Disuse-associated loss of the protease LONP1 in muscle impairs mitochondrial function and causes reduced skeletal muscle mass and strength

Abstract

Mitochondrial proteolysis is an evolutionarily conserved quality-control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a role in controlling mitochondrial function as well as skeletal muscle mass and strength in response to muscle disuse. In humans and mice, disuse-related muscle loss is associated with decreased mitochondrial LONP1 protein. Skeletal muscle-specific ablation of LONP1 in mice resulted in impaired mitochondrial protein turnover, leading to mitochondrial dysfunction. This caused reduced muscle fiber size and strength. Mechanistically, aberrant accumulation of mitochondrial-retained protein in muscle upon loss of LONP1 induces the activation of autophagy-lysosome degradation program of muscle loss. Overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known protein degraded by LONP1, in skeletal muscle induces mitochondrial dysfunction, autophagy activation, and cause muscle loss and weakness. Thus, these findings reveal a role of LONP1-dependent mitochondrial protein quality-control in safeguarding mitochondrial function and preserving skeletal muscle mass and strength, and unravel a link between mitochondrial protein quality and muscle mass maintenance during muscle disuse.

Fig. 1. Disuse-related muscle loss in mice and humans is associated with decreased mitochondrial LONP1 protein.

a Representative Western blot analysis performed with gastrocnemius (GC) muscle total protein extracts prepared from non-denervated (basal) and denervated limbs of WT mice for the indicated number of days (d) using indicated antibodies. b Quantification of LONP1/Τubulin, CLPP/Τubulin, OPA1/Tubulin, MFN1/Tubulin, MFN2/Tubulin and DRP1/Tubulin signal ratios normalized (=1.0) to basal is shown. n = 3–6 mice per group. c Representative western blot analysis performed with GC muscle total protein extracts prepared from non-immobilized (basal) and immobilized limbs of WT mice for the indicated number of days (d) using indicated antibodies. d Quantification of LONP1/Τubulin, CLPP/Τubulin, and OPA1/Tubulin signal ratios normalized (=1.0) to basal is shown. n = 3–6 mice per group. (e, f) (Left) Representative confocal images of extensor digital longus (EDL) muscles in HSA-Cre/MitoTimer mice at 5 days post denervation (e) or 10 days post immobilization (f). The scale bar represents 10 μm. (Right) Quantification of MitoTimer Red:Green ratio normalized (=1.0) to control is shown. n = 3 mice per group. g, h Supraspinatus muscle samples from 9 rotator cuff tear patients who underwent rotator cuff repair surgery were used for this analysis. g Representative Western blot analysis of LONP1, CLPP, and OPA1 expression in human supraspinatus muscles. Based on the occupation ratio, patients were divided into two groups (non-atrophy group, mean ratio 0.76; atrophy group, mean ratio 0.50). h Quantification of LONP1/Τubulin and CLPP/Τubulin data shown in (g). n = 4–5 patients per group. (i) Correlation between the protein expression of LONP1 and CLPP to the occupation ratios. n = 9 patients. Pearson’s correlation analysis was used to determine the correlation. Values represent mean ± SEM; for (b) and (d), *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding controls determined by one-way ANOVA coupled to a Fisher’s LSD post-hoc test; for (e), (f), and (h), *P < 0.05, **P < 0.01 versus corresponding controls determined by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 2. Skeletal muscle-specific ablation of LONP1 impairs muscle growth and causes muscle weakness and precocious aging.

a Representative hindlimbs and GC muscles from indicated mice at the age of 6 weeks. b, c GC and TA muscles weight in WT and LONP1 mKO mice in basal conditions (Basal) and after 15 days of denervation (Den). n = 5 mice per group. d WGA (green) staining of GC muscle from indicated mice. Scale bar: 50 μm. n = 5 mice per group. (e) Cross-sectional areas of GC myofibers. n = 5 mice per group. f LONP1 mKO mice showed decreased muscle-grip strength compared to WT controls. n = 8–12 mice per group. g Measurement of muscle tetanic contraction reveals a reduced contraction force in LONP1 mKO EDL muscle. n = 3–4 mice per group. h Bars represent mean running time and distance for 10-week-old male LONP1 mKO mice and WT controls on a motorized treadmill. n = 7 mice per group. i (Left) Schematic depicts the increments of speed over time. (Right) Respiratory exchange ratio (RER) during the course of the high-intensity exercise in indicated mice. n = 8 mice per group. j Peak ΔVO₂ (increase in oxygen consumption during exercise) is graphed. n = 8 mice per group. k Bars represent mean blood lactate levels for indicated mice following a 25-min run on a motorized treadmill. n = