10-minute incubation in a blocking solution containing 5% BSA at room temperature. Following this, the slices were incubated overnight at 4°C with MyHC-II (1:1000; Sigma, M4276) and then later were treated with primary antibodies. After another three washes with PBS, the slices were exposed to secondary antibodies at room temperature for 1 hour. Subsequently, another set of three PBS washes was performed before the slices were covered with anti-fluorescence attenuation tablets (YEASEN, D6109100) containing DAPI. The slices were stored at 4°C until they were imaged. Image J software was utilized to analyze the average fluorescence intensity of myosin heavy chain, and Prism software was employed for statistical analysis. Statistical analysis The data are presented as means ± SEMs and they were analyzed using GraphPad Prism 9 (GraphPad software, Inc., USA). Statistical analysis for multiple comparisons was performed by using one-way ANOVA followed by the post hoc Fisher’s LSD test. Between-group comparisons were assessed by the unpaired Student’s t-test. A p -value of less than 0.05 was considered to be statistically significant. Results Establishment of a natural aging mouse model exhibiting age-related loss of muscle mass and strength A comparison of life span between humans and C57BL/6J mice indicates that mice aged 18 to 24 months are comparable to humans aged 56 to 69 years[ 17 , 18 ]. Previous report has noted that a sarcopenia phenotype emerges in C57BL/6J mice at approximately 20 to 24 months of age[ 19 ]. In the present study, 19-month-old mice were administered a deno-associated viruses (AAVs) via tail vein. Muscle strength tests and additional measurements were conducted after 8 weeks of treatment, at which point the aged mice had reached 21 months of age. Compared to the adult mice (5 months old), the old mice exhibited sparse and dull hair, slow movement and laziness, in contrast to the bushy shiny hair and responsive and quick behavior observed in the younger mice. In addition, the weight of old mice was also significantly increased compared to young mice (Fig. 1 A). The impact of aging on skeletal muscle mass and muscle function was evaluated as well. Our results showed that the sum of the gastrocnemius, quadriceps and tibialis muscle weights, grip strength and forelimb suspension time were significantly lower in the old mice than in the adult mice ( Figs. 1 B-C ) . The results of H&E staining showed that aged skeletal muscle fibers exhibited apparent variations in size and arrangement, which displayed wider gaps, looser fiber arrangement, and reduced nuclei in most of the fiber sections examined when compared with the adult mice ( Fig. 1 D ) . In addition, the mean CSA of muscle fibers (dotted lines) was significantly lower in aged mice compared to adult mice (989 ± 120 vs 1423 ± 70 µm², p < 0.05) ( Fig. 1 D ) . In addition, the old mice showed a substantial rise in groin fat compared to the adult mice, whereas there was no distinction in gonadal fat mass between the adult and aged mice (Fig. 1 E). Serum concentrations of low-density lipoprotein cholesterol (LDL-C) significantly increased in the old mice while high-density lipoprotein cholesterol (HDL-C) considerably decreased when compared to the adult mice (Fig. 1 F). Taken together, these changes suggested that we had successfully established an age-related sarcopenia mice model. Successful low expression of LDLR in skeletal muscles using AAV injection In this study, we aimed to investigate the potential impact of LDLR knockdown on sarcopenia-related skeletal muscle dysfunction, and the knockdown of the LDLR gene in skeletal muscle was achieved by intravenous injections of AAV-U6 > mLdlr-U6 > mLdlr vectors containing LDLR gRNA sequences, which were regulated by the skeletal muscle-specific promoter pAAV-tMCK-Cas9 . After two months of AAV injection s , all the mice underwent a protocol to assess skeletal muscle strength at the ages of 5 months (adult mice) and 21 months (old mice), respectively. The detailed experimental design and protocol are shown in Figs. 2 A-C. The injection of the AAV transductor resulted in a notable decrease in muscle LDLR protein expression in both adult and old mice ( Fig. 3 A ) . Additionally, our results showed that there were no significant differences in LDLR protein expression in the heart, liver and intestines between the LDLR vector control group and the LDLR KD group in either adult or old mice (Supplementary Fig. S2 ). These findings suggest that AAV injection specifically reduced LDLR expression in skeletal muscle, without affecting the LDLR expression in other organs. LDLR knockdown enhances muscle strength in adult mice and attenuates aging-related loss of muscle mass and strength in aged mice To test the hypothesis that LDLR knockdown in aged muscle will attenuate sarcopenia, we measured the impact of aging in the presence of LDLR low expression on muscle mass and muscle function. Our results showed that LDLR low expression in both adult and old mice resulted in significant increases in the sum of the gastrocnemius, quadriceps and tibialis muscle weights ( Fig. 3 B ). Knockdown of LDLR significantly enhanced forelimb grip strength in old mice, while not affecting limb suspension time. In contrast, in adult mice, forelimb grip strength remained unchanged, but limb suspension time significantly increased ( Fig. 3 C ) . Interestingly, the effects of LDLR knockdown on body lipid metabolism indicators and fat mass varied across the different age groups. We observed that the serum concentrations of TG and LDL-C decreased significantly in the O-LDLR KD group, while no significant decrease being observed in the A-LDLR KD group when compared to the LDLR vector control ( Fig. 3 D ) . LDLR knockdown elicited a significant decrease in gonadal and groin fat mass in the old mice but not in the adult mice ( Fig. 3 E ). LDLR knockdown attenuated loss of type II muscle fibers and muscle atrophy in aged mice Morphology studies of the quadriceps were performed to further explore the effects of LDLR knockdown on myofiber composition. Figure 4 A illustrates the distribution of fast (Type II) and slow muscle fibers (Type I) within the soleus muscle of mice. Muscle sections were stained with MyHC-II primary antibody against fast myosin heavy chains (Type II). The representative staining is depicted, with star symbols indicating MyHC II fibers (bright green stain) and triangles indicating MyHC I fibers (dark green, no stain). Our results showed that LDLR knockdown increased the average fluorescence intensity of fast-contracting myofibers (MyHC II), and the muscle fibers’ cross-sectional shape was more regular in the old mice than that of the O-LDLR vector group ( Fig. 4 A ) . In addition, the CSA distribution of type I and type II fibers were quantified ( Figs. 4 B-C ). Our results showed that the mean CSA (dotted lines in Figs. 4 B-C) of type I fibers increased in the A-LDLR KD compared to the A-LDLR vector control group (1257 ± 137 µm² vs 840 ± 98 µm², p = 0.06). In older mice, LDLR knockdown did not affect the mean CSA of either type I or type II fibers (Figs. 4 B-C). We then evaluated the effects of LDLR knockdown on the fiber type-related gene expression. Our results showed that fast-twitch fibers-related genes (MyHC II and MyHC IIb) were significantly decreased while slow-twitch fibers-related genes (MyHC I) increased in the A-LDLR KD group, when compared to the A-LDLR vector group (Supplementary Figs. S3A-D). However, this was not the case in the old mice. The mRNA level of fast-contracting myofibers (MyHC II) significantly increased, while the mRNA level of slow-contracting myofibers (MyHC I) did not significantly change in the O-LDLR KD group when compared with the O-LDLR vector group (Supplementary Figs. 3SA-D). Collectively, these results indicate that LDLR knockdown increases the levels of slow-twitch muscle fiber-related genes in adult mice while attenuating the loss of fast-twitch muscle fiber mRNA levels in aged mice. Next, we investigated the effects of LDLR knockdown on atrophy-related genes in the quadriceps. Our results showed that LDLR knockdown significantly decreased atrogin and MuRF mRNA levels in the adult mice when compared with the A-LDLR vector group (Figs. 5 A-B). However, atrogin and MuRF mRNA levels in the old mice did not significantly change in the O-LDLR KD group when compared to the O-LDLR vector group (Figs. 5 A-B). The results of H&E staining revealed that the skeletal muscle fibers of the O-LDLR KD group had obvious differences in size and arrangement, with tight gaps, increased fiber arrangement, and more nuclei in most fiber sections when compared to the O-LDLR vector group. (Fig. 5 C). We measured the distribution of CSA of quadriceps muscle fibers based on results from H&E staining and calculated the mean CSA, represented by the dashed line. Our results showed that LDLR knockdown resulted in a significant increase in the mean CSA of quadriceps fibers in aged mice (1297 ± 29 vs 952 ± 81 µm², p < 0.05), whereas no effect was observed in adult mice (Fig. 5 C). To further investigate myogenesis- and atrophy-related proteins expression, we extracted the proteins from the quadriceps and utilized western blotting to detect the expression of MyHC - II and MuRF proteins. Our results showed that LDLR knockdown in aged muscles led to a significant increase in MyHC-II protein expression with a decrease in MuRF protein expression (Figs. 5 D-E ) . Conversely, LDLR knockdown in adult mice did not impact MyHC-II protein expression but did decrease MuRF protein expression (Figs. 5 D-E ) . Taken together, these findings indicate that low expression of LDLR in skeletal muscles mitigates the loss of type II muscle fibers and muscle atrophy in aged mice. LDLR knockdown reduced ROS levels and increased mitochondrial fusion in both adult and old mice To evaluate whether the increase in muscle mass and grip strength in response to LDLR knockdown is mediated by mitochondrial function, we quantified the level s of mitochondrial ROS, a key indicator of mitochondrial damage. Our results showed that in the absence of LDLR knockdown, mitochondrial ROS levels in the quadriceps increased significantly with age compared to those in the muscle of adult mice ( Fig. 6 A ) . However, LDLR knockdown in both adult and old mice led to significant decreases in mitochondrial ROS levels ( Fig. 6 A ) . We subsequently examined quadriceps mitochondrial morphology changes using transmission electron microscopy (TEM). Our results showed that the O-LDLR vector group is characterized by mitochondrial morphological modifications, including greater fragmentation, the reduced or absence of mitochondrial crista and disruption of the outer mitochondrial barrier. However, in the O-LDLR KD group, less mitochondrial swelling was observed ( Fig. 6 B ) . The mRNA levels of mitochondrial fusion-related genes (Mfn1 and Opa1) and mitochondrial fission-related genes (Fis1 and Drp1) were significantly decreased in the A-LDLR KD Group compared to the A-LDLR vector Group. Conversely, mRNA levels of Mfn1, Opa1, Fis1 and Drp1 exhibited an increase in the O-LDLR KD group when compared to the O-LDLR vector group (Supplementary Figs. S4A-D) . To further verify, quadriceps mitochondria were isolated, followed by extraction of mitochondrial proteins. The protein expression levels of mitochondrial fission-related proteins Drp1 ( d ynamin- r elated rotein 1) and Fis1 (fission 1) were detected, along with mitochondrial fusion-related proteins, mitofusin 2 (Mfn 2) and o ptic a trophy 1 (Opa1), using western blotting analysis. The results showed that in the A-LDLR KD group, low expression of LDLR significantly decreased mitochondrial fission-related proteins (Drp1 and Fis1) expression while increased mitochondrial fusion-related proteins (Mfn2 and Opa1) expression compared to the A-LDLR vector group ( Figs. 6 C-D ) . Similarly, in old mice, LDLR low expression resulted in a significant decrease in mitochondrial fission-related protein Drp1 (but not Fis1) and an increase in mitochondrial fusion-related protein Mfn2 (but not Opa1) in the O-LDLR KD group compared to the O-LDLR vector group ( Figs. 6 C-D ) . Taken together, these findings indicate that low expression of LDLR in the skeletal muscle lowered ROS levels and promoted mitochondrial fusion, potentially enhancing muscle function. LDLR knockdown suppresses skeletal muscle ferroptosis Lipid peroxidation, mediated by ROS, can induce ferroptosis. Acyl-CoA synthetase long-chain family member 4 (ACSL4), a crucial compound necessary for initiating lipid peroxidation and dictating sensitivity to ferroptosis, catalyzes the esterification of free polyunsaturated fatty acids (PUFAs) to produce PUFA-CoA[ 20 , 21 ]. Conversely, SLC7A11 and GPX4 play vital roles in resisting lipid peroxidation[ 22 , 23 ]. Additionally, the transferrin receptor (TFRC) facilitates the accumulation and transportation of ferrous iron, leading to iron-dependent ROS activation and ultimately ferroptosis[ 24 ]. Recently, researchers have focused on the role of ferroptosis in various skeletal muscle diseases, highlighting its significance in both physiological and pathological processes, including sarcopenia and rhabdomyosarcoma[ 25 – 27 ]. In the present study, western blotting analysis revealed that the expression of ACSL4 was significantly increased in the aged mice when compared to the adult mice (Supplementary Figs. S5A-B) . However, there was no significant variation tendency in SLC7A11 and GPX4 levels in the old mice compared to the adult mice (Supplementary Figs. S5C-F). Further investigation was conducted on the impact of LDLR-low expression on the ferroptosis pathway. Our results showed that LDLR-low expression suppresses ferroptosis, as evidenced by elevated protein expression of GPX4 and SLC7A11, along with decreased protein levels of ACSL4 and TFRC in the A-LDLR KD group when compared with the A-LDLR vector group ( Figs. 7 A-D ) . Similarly, in old mice, LDLR-low expression led to higher levels of SLC7A11 (but not GPX4) and lower levels of ACSL4 and TFRC in the O-LDLR KD group when compared with the O-LDLR vector group ( Figs. 7 A-D ) . Thus, these results suggested that LDLR-low expression in the quadriceps muscle suppresses ferroptosis. Discussion Age-related sarcopenia, characterized by muscle loss, increases the risk of fractures, impairs physical function and lowers quality of life[ 28 ]. Our findings align with previous research, demonstrating that elderly mice have lower muscle mass and grip strength compared to young adult mice. In addition, we found that lipofuscin, mitochondrial ROS, and ferroptosis-related protein ACSL4 were significantly increased in the quadriceps muscle of elderly mice. Although LDLR accumulation in the skeletal muscles was found in the old rats, the connection between LDLR in skeletal muscle and age-related sarcopenia remains poorly understood. Here, we provide the first evidence that LDLR-specific knockdown in skeletal muscle attenuates aging-related sarcopenia potentially linked to enhanced mitochondrial fusion and suppression of ferroptosis (Fig. 8 ). As a key site of glucose and lipid oxidation, skeletal muscle plays an important role in controlling whole-body energy expenditure[ 29 ]. Previous studies ha ve focused on the relationship between skeletal muscle and cholesterol, emphasizing the potential of skeletal muscle in reducing cholesterol levels and preventing atherosclerotic cardiovascular disease. Knocking out the LDLR in mice (Ldlr-/- mice) is known to inhibit the uptake of LDL, resulting in elevated plasma LDL-C levels. Notably, this study specifically targeted the knockdown of skeletal muscle LDLR while leaving LDLR levels in other organs and tissues unaffected. Skeletal muscle serves as a crucial site for carbohydrate and lipid metabolism. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear transcriptional coactivator, regulates mitochondrial biogenesis and plays a role in carbohydrate and lipid metabolism. Previous studies have shown that muscle-specific overexpression of PGC-1α increases several phospholipid species in glycolytic muscle[ 30 ], enhances oxidase levels[ 31 ], and increases mitochondrial preference for lipid substrates[ 32 ]. These findings suggest that PGC-1α may promote lipid utilization in skeletal muscle. Therefore, we performed additional experiments to measure the mRNA levels of PGC-1α in skeletal muscles. Our results showed that PGC-1α mRNA levels were significantly increased in the O-LDLR KD group when compared to the O-LDLR vector group (Supplementary Figure S3). Therefore, we speculate that skeletal muscle-specific LDLR knockdown reduced serum TG and LDL-C levels, potentially linked to the observed increase in PGC-1α levels. However, this hypothesis requires further verification in future studies. Additionally, the improvement in locomotor function appears to be a secondary effect of the decreased serum TG and LDL-C levels in the aged mice. Study have shown that cholesterol could hinder satellite cell myogenic development[ 33 ]. Although the side effects of statin-induced muscle myopathy have been noted, the mechanism of action remains unclear. It was reported that after being treated chronically with statins, LDLR was increasingly expressed in the gastrocnemius muscle in adult male rats[ 13 ], which may provide the key link between LDLR and myopathy. In this study, we found that LDLR skeletal muscle-specific knockdown in aged mice increased muscle mass, limb grip strength and the myofiber CSA while decreasing lipofuscin in the quadriceps muscle. During sarcopenia, skeletal muscle fibers, especially type II muscle fibers (MyHC II), are susceptible to atrophy in aged mice in aged mice[ 34 ]. Interestingly, the O-LDLR KD group exhibited a higher augmentation in fast-contracting myofibers (MyHC II) compared to the control group, along with a notable decrease in the expression of the atrophy-related protein MuRF. In this study, adult and aged mice exhibited differences in muscle fiber types and associated muscle strength following LDLR knockdown. In age-related muscle atrophy, there was a preferential loss of glycolytic type II muscle fibers, while oxidative type I muscle fibers were typically preserved[ 35 , 36 ]. Our results showed that LDLR knockout led to an increased expression of MyHC II (type II, fast filament) mRNA and protein in aged mice, whereas MyHC I (type I, slow filament) mRNA levels remained unchanged. Although the mean CSA of type I and type II fibers did not differ, the mean CSA of quadriceps fibers increased. These changes corresponded to enhanced strength related to fast-twitch fibers, as evidenced by improved forelimb grip strength. However, endurance of slow-twitch fibers, measured by limb suspension time, remained unchanged in the old mice following LDLR knockdown. In adult mice, LDLR knockout increased the mRNA level of MyHC I while decreasing that of MyHC II. The mean CSA of muscle fibers, primarily type I fibers, increased, leading to enhanced slow-twitch endurance (limb suspension time), while the corresponding force of fast muscles (forelimb grip strength) was not affected. Mitochondria in muscle cells may undergo a decrease in efficiency and energy production capacity with age, which could play a role in sarcopenia[ 37 ]. The mitochondrial free radical theory of aging suggests that age-related mitochondrial dysfunction results in higher levels of ROS, leading to further mitochondrial damage and overall cellular deterioration[ 38 ]. Our data suggest that LDLR knockdown reduces the formation of ROS associated with aging ( Fig. 6 A ) . Increased levels of mitochondrial ROS may lead to oxidative modification of cardiolipin, a fusogenic lipid, and affect the oligomerization of GTPases that control mitochondrial membrane fusion[ 39 ]. Mitochondria exhibit a complex and dynamic architecture in various cell type, particularly in skeletal muscle[ 40 ]. Mitochondria form a dynamic network capable of fusion and fission events, collectively known as mitochondrial dynamics. The regulation of these events is mediated by Mfn1 and Mfn2, Opa-1 and Drp1, and mitochondrial Fis1 proteins[ 14 ]. Studies have shown that morphological changes in the mitochondria have also been observed with age, including less uniformity and more fragmentation as well as a swollen appearance[ 41 , 42 ]. Our results are in line with those observations (Fig. 6 B). Mitochondrial morphology is regulated by fusion and fission processes, which are essential for maintaining organelle homeostasis and normal cellular function s . Dysfunction in mitochondrial dynamics has been implicated in myopathy, where increased fission leads to fragmented mitochondria and muscle wasting[ 43 ]. Additionally, aging-related skeletal muscle atrophy is associated with mitochondrial fission[ 44 , 45 ]. Generally, cholesterol carried by LDL is internalized into cells via the LDL receptor (LDLR) through a clathrin-dependent mechanism. LDL-C is subsequently transported to lysosomes for hydrolysis, releasing cholesterol, while LDLR is recycled back to the plasma membrane[ 6 ]. However, a recent study has reported that LDL-LDLR vesicle can bypass lysosomes and be directly transported to mitochondria, where LDL-C is hydrolyzed to release cholesterol for steroidogenesis[ 14 ]. In the context of aging, the accumulation of cholesterol in mitochondria beyond physiological levels may impair mitochondrial function and trigger oxidative stress and cell death[ 46 ]. Following LDLR knockdown, skeletal muscle mitochondrial ROS levels and the mitochondrial fission-related protein, Drp1, were significantly decreased, while the mitochondrial fusion-related protein, Mfn2, was significantly increased in the old mice (Figs. 6 C-D). This suggests that LDLR knockdown may enhance mitochondrial fusion and reduce ROS levels in the mitochondria within the skeletal muscles of older mice. Although the mechanisms underlying these observations are not entirely clear, several concurrent scenarios may explain this condition. As aging advances, the expression of PGC-1α, a crucial regulator of mitochondrial biogenesis, decreases. This decline increases oxidative stress in skeletal muscle, resulting in elevated ROS levels that damage mitochondrial function, reduce mitochondrial membrane fusion, and increase mitochondrial fission. In contrast, LDLR knockdown appears to enhance the expression of PGC-1a in aged mice (Supplementary Figure S3), thereby preserving mitochondrial function and dynamics. The accumulation of ROS and decreased endogenous antioxidant mechanisms are key factors in the progression of sarcopenia[ 47 ]. Ferroptosis, a form of cell death regulated by iron metabolism, antioxidant processes and lipid metabolism, has emerged as a significant player in skeletal muscle disorders such as sarcopenia and rhabdomyolysis[ 48 ]. ACSL4, a crucial enzyme necessary for initiating lipid peroxidation, has been identified as a crucial player in the execution of ferroptosis[ 49 ]. Consistent with previous studies , our findings demonstrated a significant increase in ACSL4 levels in the quadriceps muscle of aged mice, suggesting the occurrence of ferroptosis (Supplemental Fig. S5) . Interestingly, LDLR skeletal muscle-specific knockdown mice exhibited elevated levels of SLC7A11, known for its crucial role in combating lipid peroxidation, and decreased levels of ACSL4. This indicates that LDLR-specific knockdown may offer protection against ferroptosis in skeletal muscle. The present study has identified several limitations that should be considered in future research. It is important to recognize the different types of sarcopenia, including age-related sarcopenia, obese sarcopenia and cachexia-induced sarcopenia. Findings from studies conducted on older mice may not directly translate to observations in clinical patients. Additionally, this study only focused on in vivo research, and it would be beneficial to include in vitro studies to further validate the conclusions drawn. Moreover, we only measured in vivo muscle strength tests such as grip strength and limb suspension tests. Future studies should include ex vivo muscle strength assessment to evaluate the contractile function of isolated skeletal muscles. Last but not the least, while the effects of LDLR knockdown on mitochondrial dynamics and ferroptosis were observed in the older mice; however, further experiments are necessary to confirm the causal relationship between these factors and to fully elucidate the mechanism by which LDLR contributes to the treatment of sarcopenia. Conclusions The present study demonstrated for the first time that LDLR plays a vital role in regulating age-related sarcopenia, which is associated with skeletal muscle mitochondrial dynamic process and ferroptosis (Fig. 8 ) . LDLR-specific knockdown in skeletal muscle s can attenuate muscle atrophy and loss of strength in aged mice. Further clarification of the relationship between LDLR and sarcopenia will provide new therapeutic strategies and targets for age-related sarcopenia and subsequent clinical complications. Abbreviations HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TG: Triacylglycerol; DCFH-DA: Dichlorofluorescein diacetate; LDLR: Low-density lipoprotein Receptor; FFPE: Formalin-fixed Paraffin Embedded; PVDF: Polyvinylidene fluoride; BCA: Bicinchoninic Acid; Atrogin: Atrogin 1 recombinant protein; MuRF: Muscle RING-finger protein-1; MyHC I: Myosin heavy chain Type I MyHC II: Myosin heavy chain Type II; OPA1: Optic atrophy 1; Mfn2: Mitofusin-2; Drp1：Dynamin-related protein1; Fis1：Mitochondrial fission 1 protein; VDAC: Voltage-dependent anion channel 1; TFRC: Transferrin receptor; ACSL4: Acyl-CoA Synthetase Long-Chain Family Member 4; GPX4: Glutathione Peroxidase 4; SLC7A11: Solute Carrier Family 7 Member 11; HE staining: Hematoxylin and Eosin (H&E) staining; AAV: Adenovirus Associated Virus; A-LDLR vector : Adult mice injected with empty vector as control; A-LDLR KD : Adult mice injected with AAV vectors to knockdown LDLR in skeletal muscle; O-LDLR vector : Old mice injected with empty vector as control; O-LDLR KD : Old mice injected with AAV vectors to knockdown LDLR in skeletal muscle. Declarations Author contributions JW and SL : Conceptualization, funding acquisition, and resources; ZF , TF , XZ , YF, YL , YL , BL , LH : Performed experiments; SRS: Writing-review and editing； ZF , TF , XZ : Formal analysis; ZF : Writing-original draft; JW and SL : Conceptualization, Writing-review and editing. Funding This study was supported by the National Natural Science Foundation of China (No. 81560239 to JW), the Baise scientific research and technology development plan of regionally frequently occurring diseases, China (No. 20224129 to SL), the basic scientific research ability improvement project of young and middle-aged university teachers in Guangxi, China (No. 2019KY0568 to SL); the Natural Science Foundation of Youjiang Medical University for Nationalities (No. yy2018ky001 to SL),the 2023 Innovation Project of Youjiang Medical University for Nationalities Graduate Education (No. YXCXJH2023024) and the high-level Talent Program of Youjiang Medical University for Nationalities (No. yy2024rcky003). Availability of data and materials The data used in this study are available from the corresponding authors upon reasonable request. Conflict of Interest The authors declare that they have no competing interests. References Dhillon RJ, Hasni S. Pathogenesis and Management of Sarcopenia. 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1","display":"","copyAsset":false,"role":"figure","size":575319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of a sarcopenic animal model.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e. Quantification of the impact of aging on body weight; \u003cstrong\u003e(B)\u003c/strong\u003e. \u003cstrong\u003eThe left image \u003c/strong\u003ere\u003cstrong\u003epresents the appearance of the quadriceps muscle. The bar graph counts the ratio of total muscle mass (for the quadriceps, gastrocnemius and tibialis anterior) to body weight in the adult and old mice; (C). The grip strength and forelimb suspension time were normalized to body weight; (D). Above: r\u003c/strong\u003eepresentative hematoxylin-eosin (H\u0026amp;E) staining of the gastrocnemius muscle (scale = 20 μm), ①nucleus, ②myocyte, ③lipofuscin; \u003cstrong\u003eBelow: the distribution of CSA of quadriceps muscle fibers (the dotted lines represent the mean fiber CSA\u003c/strong\u003e). \u003cstrong\u003e(E)\u003c/strong\u003e. Quantification of the impact of aging on groin fat mass and gonadal fat mass; \u003cstrong\u003e(F)\u003c/strong\u003e. Serum concentrations \u003cstrong\u003elevels \u003c/strong\u003eof TG, LDL-C and HDL-C. * \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. \u003cstrong\u003eAbbreviations: CSA: cross-sectional area;\u003c/strong\u003eHDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TG: Triacylglycerol.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/c99e1a95e1f49502c5d83485.png"},{"id":92871175,"identity":"007fb9ce-d504-482f-a9f5-7075b9dca7e0","added_by":"auto","created_at":"2025-10-06 14:07:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":494973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design.\u003c/strong\u003e \u003cstrong\u003e(A). The LDLR gRNA sequence and \u003c/strong\u003es\u003cstrong\u003etructural location;\u003c/strong\u003e \u003cstrong\u003e(B)\u003c/strong\u003e. \u003cstrong\u003eThe localization and sequence of the tMCK promoter fragment;\u003c/strong\u003e \u003cstrong\u003e(C).\u003c/strong\u003e \u003cstrong\u003eThe detailed experimental protocol. \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eAbbreviations: AAV：Adeno-associated viruses, MCK: muscle creatine kinase.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/385dc836f9d81fdaff1f43ae.png"},{"id":92871176,"identity":"56052a9f-4cf7-4a23-97a1-39f4f891fd43","added_by":"auto","created_at":"2025-10-06 14:07:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":487191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLR knockdown enhances muscle strength in adult mice and attenuates aging-related sarcopenia.\u003c/strong\u003e \u0026nbsp;\u003cstrong\u003e(A). Detection and quantification of LDLR expression in quadriceps by western blotting analysis\u003c/strong\u003e; \u003cstrong\u003e(B).\u003c/strong\u003e \u003cstrong\u003eThe left image represents the appearance of the quadriceps muscle.\u0026nbsp; The bar graph counts the ratio of total muscle mass (for the quadriceps, gastrocnemius and tibialis anterior) to body weight. (C). The grip strength and forelimb suspension time were normalized to body weight; \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003e(D).\u003c/strong\u003e The impact of LDLR knockdown on serum \u003cstrong\u003elevels of \u003c/strong\u003eTG and LDL-C; \u003cstrong\u003e(E).\u003c/strong\u003e The impact of LDLR knockdown on \u003cstrong\u003ethe \u003c/strong\u003egroin and gonadal fat mass\u003cstrong\u003ees\u003c/strong\u003e.\u0026nbsp; * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u0026nbsp; \u003cstrong\u003eAbbreviations: \u003c/strong\u003eLDL-C: Low-density\u0026nbsp;lipoprotein cholesterol; TG: Triacylglycerol; A-LDLR\u003csup\u003evector\u003c/sup\u003e: Adult mice injected with empty vector as control; A-LDLR\u003csup\u003eKD\u003c/sup\u003e: Adult mice injected with AAV vectors to knockdown LDLR in skeletal muscle; O-LDLR\u003csup\u003evector\u003c/sup\u003e: Old mice injected with empty vector as control; O-LDLR\u003csup\u003eKD\u003c/sup\u003e: Old mice injected with AAV vectors to knockdown LDLR in skeletal muscle.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/15fc8bf156519896dd91f430.png"},{"id":92871182,"identity":"3e502d21-ffbc-4c1a-a97f-36ffc2510cfd","added_by":"auto","created_at":"2025-10-06 14:07:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1071174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLR knockdown attenuated loss of type II muscle fibers.\u0026nbsp; (A).\u003c/strong\u003e Representative images of MyHC\u003cstrong\u003e II\u003c/strong\u003e fluorescence (green; marking \u003cstrong\u003ethe \u003c/strong\u003emyofiber\u003cstrong\u003es\u003c/strong\u003e) coupled with DAPI staining (blue; marking \u003cstrong\u003ethe \u003c/strong\u003enuclei) obtained from cross-sections of the \u003cstrong\u003esoleus \u003c/strong\u003emuscle (black scale bar=50μm), star s\u003cstrong\u003eymbols\u003c/strong\u003e indicate MyHC II-fibers (\u003cstrong\u003ebright\u003c/strong\u003e green \u003cstrong\u003estaining\u003c/strong\u003e), triangles indicate MyHC I-fibers (dark green, \u003cstrong\u003eno staining\u003c/strong\u003e);\u003cstrong\u003e (B-C). The cross-sectional area distribution of type I and type II fibers, the dotted lines represent the mean fiber CSA. Abbreviations:\u003c/strong\u003e \u003cstrong\u003eCSA: cross-sectional area; MyHC II: Myosin Heavy Chain Type II\u003c/strong\u003e; \u003cstrong\u003eMyHC I: Myosin Heavy Chain Type I; \u003c/strong\u003eA-LDLR\u003csup\u003evector\u003c/sup\u003e: Adult mice injected with empty vector as control; A-LDLR\u003csup\u003eKD\u003c/sup\u003e: Adult mice injected with AAV vectors to knockdown LDLR in skeletal muscle; O-LDLR\u003csup\u003evector\u003c/sup\u003e: Old mice injected with empty vector as control; O-LDLR\u003csup\u003eKD\u003c/sup\u003e: Old mice injected with AAV vectors to knockdown LDLR in skeletal muscle.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/26ae579c8e3ea246e7d8b5a2.png"},{"id":92874950,"identity":"6c88cf37-6e27-47e4-a5e0-63517be8df6e","added_by":"auto","created_at":"2025-10-06 14:31:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":619824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLR knockdown affects atrophy in the skeletal muscle. \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003e(A-B). \u003c/strong\u003eQuantitative real-time PCR analysis of \u003cstrong\u003eatrogin \u003c/strong\u003emRNA levels and MuRF mRNA levels in the quadriceps; \u003cstrong\u003e(C). \u003c/strong\u003eRepresentative hematoxylin-eosin staining of the quadriceps muscle (scale =20μm); ①nucleus ② myocyte ③ lipofuscin. \u003cstrong\u003eRight:\u003c/strong\u003e \u003cstrong\u003ethe cross-sectional area distribution of muscle fibers, the dotted lines represent the mean fiber CSA. (D-E)\u003c/strong\u003e. \u003cstrong\u003eDetection and quantification of MyHC II and MuRF expression in quadriceps by western blotting analysis\u003c/strong\u003e;\u0026nbsp; * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u0026nbsp; \u003cstrong\u003eAbbreviations: \u003c/strong\u003eAtrogin: Atrogin 1 Recombinant Protein; MuRF: Muscle RING-finger protein-1;\u003cstrong\u003e MyHC II: Myosin Heavy Chain Type II\u003c/strong\u003e; A-LDLR\u003csup\u003evector\u003c/sup\u003e: Adult mice injected with empty vector as control; A-LDLR\u003csup\u003eKD\u003c/sup\u003e: Adult mice injected with AAV vectors to knockdown LDLR in skeletal muscle; O-LDLR\u003csup\u003evector\u003c/sup\u003e: Old mice injected with empty vector as control; O-LDLR\u003csup\u003eKD\u003c/sup\u003e: Old mice injected with AAV vectors to knockdown LDLR in skeletal muscle.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/e7bcdc48edbacc849d689520.png"},{"id":92871185,"identity":"1439f560-9115-4866-9233-aa9e6ca3669f","added_by":"auto","created_at":"2025-10-06 14:07:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":516875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLR knockdown reduced ROS levels and increased mitochondrial fusion in aged mice.\u0026nbsp; (A). \u003c/strong\u003eMuscle mitochondrial reactive oxygen species levels; \u003cstrong\u003e(B).\u003c/strong\u003e Representative transmission electron micrographs of mitochondria;\u003cstrong\u003e (C). Detection and quantification of Drp1 and Fis1 expression in quadriceps by western blotting analysis\u003c/strong\u003e; \u003cstrong\u003e(D).\u003c/strong\u003e \u003cstrong\u003eDetection and quantification of Mfn2 and OPA1 expression in quadriceps by western blotting analysis\u003c/strong\u003e; * \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003e\u0026nbsp;Abbreviations:\u003c/strong\u003e OPA1: Optic Atrophy 1; Mfn2: Mitofusin-2; Drp1: Dynamin-related protein1; Fis1: Mitochondrial fission 1 protein; VDAC: Voltage-dependent anion channels; A-LDLR\u003csup\u003evector\u003c/sup\u003e: Adult mice injected with empty vector as control; A-LDLR\u003csup\u003eKD\u003c/sup\u003e: Adult mice injected with AAV vectors to knockdown LDLR in skeletal muscle; O-LDLR\u003csup\u003evector\u003c/sup\u003e: Old mice injected with empty vector as control; O-LDLR\u003csup\u003eKD\u003c/sup\u003e: Old mice injected with AAV vectors to knockdown LDLR in skeletal muscle.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/7daf990ddf21350dc09739ce.png"},{"id":92872296,"identity":"153db379-0403-4876-a710-c9effb07ee3c","added_by":"auto","created_at":"2025-10-06 14:15:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":360062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLR knockdown suppresses skeletal muscle ferroptosis. (A). \u003c/strong\u003eRepresentative bands of GPX4, SLC7A11 and α-actin from the quadriceps muscle \u003cstrong\u003eprotein \u003c/strong\u003eextracts; \u003cstrong\u003e(B). Quantification of protein expression of GPX4 and SLC7A11\u003c/strong\u003e; \u003cstrong\u003e(C).\u003c/strong\u003eRepresentative bands of ACSL4, TFRC and α-actin from the quadriceps muscle \u003cstrong\u003eprotein \u003c/strong\u003eextracts;\u003cstrong\u003e (D).\u003c/strong\u003e \u003cstrong\u003eQuantification of protein expression of ACSL4 and TFRC\u003c/strong\u003e. \u003cstrong\u003eAbbreviations:\u003c/strong\u003e GPX4: Glutathione peroxidase 4; ACSL4: Acyl-CoA Synthetase Long-Chain Family Member 4; SLC7A11: Solute Carrier Family 7 Member 11; TFRC: Transferrin receptor.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/0b6321893e477d84d32abc62.png"},{"id":92871190,"identity":"dae01dba-c5e2-4211-afe3-a4a305ff5f0d","added_by":"auto","created_at":"2025-10-06 14:07:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":560486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDLR knockdown attenuates sarcopenia in aged mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn the context of aging, the accumulation of cholesterol in mitochondria at levels exceeding physiological norms may impair mitochondrial function. \u0026nbsp;The release of ROS from damaged mitochondria triggers the activation of ACSL4-mediated ferroptosis. Although other factors may contribute, an increase in the expression of MuRF and a decrease in the expression of MyHC II have been observed. Ultimately, this leads to aging-related loss of muscle mass and strength. Notably, specific knockdown of LDLR in skeletal muscle showed to ameliorate muscle mass loss and functional decline, including increased MyHC II protein levels and decreased MuRF protein levels. These effects may be associated with the promotion of mitochondrial fusion and inhibition of ferroptosis.\u003c/strong\u003e \u0026nbsp;\u003cstrong\u003eSolid lines indicate direct effects; dashed lines indicate mediated (indirect) effects；Plus sign (+) means promotion; minus sign (-) means relief.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/39578c9c9d3cfc14834b9578.png"},{"id":92876071,"identity":"142e5b2b-60f3-4be0-80b1-5abb2c19cb88","added_by":"auto","created_at":"2025-10-06 14:39:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7303529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/653f31d9-19da-4bc5-99e4-6e07e6d6643a.pdf"},{"id":92872281,"identity":"66a8ef65-f5e0-410e-8e59-208a714f5ed1","added_by":"auto","created_at":"2025-10-06 14:15:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":786551,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/3eceb1fb2461c6380755ce15.docx"},{"id":92873447,"identity":"e148f8a8-319b-4d7d-9b93-276e2063ee12","added_by":"auto","created_at":"2025-10-06 14:23:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1383136,"visible":true,"origin":"","legend":"","description":"","filename":"Originalwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6891536/v1/6116bca37c47370d7bb15079.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Knockdown of low-density lipoprotein receptors in skeletal muscle attenuates aging-related sarcopenia associated with mitochondrial fusion and ferroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAge-related sarcopenia, a progressive loss of skeletal muscle mass and strength occurring with aging, frequently results in disabilities, loss of independence and death[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The prevalence of sarcopenia in the elderly population will rise as the global population ages[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and there is an urgent need to develop effective preventive and therapeutic strategies against this condition. Previous studies have found that abnormal lipid metabolism was closely related to muscle dysfunction and poor physical performance in older people with sarcopenia[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The low-density lipoprotein (LDL) receptor (LDLR) is a cell-surface glycoprotein that facilitates the internalization of LDL into cells via endocytosis. In general, the cholesterol carried by LDL is specifically transported into the mitochondria, where the biosynthesis of steroid hormones occurs[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, increased cholesterol levels within mitochondria can impair mitochondrial function[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In order to maintain their normal morphology and functions, mitochondria constantly undergo the two essential processes of fusion and fission. The balance of mitochondrial fusion and fission is critical for preserving optimal muscle function, as increased mitochondrial fission has been associated with the progression of skeletal muscle atrophy[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStatins, a group of lipid-lowering drugs for treating hypercholesterolemia, inhibit cellular cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl (HMG) coenzyme-A reductase, and up-regulating the LDLRs in the liver and peripheral tissues, resulting in increased blood LDL cholesterol (LDL-C) clearance[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, side effects of statins on muscle tissues are well-known and range from myalgia and cramping to an increase in creatine kinase (CK), myopathy, rhabdomyolysis and exacerbation of myasthenia gravis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Marco and colleagues[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]reported that LDLR was increasingly expressed in the gastrocnemius muscle in adult male rats after being treated chronically with statins, which may provide the key link between LDLR and statins-induced myopathy. Although LDLR accumulation in the skeletal muscle\u003cb\u003es\u003c/b\u003e was found in male and female old rats[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the underlying molecular mechanisms have not been clarified. Interestingly, a recent study reported that mitochondrial outer membrane protein phospholipase D6 (PLD6) hydrolyzes cardiolipin into phosphatidic acid facilitating the fusion of LDL-LDLR vesicles with mitochondria and accelerates the degradation of LDLR[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This revealed a novel link between mitochondrion and LDLR homeostasis.\u003c/p\u003e\u003cp\u003eAn interesting question arises as to whether LDLR accumulation in the aging skeletal muscle represents a potential mechanism that could lead to mitochondrial dysfunction and, in turn, to sarcopenia. However, the relationship between LDLR in the skeletal muscle and age-related sarcopenia has not been investigated. In this study, we speculated that a low expression of LDLR in skeletal muscles may attenuate aging-related skeletal muscle mass loss and muscle dysfunction. Therefore, the aim of this study was to investigate the effects of LDLR knockdown on skeletal muscle and to uncover the underlying mechanisms involved in this process.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eProcedures on animals and AAV injections\u003c/h2\u003e\u003cp\u003eTow-month-old C57BL/6J male mice were purchased from Viton Lihua Laboratory Animal Technology Company (Beijing, China), and sixteen-month-old C57BL/6J male mice were purchased from Ziyuan Laboratory Animal Technology Company (Hangzhou, China). 3 to 4 mice were raised in each cage and maintained under the conditions of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and relative humidity of 50\u0026ndash;60%, with a light/dark cycle of 12:12 hours, and they were fed with standard feed and water ad libitum. Adeno-associated viruses (AAVs) were administered via the tail vein after adaptation feeding until the adult C57BL/6 mice reached 3 months and the aged animals reached 19 months. All AAVs used in this study were of Serotype 9, which is a serotype with high levels of luciferase expression in the lower extremity skeletal muscles, and they exhibit a fast expression rate and maintain stable tissue expression[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe construction of a plasmid AAV-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr backbone-based LDLR knockdown vector (LDLR\u003csup\u003eKD\u003c/sup\u003e) was achieved by modifying the AAV-U6gRNA1-U6gRNA2-TnT-Cre adeno-associated viral vector (Addgene, #87682). The pAAV-tMCK-Cas9 vector was modified from the pAAV-nEFCas9 plasmid (Addgene, #87115) by inserting skeletal-muscle-specific muscle creatine kinase (MCK) promoter. The plasmids AAV-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr and pAAV-tMCK-Cas9 were combined with autoclaved PBS as the solvent at a ratio of 1:1, with each vector having a titer of 1 x 10\u003csup\u003e12\u003c/sup\u003e. This mixture necessitates only a single injection to effectively knock down LDLR in the skeletal muscles of both adult and aged mice. Empty vectors AAV-U6-U6 without carrying LDLR gRNA sequences were prepared simultaneously as control. Adult mice were randomly divided into two subgroups: the A-LDLR\u003csup\u003eKD\u003c/sup\u003e (adult mice co-injected with AAV-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr and pAAV-tMCK-Cas9 vectors to knockdown LDLR in the skeletal muscles, n\u0026thinsp;=\u0026thinsp;11) group, and the A-LDLR\u003csup\u003evector\u003c/sup\u003e (adult mice co-injected with empty vector AAV-U6-U6 and pAAV-tMCK-Cas9 as control, n\u0026thinsp;=\u0026thinsp;9) group. Old mice were then randomly divided into two subgroups: the O-LDLR\u003csup\u003eKD\u003c/sup\u003e (old mice injected with AAV-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr and pAAV-tMCK-Cas9 vectors to knockdown LDLR in the skeletal muscles, n\u0026thinsp;=\u0026thinsp;8) group, and the O-LDLR\u003csup\u003evector\u003c/sup\u003e (old mice injected with empty vector AAV-U6-U6 and pAAV-tMCK-Cas9 as control, n\u0026thinsp;=\u0026thinsp;6) group.\u003c/p\u003e\u003cp\u003eThe mice that had completed their course of tail vein injections, were maintained and they were housed in an SPF environment with the same feed and drinking patterns. Particular attention was paid to observing whether the wound became infected, and the tail tip was ischemia. After 8 weeks of rearing, a muscle strength test was performed at 12 hours before death. All animal procedures were approved by the Institutional Animal Care and Use Committee of Youjiang Medical University for Nationalities (Approval number: 2022100801).The licensing committee approving the experiments, including any relevant details. The study was performed with regulations and by the ARRIVE guidelines. We confirm that all methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eForelimb grip strength\u003c/h3\u003e\n\u003cp\u003ePrior to testing, mice were allowed to acclimate to the handgrip tester for 5 minutes. The forelimbs of the mouse were positioned on the sensing bar of the grip dynamometer. Once the forelimbs grasped the crossbar, the mouse's tail was pulled backwards horizontally until the forelimbs were released. The grasp force meter automatically recorded the maximum grasping force exerted by the mouse on the crossbar. The ratio of grip strength to body weight was used to calculate the relative grip strength, allowing for normalization of the grip strength measurement.\u003c/p\u003e\u003cp\u003eForelimb suspension test\u003c/p\u003e\u003cp\u003eTo assess the muscular endurance of mice, we conducted a forelimb suspension test. The mouse's tail was pinched to ensure that its forelimbs grasped the suspension wire securely. After confirming that the forelimbs were firmly gripping the rod, the mouse's tail was released. Immediately after releasing the mice, a timer was initiated to measure the duration from the moment the mouse's forelimbs were suspended from the wire until they fell. Three trials were conducted for each mouse, with a 5-minute interval between trials. The relative suspension time was calculated as the ratio of forelimb suspension time to body weight.\u003c/p\u003e\n\u003ch3\u003eBiochemical measurements\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected via the retro-orbital sinus in animals under terminal anesthesia. The isolated plasma was centrifuged at 1500rpm for 20 minutes at 4\u0026deg;C and kept at 80\u0026deg;C until the assays were performed. LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triacylglycerol (TG) levels were determined by the colorimetric method (Elabscience) following the manufacturer's protocols.\u003c/p\u003e\n\u003ch3\u003eTissue Preparation and Collection\u003c/h3\u003e\n\u003cp\u003eUnder baseline conditions, the muscle tissues were extracted from adult (5-month-old) and old (21-month-old) mice. The quadriceps muscles were immediately dissected and cleaned to remove blood and fur before being snap-frozen in liquid nitrogen and stored at -80 ℃ before the examination.\u003c/p\u003e\n\u003ch3\u003eSeparation of mitochondria\u003c/h3\u003e\n\u003cp\u003eMitochondrial separation was carried out following the manufacturer's instructions (Beyotime). Briefly, 120 mg of the quadriceps muscle was minced and homogenized in an EP tube containing a cold isolation buffer together with pancreatic extracts. After rotating the mitochondrial suspension at 600g for 10 minutes, the supernatants were centrifuged at 11000g for 10 minutes. The mitochondria obtained were frozen at -80 ℃ for future analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMitochondria ROS measurement\u003c/h2\u003e\u003cp\u003eThe Reactive Oxygen Species Assay Kit was used to determine mitochondrial ROS levels (Beyotime, China). Measurement of mitochondrial ROS was performed as described previously[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Total Mitochondrial ROS was measured using dichlorofluorescein diacetate from mitochondria (DCFH-DA, Beyotime). Briefly, the mitochondrial storage solution containing mitochondria was incubated at 37\u0026deg;C for 20 minutes together with 10 \u0026micro;M DCFH-DA and the fluorescence was measured on a microplate reader.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eElectron microscopy\u003c/h3\u003e\n\u003cp\u003eThe isolated mitochondria were collected and fixed in 2.5% glutaraldehyde (H7650, Japan, Hitachi). Subsequently, the mitochondrial pellets were treated with 1% osmic acid for 2 hours at ambient temperature. The pellets then underwent dehydration through a series of increasing ethanol concentrations. Following dehydration, the mitochondria were embedded in Epon-Araldite resin. Finally, ultrathin sections were mounted onto copper grids, stained with uranyl acetate and lead citrate, and examined via transmission electron microscopy (H-7650, Hitachi, Japan).\u003c/p\u003e\n\u003ch3\u003eRNA extraction and quantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eThe RNA was extracted from skeletal muscles by using TIANGEN reagent and following the manufacturer's instructions. The concentration and purity of RNA were assessed by measuring the absorption at 260/280 nm. To synthesize cDNA, 1g of total RNA was reverse transcribed with HiScript\u0026reg; III RT Super Mix for qPCR (+\u0026thinsp;gDNA wiper) cDNA reverse transcription reagent (R323-01, Vazyme, China). Real-time PCR was carried out using Light Cycler96 (Roche Diagnostics) and Brilliant III Ultra-Fast SYBR QPCR master mix amplification kit (B21202, Bimake, China). The primers utilized are listed in Supplementary \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. Each reaction contained 0.5\u0026micro;L of both forward and reverse primers, 2\u0026micro;L of cDNA, with a final volume of 10\u0026micro;L. All primers exhibited optimal amplification efficiencies (within the range of 90\u0026ndash;110%). A control PCR for the housekeeping gene, alpha-actin, was performed. The thermocycling conditions included initial denaturation at 95˚C for 5 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 15 s, annealing at 60\u0026deg;C for 60 s, extension at 72\u0026deg;C for 15 s, and a final extension at 95\u0026deg;C, 60\u0026deg;C for 1 min, and 95\u0026deg;C. The expression data were normalized to alpha-actin and presented as 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e units. The PCR products were verified by using a melting curve analysis.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eSkeletal tissues were homogenized in a lysate buffer that contained a high concentration of RIPA buffer (R0010, Solarbio), 1mM PMSF (R0100, Solarbio), and protein phosphatase inhibitors (P1260, Solarbio). To remove insoluble material, the tissue lysates were centrifuged at 1,000 rpm for 10 minutes at 4\u0026deg;C. The concentration of protein was determined using the Bicinchoninic Acid (BCA) Assay (P0010, Beyotime). 20\u0026micro;g of proteins were loaded onto 10\u0026ndash;12% gels, separated via SDS-PAGE, and transferred onto polyvinyl fluoride membranes (LABSELECT TM-PVDF-S-45, PVDF membrane, 0.45\u0026micro;m, China). The membranes were incubated for 2 hours at room temperature in a blocking solution composed of Tris-buffered saline with 5% non-fat dried milk, 1% BSA and 0.1% Tween 20. Following this, the membranes were subjected to overnight incubation at 4\u0026deg;C with primary antibodies. Anti-LDL receptor antibody (1:500; ab52818; Abcam), alpha-actin polyclonal antibody (1:2000; 23660-1-AP; Proteintech), MuRF1 (C-11) (1:500; sc-398608; Santa Cruz), MyHC-2X/D (1:1000; M4276; Sigma-Aldrich), Drp1 (1:1000; ab184247; Abcam), OPA1 (D-9) (1:500; sc-393296-I; Santa Cruz), Mfn2 (XX-1) (1:400; sc-100560; Santa Cruz), Fis1(B-5) (1:300; sc-376447-i; Santa Cruz), D73D12(VDAC) (1:1000; 4661T; CST), SLC7A11(1:1000; PA1-16893; Invitrogen), GPX4 (1:1000; 14432-1-AP; Proteintech), ACSL4 (1:500; sc-365230; Santa Cruz), TFRC (1:500; BA0462-2; Boster), MyHC II (1:1000; M4276, clone MY-32; Sigma-Aldrich) were used as primary antibodies in this study. Membranes were washed (3\u0026times;10min) in TBS-T and subsequently incubated with the appropriate HRP-conjugated secondary antibodies. These included: HRP goat-anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (1:2000; AS014; ABCLONAL) and goat anti-mouse IgG secondary antibody HRP-conjugated (1:1000; #L3032; SAB). Both antibodies were diluted in secondary antibody diluent (P0258, Beyotime) and incubated with the membranes for 1 hour at room temperature. Membranes were then washed (3\u0026times;5min) in TBST. Protein bands were visualized using an enhanced chemiluminescence substrate (PW30701S, Monad) and imaged with the automatic chemiluminescence image analysis system (Tanon-5200Multi). Protein content was normalized with alpha-actin. Image J (NIH image) was used to analyze the gray value and the ratio between the target protein and the standard protein was calculated. GraphPad Prism (version 8.0) was used to analyze the statistical difference.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHematoxylin and Eosin (H\u0026amp;E) staining\u003c/h2\u003e\u003cp\u003eThe muscle tissues were fixed in 10% neutral formaldehyde and then they were dehydrated by a fully automatic dehydrator, followed by embedding in paraffin wax, and sectioned. These were stained with hematoxylin for 15 minutes, washed with running water for 3minutes, graded with 3% saline for 5 seconds, and blue washed again for 15 minutes. After the sections underwent staining with eosin for 3 minutes, the sections were immersed in 70, 80, 90, 95 and 100% ethyl alcohol, respectively, each for 5 minutes, and subsequently in xylene twice, each time for 10 minutes. The sections were sealed with neutral balsam and then imaged using a microscope. Each section was magnified under 40x microscopy to observe the gross lesions of all tissues, and then 3 magnified microscopic images wer\u003cb\u003ee\u003c/b\u003e collected by selecting the area to be observed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMuscle fiber area and distribution\u003c/h2\u003e\u003cp\u003eTo determine the fiber area distribution, we utilized Image J software to trace the outlines of approximately 200 muscle fibers in the skeletal muscle from each HE-stained sample. Additionally, we traced approximately 80\u0026ndash;100 muscle fibers from each immunofluorescence sample, which included both type I (slow-twitch) and type II (fast-twitch) fibers. The cross-sectional area (CSA) of these fibers was measured. The total fiber area was divided by the total number of fibers to calculate the average fiber CSA and analyzed the fiber type area distribution. The results were expressed in square micrometers (\u0026micro;m\u0026sup2;).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eImmunofluorescence assays were conducted on the quadriceps muscle by following standard protocols. The muscle tissue\u003cb\u003es\u003c/b\u003e were sliced at a thickness of 3\u0026micro;m for immunofluorescence staining. After being fixed in 95% alcohol for 20 minutes, the tissue sections underwent three washes with phosphate-buffered saline (PBS) and were then subjected to a 10-minute incubation in a blocking solution containing 5% BSA at room temperature. Following this, the slices were incubated overnight at 4\u0026deg;C with MyHC-II (1:1000; Sigma, M4276) and then later were treated with primary antibodies. After another three washes with PBS, the slices were exposed to secondary antibodies at room temperature for 1 hour. Subsequently, another set of three PBS washes was performed before the slices were covered with anti-fluorescence attenuation tablets (YEASEN, D6109100) containing DAPI. The slices were stored at 4\u0026deg;C until they were imaged. Image J software was utilized to analyze the average fluorescence intensity of myosin heavy chain, and Prism software was employed for statistical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs and they were analyzed using GraphPad Prism 9 (GraphPad software, Inc., USA). Statistical analysis for multiple comparisons was performed by using one-way ANOVA followed by the \u003cem\u003epost hoc\u003c/em\u003e Fisher\u0026rsquo;s LSD test. Between-group comparisons were assessed by the unpaired Student\u0026rsquo;s t-test. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered to be statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eEstablishment of a natural aging mouse model exhibiting age-related loss of muscle mass and strength\u003c/p\u003e\u003cp\u003eA comparison of life span between humans and C57BL/6J mice indicates that mice aged 18 to 24 months are comparable to humans aged 56 to 69 years[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Previous report has noted that a sarcopenia phenotype emerges in C57BL/6J mice at approximately 20 to 24 months of age[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the present study, 19-month-old mice were administered a deno-associated viruses (AAVs) via tail vein. Muscle strength tests and additional measurements were conducted after 8 weeks of treatment, at which point the aged mice had reached 21 months of age. Compared to the adult mice (5 months old), the old mice exhibited sparse and dull hair, slow movement and laziness, in contrast to the bushy shiny hair and responsive and quick behavior observed in the younger mice. In addition, the weight of old mice was also significantly increased compared to young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The impact of aging on skeletal muscle mass and muscle function was evaluated as well. Our results showed that the sum of the gastrocnemius, quadriceps and tibialis muscle weights, grip strength and forelimb suspension time were significantly lower in the old mice than in the adult mice \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e. The results of H\u0026amp;E staining showed that aged skeletal muscle fibers exhibited apparent variations in size and arrangement, which displayed wider gaps, looser fiber arrangement, and reduced nuclei in most of the fiber sections examined when compared with the adult mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. In addition, the mean CSA of muscle fibers (dotted lines) was significantly lower in aged mice compared to adult mice (989\u0026thinsp;\u0026plusmn;\u0026thinsp;120 vs 1423\u0026thinsp;\u0026plusmn;\u0026thinsp;70 \u0026micro;m\u0026sup2;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. In addition, the old mice showed a substantial rise in groin fat compared to the adult mice, whereas there was no distinction in gonadal fat mass between the adult and aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Serum concentrations of low-density lipoprotein cholesterol (LDL-C) significantly increased in the old mice while high-density lipoprotein cholesterol (HDL-C) considerably decreased when compared to the adult mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Taken together, these changes suggested that we had successfully established an age-related sarcopenia mice model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSuccessful low expression of LDLR in skeletal muscles using AAV injection\u003c/h2\u003e\u003cp\u003eIn this study, we aimed to investigate the potential impact of LDLR knockdown on sarcopenia-related skeletal muscle dysfunction, and the knockdown of the LDLR gene in skeletal muscle was achieved by intravenous injections of AAV-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr-U6\u0026thinsp;\u0026gt;\u0026thinsp;mLdlr vectors containing LDLR gRNA sequences, which were regulated by the skeletal muscle-specific promoter \u003cb\u003epAAV-tMCK-Cas9\u003c/b\u003e. After two months of AAV injection\u003cb\u003es\u003c/b\u003e, all the mice underwent a protocol to assess skeletal muscle strength at the ages of 5 months (adult mice) and 21 months (old mice), respectively. The detailed experimental design and protocol are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C. The injection of the AAV transductor resulted in a notable decrease in muscle LDLR protein expression in both adult and old mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Additionally, our results showed that there were no significant differences in LDLR protein expression in the heart, liver and intestines between the LDLR\u003csup\u003evector\u003c/sup\u003e control group and the LDLR\u003csup\u003eKD\u003c/sup\u003e group in either adult or old mice \u003cb\u003e(Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/b\u003e These findings suggest that AAV injection specifically reduced LDLR expression in skeletal muscle, without affecting the LDLR expression in other organs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLDLR knockdown enhances muscle strength in adult mice and attenuates aging-related loss of muscle mass and strength in aged mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo test the hypothesis that LDLR knockdown in aged muscle will attenuate sarcopenia, we measured the impact of aging in the presence of LDLR low expression on muscle mass and muscle function. Our results showed that LDLR low expression in both adult and old mice resulted in significant increases in the sum of the gastrocnemius, quadriceps and tibialis muscle weights \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e Knockdown of LDLR significantly enhanced forelimb grip strength in old mice, while not affecting limb suspension time. In contrast, in adult mice, forelimb grip strength remained unchanged, but limb suspension time significantly increased\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Interestingly, the effects of LDLR knockdown on body lipid metabolism indicators and fat mass varied across the different age groups. We observed that the serum concentrations of TG and LDL-C decreased significantly in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group, while no significant decrease \u003cb\u003ebeing\u003c/b\u003e observed in the A-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared to the LDLR\u003csup\u003evector\u003c/sup\u003e control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. LDLR knockdown elicited a significant decrease in gonadal and groin fat mass in the old mice but not in the adult mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eLDLR knockdown attenuated loss of type II muscle fibers and muscle atrophy in aged mice\u003c/h2\u003e\u003cp\u003eMorphology studies of the quadriceps were performed to further explore the effects of LDLR knockdown on myofiber composition. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA illustrates the distribution of fast (Type II) and slow muscle fibers (Type I) within the soleus muscle of mice. Muscle sections were stained with MyHC-II primary antibody against fast myosin heavy chains (Type II). The representative staining is depicted, with star symbols indicating MyHC II fibers (bright green stain) and triangles indicating MyHC I fibers (dark green, no stain). Our results showed that LDLR knockdown increased the average fluorescence intensity of fast-contracting myofibers (MyHC II), and the muscle fibers\u0026rsquo; cross-sectional shape was more regular in the old mice than that of the O-LDLR\u003csup\u003evector\u003c/sup\u003e group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. In addition, the CSA distribution of type I and type II fibers were quantified \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C\u003cb\u003e).\u003c/b\u003e Our results showed that the mean CSA (dotted lines in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C) of type I fibers increased in the A-LDLR\u003csup\u003eKD\u003c/sup\u003e compared to the A-LDLR\u003csup\u003evector\u003c/sup\u003e control group (1257\u0026thinsp;\u0026plusmn;\u0026thinsp;137 \u0026micro;m\u0026sup2; vs 840\u0026thinsp;\u0026plusmn;\u0026thinsp;98 \u0026micro;m\u0026sup2;, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06). In older mice, LDLR knockdown did not affect the mean CSA of either type I or type II fibers (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then evaluated the effects of LDLR knockdown on the fiber type-related gene expression. Our results showed that fast-twitch fibers-related genes (MyHC II and MyHC IIb) were significantly decreased while slow-twitch fibers-related genes (MyHC I) increased in the A-LDLR\u003csup\u003eKD\u003c/sup\u003e group, when compared to the A-LDLR\u003csup\u003evector\u003c/sup\u003e group (Supplementary Figs. S3A-D). However, this was not the case in the old mice. The mRNA level of fast-contracting myofibers (MyHC II) significantly increased, while the mRNA level of slow-contracting myofibers (MyHC I) did not significantly change in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared with the O-LDLR\u003csup\u003evector\u003c/sup\u003e group (Supplementary Figs.\u0026nbsp;3SA-D). Collectively, these results indicate that LDLR knockdown increases the levels of slow-twitch muscle fiber-related genes in adult mice while attenuating the loss of fast-twitch muscle fiber mRNA levels in aged mice.\u003c/p\u003e\u003cp\u003eNext, we investigated the effects of LDLR knockdown on atrophy-related genes in the quadriceps. Our results showed that LDLR knockdown significantly decreased atrogin and MuRF mRNA levels in the adult mice when compared with the A-LDLR\u003csup\u003evector\u003c/sup\u003e group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). However, atrogin and MuRF mRNA levels in the old mice did not significantly change in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared to the O-LDLR\u003csup\u003evector\u003c/sup\u003e group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). The results of H\u0026amp;E staining revealed that the skeletal muscle fibers of the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group had obvious differences in size and arrangement, with tight gaps, increased fiber arrangement, and more nuclei in most fiber sections when compared to the O-LDLR\u003csup\u003evector\u003c/sup\u003e group. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We measured the distribution of CSA of quadriceps muscle fibers based on results from H\u0026amp;E staining and calculated the mean CSA, represented by the dashed line. Our results showed that LDLR knockdown resulted in a significant increase in the mean CSA of quadriceps fibers in aged mice (1297\u0026thinsp;\u0026plusmn;\u0026thinsp;29 vs 952\u0026thinsp;\u0026plusmn;\u0026thinsp;81 \u0026micro;m\u0026sup2;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no effect was observed in adult mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To further investigate myogenesis- and atrophy-related proteins expression, we extracted the proteins from the quadriceps and utilized western blotting to detect the expression of MyHC\u003cb\u003e-\u003c/b\u003eII and MuRF proteins. Our results showed that LDLR knockdown in aged muscles led to a significant increase in MyHC-II protein expression with a decrease in MuRF protein expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e. Conversely, LDLR knockdown in adult mice did not impact MyHC-II protein expression but did decrease MuRF protein expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e. Taken together, these findings indicate that low expression of LDLR in skeletal muscles mitigates the loss of type II muscle fibers and muscle atrophy in aged mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eLDLR knockdown reduced ROS levels and increased mitochondrial fusion in both adult and old mice\u003c/h2\u003e\u003cp\u003eTo evaluate whether the increase in muscle mass and grip strength in response to LDLR knockdown is mediated by mitochondrial function, we quantified the level\u003cb\u003es\u003c/b\u003e of mitochondrial ROS, a key indicator of mitochondrial damage. Our results showed that in the absence of LDLR knockdown, mitochondrial ROS levels in the quadriceps increased significantly with age compared to those in the muscle of adult mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. However, LDLR knockdown in both adult and old mice led to significant decreases in mitochondrial ROS levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We subsequently examined quadriceps mitochondrial morphology changes using transmission electron microscopy (TEM). Our results showed that the O-LDLR\u003csup\u003evector\u003c/sup\u003e group is characterized by mitochondrial morphological modifications, including \u003cb\u003egreater\u003c/b\u003e fragmentation, the reduced or absence of mitochondrial crista and disruption of the outer mitochondrial barrier. However, in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group, less mitochondrial swelling was observed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The mRNA levels of mitochondrial fusion-related genes (Mfn1 and Opa1) and mitochondrial fission-related genes (Fis1 and Drp1) were significantly decreased in the A-LDLR\u003csup\u003eKD\u003c/sup\u003e Group compared to the A-LDLR\u003csup\u003evector\u003c/sup\u003e Group. Conversely, mRNA levels of Mfn1, Opa1, Fis1 and Drp1 exhibited an increase in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared to the O-LDLR\u003csup\u003evector\u003c/sup\u003e group \u003cb\u003e(Supplementary Figs. S4A-D)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further verify, quadriceps mitochondria were isolated, followed by extraction of mitochondrial proteins. The protein expression levels of mitochondrial fission-related proteins Drp1 (\u003cb\u003ed\u003c/b\u003eynamin-\u003cb\u003er\u003c/b\u003eelated rotein 1) and Fis1 (fission 1) were detected, along with mitochondrial fusion-related proteins, mitofusin 2 (Mfn 2) and \u003cb\u003eo\u003c/b\u003eptic \u003cb\u003ea\u003c/b\u003etrophy 1 (Opa1), using western blotting analysis. The results showed that in the A-LDLR\u003csup\u003eKD\u003c/sup\u003e group, low expression of LDLR significantly decreased mitochondrial fission-related proteins (Drp1 and Fis1) expression while increased mitochondrial fusion-related proteins (Mfn2 and Opa1) expression compared to the A-LDLR\u003csup\u003evector\u003c/sup\u003e group \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. Similarly, in old mice, LDLR low expression resulted in a significant decrease in mitochondrial fission-related protein Drp1 (but not Fis1) and an increase in mitochondrial fusion-related protein Mfn2 (but not Opa1) in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group compared to the O-LDLR\u003csup\u003evector\u003c/sup\u003e group \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. Taken together, these findings indicate that low expression of LDLR in the skeletal muscle lowered ROS levels and promoted mitochondrial fusion, potentially enhancing muscle function.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eLDLR knockdown suppresses skeletal muscle ferroptosis\u003c/h2\u003e\u003cp\u003eLipid peroxidation, mediated by ROS, can induce ferroptosis. Acyl-CoA synthetase long-chain family member 4 (ACSL4), a crucial compound necessary for initiating lipid peroxidation and dictating sensitivity to ferroptosis, catalyzes the esterification of free polyunsaturated fatty acids (PUFAs) to produce PUFA-CoA[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Conversely, SLC7A11 and GPX4 play vital roles in resisting lipid peroxidation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, the transferrin receptor (TFRC) facilitates the accumulation and transportation of ferrous iron, leading to iron-dependent ROS activation and ultimately ferroptosis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recently, researchers have focused on the role of ferroptosis in various skeletal muscle diseases, highlighting its significance in both physiological and pathological processes, including sarcopenia and rhabdomyosarcoma[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, western blotting analysis revealed that the expression of ACSL4 was significantly increased in the aged mice when compared to the adult mice \u003cb\u003e(Supplementary Figs. S5A-B)\u003c/b\u003e. However, there was no significant variation tendency in SLC7A11 and GPX4 levels in the old mice compared to the adult mice \u003cb\u003e(Supplementary Figs. S5C-F).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFurther investigation was conducted on the impact of LDLR-low expression on the ferroptosis pathway. Our results showed that LDLR-low expression suppresses ferroptosis, as evidenced by elevated protein expression of GPX4 and SLC7A11, along with decreased protein levels of ACSL4 and TFRC in the A-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared with the A-LDLR\u003csup\u003evector\u003c/sup\u003e group \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D\u003cb\u003e)\u003c/b\u003e. Similarly, in old mice, LDLR-low expression led to higher levels of SLC7A11 (but not GPX4) and lower levels of ACSL4 and TFRC in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared with the O-LDLR\u003csup\u003evector\u003c/sup\u003e group \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D\u003cb\u003e)\u003c/b\u003e. Thus, these results suggested that LDLR-low expression in the quadriceps muscle suppresses ferroptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAge-related sarcopenia, characterized by muscle loss, increases the risk of fractures, impairs physical function and lowers quality of life[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our findings align with previous research, demonstrating that elderly mice have lower muscle mass and grip strength compared to young adult mice. In addition, we found that lipofuscin, mitochondrial ROS, and ferroptosis-related protein ACSL4 were significantly increased in the quadriceps muscle of elderly mice. Although LDLR accumulation in the skeletal muscles was found in the old rats, the connection between LDLR in skeletal muscle and age-related sarcopenia remains poorly understood. Here, we provide the first evidence that LDLR-specific knockdown in skeletal muscle attenuates aging-related sarcopenia potentially linked to enhanced mitochondrial fusion and suppression of ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs a key site of glucose and lipid oxidation, skeletal muscle plays an important role in controlling whole-body energy expenditure[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies ha\u003cb\u003eve\u003c/b\u003e focused on the relationship between skeletal muscle and cholesterol, emphasizing the potential of skeletal muscle in reducing cholesterol levels and preventing atherosclerotic cardiovascular disease. Knocking out the LDLR in mice (Ldlr-/- mice) is known to inhibit the uptake of LDL, resulting in elevated plasma LDL-C levels. Notably, this study specifically targeted the knockdown of skeletal muscle LDLR while leaving LDLR levels in other organs and tissues unaffected. Skeletal muscle serves as a crucial site for carbohydrate and lipid metabolism. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear transcriptional coactivator, regulates mitochondrial biogenesis and plays a role in carbohydrate and lipid metabolism. Previous studies have shown that muscle-specific overexpression of PGC-1α increases several phospholipid species in glycolytic muscle[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], enhances oxidase levels[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and increases mitochondrial preference for lipid substrates[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These findings suggest that PGC-1α may promote lipid utilization in skeletal muscle. Therefore, we performed additional experiments to measure the mRNA levels of PGC-1α in skeletal muscles. Our results showed that PGC-1α mRNA levels were significantly increased in the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group when compared to the O-LDLR\u003csup\u003evector\u003c/sup\u003e group (Supplementary Figure S3). Therefore, we speculate that skeletal muscle-specific LDLR knockdown reduced serum TG and LDL-C levels, potentially linked to the observed increase in PGC-1α levels. However, this hypothesis requires further verification in future studies. Additionally, the improvement in locomotor function appears to be a secondary effect of the decreased serum TG and LDL-C levels in the aged mice.\u003c/p\u003e\u003cp\u003eStudy have shown that cholesterol could hinder satellite cell myogenic development[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although the side effects of statin-induced muscle myopathy have been noted, the mechanism of action remains unclear. It was reported that after being treated chronically with statins, LDLR was increasingly expressed in the gastrocnemius muscle in adult male rats[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which may provide the key link between LDLR and myopathy. In this study, we found that LDLR skeletal muscle-specific knockdown in aged mice increased muscle mass, limb grip strength and the myofiber CSA while decreasing lipofuscin in the quadriceps muscle. During sarcopenia, skeletal muscle fibers, especially type II muscle fibers (MyHC II), are susceptible to atrophy in aged mice in aged mice[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Interestingly, the O-LDLR\u003csup\u003eKD\u003c/sup\u003e group exhibited a higher augmentation in fast-contracting myofibers (MyHC II) compared to the control group, along with a notable decrease in the expression of the atrophy-related protein MuRF. In this study, adult and aged mice exhibited differences in muscle fiber types and associated muscle strength following LDLR knockdown. In age-related muscle atrophy, there was a preferential loss of glycolytic type II muscle fibers, while oxidative type I muscle fibers were typically preserved[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our results showed that LDLR knockout led to an increased expression of MyHC II (type II, fast filament) mRNA and protein in aged mice, whereas MyHC I (type I, slow filament) mRNA levels remained unchanged. Although the mean CSA of type I and type II fibers did not differ, the mean CSA of quadriceps fibers increased. These changes corresponded to enhanced strength related to fast-twitch fibers, as evidenced by improved forelimb grip strength. However, endurance of slow-twitch fibers, measured by limb suspension time, remained unchanged in the old mice following LDLR knockdown. In adult mice, LDLR knockout increased the mRNA level of MyHC I while decreasing that of MyHC II. The mean CSA of muscle fibers, primarily type I fibers, increased, leading to enhanced slow-twitch endurance (limb suspension time), while the corresponding force of fast muscles (forelimb grip strength) was not affected.\u003c/p\u003e\u003cp\u003eMitochondria in muscle cells may undergo a decrease in efficiency and energy production capacity with age, which could play a role in sarcopenia[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The mitochondrial free radical theory of aging suggests that age-related mitochondrial dysfunction results in higher levels of ROS, leading to further mitochondrial damage and overall cellular deterioration[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our data suggest that LDLR knockdown reduces the formation of ROS associated with aging \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Increased levels of mitochondrial ROS may lead to oxidative modification of cardiolipin, a fusogenic lipid, and affect the oligomerization of GTPases that control mitochondrial membrane fusion[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Mitochondria exhibit a complex and dynamic architecture in various cell type, particularly in skeletal muscle[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Mitochondria form a dynamic network capable of fusion and fission events, collectively known as mitochondrial dynamics. The regulation of these events is mediated by Mfn1 and Mfn2, Opa-1 and Drp1, and mitochondrial Fis1 proteins[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have shown that morphological changes in the mitochondria have also been observed with age, including less uniformity and more fragmentation as well as a swollen appearance[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our results are in line with those observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Mitochondrial morphology is regulated by fusion and fission processes, which are essential for maintaining organelle homeostasis and normal cellular function\u003cb\u003es\u003c/b\u003e. Dysfunction in mitochondrial dynamics has been implicated in myopathy, where increased fission leads to fragmented mitochondria and muscle wasting[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, aging-related skeletal muscle atrophy is associated with mitochondrial fission[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGenerally, cholesterol carried by LDL is internalized into cells via the LDL receptor (LDLR) through a clathrin-dependent mechanism. LDL-C is subsequently transported to lysosomes for hydrolysis, releasing cholesterol, while LDLR is recycled back to the plasma membrane[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, a recent study has reported that LDL-LDLR vesicle can bypass lysosomes and be directly transported to mitochondria, where LDL-C is hydrolyzed to release cholesterol for steroidogenesis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the context of aging, the accumulation of cholesterol in mitochondria beyond physiological levels may impair mitochondrial function and trigger oxidative stress and cell death[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Following LDLR knockdown, skeletal muscle mitochondrial ROS levels and the mitochondrial fission-related protein, Drp1, were significantly decreased, while the mitochondrial fusion-related protein, Mfn2, was significantly increased in the old mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). This suggests that LDLR knockdown may enhance mitochondrial fusion and reduce ROS levels in the mitochondria within the skeletal muscles of older mice. Although the mechanisms underlying these observations are not entirely clear, several concurrent scenarios may explain this condition. As aging advances, the expression of PGC-1α, a crucial regulator of mitochondrial biogenesis, decreases. This decline increases oxidative stress in skeletal muscle, resulting in elevated ROS levels that damage mitochondrial function, reduce mitochondrial membrane fusion, and increase mitochondrial fission. In contrast, LDLR knockdown appears to enhance the expression of PGC-1a in aged mice (Supplementary Figure S3), thereby preserving mitochondrial function and dynamics.\u003c/p\u003e\u003cp\u003eThe accumulation of ROS and decreased endogenous antioxidant mechanisms are key factors in the progression of sarcopenia[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Ferroptosis, a form of cell death regulated by iron metabolism, antioxidant processes and lipid metabolism, has emerged as a significant player in skeletal muscle disorders such as sarcopenia and rhabdomyolysis[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. ACSL4, a crucial enzyme necessary for initiating lipid peroxidation, has been identified as a crucial player in the execution of ferroptosis[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Consistent with previous \u003cb\u003estudies\u003c/b\u003e, our findings demonstrated a significant increase in ACSL4 levels in the quadriceps muscle of aged mice, suggesting the occurrence of ferroptosis \u003cb\u003e(Supplemental Fig. S5)\u003c/b\u003e. Interestingly, LDLR skeletal muscle-specific knockdown mice exhibited elevated levels of SLC7A11, known for its crucial role in combating lipid peroxidation, and decreased levels of ACSL4. This indicates that LDLR-specific knockdown may offer protection against ferroptosis in skeletal muscle.\u003c/p\u003e\u003cp\u003eThe present study has identified several limitations that should be considered in future research. It is important to recognize the different types of sarcopenia, including age-related sarcopenia, obese sarcopenia and cachexia-induced sarcopenia. Findings from studies conducted on older mice may not directly translate to observations in clinical patients. Additionally, this study only focused on \u003cem\u003ein vivo\u003c/em\u003e research, and it would be beneficial to include \u003cem\u003ein vitro\u003c/em\u003e studies to further validate the conclusions drawn. Moreover, we only measured in vivo muscle strength tests such as grip strength and limb suspension tests. Future studies should include ex vivo muscle strength assessment to evaluate the contractile function of isolated skeletal muscles. Last but not the least, while the effects of LDLR knockdown on mitochondrial dynamics and ferroptosis were observed in the older mice; however, further experiments are necessary to confirm the causal relationship between these factors and to fully elucidate the mechanism by which LDLR contributes to the treatment of sarcopenia.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study demonstrated for the first time that LDLR plays a vital role in regulating age-related sarcopenia, which is associated with skeletal muscle mitochondrial dynamic process and ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. LDLR-specific knockdown in skeletal muscle\u003cb\u003es\u003c/b\u003e can attenuate muscle atrophy and loss of strength in aged mice. Further clarification of the relationship between LDLR and sarcopenia will provide new therapeutic strategies and targets for age-related sarcopenia and subsequent clinical complications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TG: Triacylglycerol; DCFH-DA: Dichlorofluorescein diacetate; LDLR: Low-density lipoprotein Receptor; FFPE: Formalin-fixed Paraffin Embedded; PVDF: Polyvinylidene fluoride; BCA: Bicinchoninic Acid; Atrogin: Atrogin 1 recombinant protein; MuRF: Muscle RING-finger protein-1; MyHC I: Myosin heavy chain Type I MyHC II: Myosin heavy chain Type II; OPA1: Optic atrophy 1; Mfn2: Mitofusin-2; Drp1：Dynamin-related protein1; Fis1：Mitochondrial fission 1 protein; VDAC: Voltage-dependent anion channel 1; TFRC: Transferrin receptor; ACSL4: Acyl-CoA Synthetase Long-Chain Family Member 4; GPX4: Glutathione Peroxidase 4; SLC7A11: Solute Carrier Family 7 Member 11; HE staining: Hematoxylin and Eosin (H\u0026amp;E) staining; AAV: Adenovirus Associated Virus;\u0026nbsp;A-LDLR\u003csup\u003evector\u003c/sup\u003e: Adult mice injected with empty vector as control; A-LDLR\u003csup\u003eKD\u003c/sup\u003e: Adult mice injected with AAV vectors to knockdown LDLR in skeletal muscle; O-LDLR\u003csup\u003evector\u003c/sup\u003e: Old mice injected with empty vector as control; O-LDLR\u003csup\u003eKD\u003c/sup\u003e: Old mice injected with AAV vectors to knockdown LDLR in skeletal muscle.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJW\u003c/strong\u003e and \u003cstrong\u003eSL\u003c/strong\u003e: Conceptualization, funding acquisition, and resources; \u003cstrong\u003eZF\u003c/strong\u003e, \u003cstrong\u003eTF\u003c/strong\u003e, \u003cstrong\u003eXZ\u003c/strong\u003e, \u003cstrong\u003eYF, YL\u003c/strong\u003e, \u003cstrong\u003eYL\u003c/strong\u003e, \u003cstrong\u003eBL\u003c/strong\u003e, \u003cstrong\u003eLH\u003c/strong\u003e: Performed experiments; SRS: Writing-review and editing；\u003cstrong\u003eZF\u003c/strong\u003e, \u003cstrong\u003eTF\u003c/strong\u003e, \u003cstrong\u003eXZ\u003c/strong\u003e: Formal analysis; \u003cstrong\u003eZF\u003c/strong\u003e: Writing-original draft; \u003cstrong\u003eJW\u003c/strong\u003e and \u003cstrong\u003eSL\u003c/strong\u003e: Conceptualization, Writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 81560239 to JW), the Baise scientific research and technology development plan of regionally frequently occurring diseases, China (No. 20224129 to SL), the basic scientific research ability improvement project of young and middle-aged university teachers in Guangxi, China (No. 2019KY0568 to SL); the Natural Science Foundation of Youjiang Medical University for Nationalities (No. yy2018ky001 to SL),the 2023 Innovation Project of Youjiang Medical University for Nationalities Graduate Education (No. YXCXJH2023024) and the high-level Talent Program of Youjiang Medical University for Nationalities (No. yy2024rcky003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDhillon RJ, Hasni S. 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Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner. \u003cem\u003eJ Biol Chem\u003c/em\u003e. Dec 18 2015;290(51):30514-29.\u003c/li\u003e\n \u003cli\u003eShang GK, Han L, Wang ZH, et al. Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles. \u003cem\u003eJ Cachexia Sarcopenia Muscle\u003c/em\u003e. Aug 2020;11(4):1104-1120.\u003c/li\u003e\n \u003cli\u003eYoshida N, Endo J, Kinouchi K, et al. (Pro)renin receptor accelerates development of sarcopenia via activation of Wnt/YAP signaling axis. \u003cem\u003eAging Cell\u003c/em\u003e. Oct 2019;18(5):e12991.\u003c/li\u003e\n \u003cli\u003eWang R, Li K, Sun L, et al. L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens. \u003cem\u003eAnim Nutr\u003c/em\u003e. Sep 2022;10:68-85.\u003c/li\u003e\n \u003cli\u003eGonzalez-Freire M, Adelnia F, Moaddel R, Ferrucci L. Searching for a mitochondrial root to the decline in muscle function with ageing. \u003cem\u003eJ Cachexia Sarcopenia Muscle\u003c/em\u003e. Jun 2018;9(3):435-440.\u003c/li\u003e\n \u003cli\u003eL\u0026oacute;pez-Ot\u0026iacute;n C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. \u003cem\u003eCell\u003c/em\u003e. Jun 6 2013;153(6):1194-217.\u003c/li\u003e\n \u003cli\u003eSingh SB, Ornatowski W, Vergne I, et al. Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria. \u003cem\u003eNat Cell Biol\u003c/em\u003e. Dec 2010;12(12):1154-65.\u003c/li\u003e\n \u003cli\u003eDantas WS, Zunica ERM, Heintz EC, et al. Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control. \u003cem\u003eJ Cachexia Sarcopenia Muscle\u003c/em\u003e. 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Lower Cardiorespiratory Fitness Is Associated With Right Ventricular Geometry and Function - The Sedentary\u0026apos;s Heart: SHIP. \u003cem\u003eJ Am Heart Assoc\u003c/em\u003e. Nov 16 2021;10(22):e021116.\u003c/li\u003e\n \u003cli\u003eCartee GD, Hepple RT, Bamman MM, Zierath JR. Exercise Promotes Healthy Aging of Skeletal Muscle. \u003cem\u003eCell Metab\u003c/em\u003e. Jun 14 2016;23(6):1034-1047.\u003c/li\u003e\n \u003cli\u003eGoicoechea L, Conde de la Rosa L, Torres S, Garc\u0026iacute;a-Ruiz C, Fern\u0026aacute;ndez-Checa JC. Mitochondrial cholesterol: Metabolism and impact on redox biology and disease. \u003cem\u003eRedox Biol\u003c/em\u003e. May 2023;61:102643.\u003c/li\u003e\n \u003cli\u003eScicchitano BM, Pelosi L, Sica G, Musar\u0026ograve; A. The physiopathologic role of oxidative stress in skeletal muscle. \u003cem\u003eMech Ageing Dev\u003c/em\u003e. Mar 2018;170:37-44.\u003c/li\u003e\n \u003cli\u003eZhang Y, Huang X, Qi B, et al. Ferroptosis and musculoskeletal diseases: \u0026quot;Iron Maiden\u0026quot; cell death may be a promising therapeutic target. \u003cem\u003eFront Immunol\u003c/em\u003e. 2022;13:972753.\u003c/li\u003e\n \u003cli\u003eDoll S, Proneth B, Tyurina YY, et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. \u003cem\u003eNat Chem Biol\u003c/em\u003e. Jan 2017;13(1):91-98.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sarcopenia, Aging, LDLR, Skeletal muscle, Mitochondrial dynamics, Ferroptosis ","lastPublishedDoi":"10.21203/rs.3.rs-6891536/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6891536/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e 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. \u0026nbsp;Lipid accumulation is suggested to be a potential mechanism that may contribute to mitochondrial dysfunction and subsequently lead to sarcopenia. \u0026nbsp;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.\u0026nbsp; This study aimed to investigate the effects of LDLR knockdown on skeletal muscle.\u003c/p\u003e\n\u003cp\u003eMethods: Wild-type and LDLR skeletal muscle-specific knockdown mice were randomly divided into adult and old groups. \u0026nbsp;The control group consisted of adult and old mice that were injected with AAV-gRNA empty vector virus. \u0026nbsp;The grip strength was measured before sacrifice.\u0026nbsp; Following scarification, skeletal muscles were collected for atrophy assessment using histopathological and immunofluorescent methods. \u0026nbsp;Mitochondria were isolated from skeletal muscle and their morphology and ROS levels were assessed. \u0026nbsp;LDLR expression, atrophy-related proteins, mitochondrial fission, and fusion-related proteins, and ferroptosis pathway were measured by western blotting.\u003c/p\u003e\n\u003cp\u003eResults: In aged mice, there was a significant decrease in muscle mass normalized to body weight (1.3±0.04 vs 1.5±0.05 %, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and forelimb grip strength (2.01±0.13 vs 2.38±0.08 g/g, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) as well as increased levels of lipofuscin, mitochondrial ROS (3924±369 vs 2527±326 a.u., \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) and the ferroptosis-related protein, ACSL4, in the quadriceps muscle, when compared to adult mice.\u0026nbsp; Following LDLR knockdown, there was an increase in muscle mass normalized to body weight (1.50±0.02 vs 1.36±0.03%, \u003cem\u003ep\u003c/em\u003e \u0026lt; 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,\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05) in LDLR knockdown aged mice (O-LDLR\u003csup\u003eKD \u003c/sup\u003egroup), when compared to the old mice injected with empty vector (O-LDLR\u003csup\u003evector \u003c/sup\u003egroup). \u0026nbsp;Additionally, lipofuscin levels and the atrophy-related protein, MuRF, were decreased in the O-LDLR\u003csup\u003eKD \u003c/sup\u003egroup compared to the control\u003csup\u003e \u003c/sup\u003egroup. \u0026nbsp;Mitochondrial ROS\u0026nbsp; and the Drp1 mitochondrial fission protein (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) levels were significantly decreased, while the Mfn2 mitochondrial fusion protein levels increased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u0026nbsp;Among the ferroptosis-related markers, ACSL4 showed a marked decrease (\u003cem\u003ep\u003c/em\u003e＜0.01), while SLC7A11 increased (\u003cem\u003ep\u003c/em\u003e＜0.05) in the O-LDLR\u003csup\u003eKD \u003c/sup\u003egroup compared to the O-LDLR\u003csup\u003evector \u003c/sup\u003egroup.\u003c/p\u003e\n\u003cp\u003eConclusions: 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.\u003c/p\u003e","manuscriptTitle":"Knockdown of low-density lipoprotein receptors in skeletal muscle attenuates aging-related sarcopenia associated with mitochondrial fusion and ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 14:07:36","doi":"10.21203/rs.3.rs-6891536/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-09T07:29:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T22:02:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T08:48:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181612191453933261910462224063376280513","date":"2025-09-29T16:51:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233803890206666535479270461255981240806","date":"2025-09-24T15:54:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-24T06:22:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-23T17:37:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-07T13:20:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-27T03:18:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-27T03:14:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afab645d-b797-4225-832e-2319d7a7ddfd","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":55757249,"name":"Health sciences/Medical research"},{"id":55757250,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-01-17T01:23:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 14:07:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6891536","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6891536","identity":"rs-6891536","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}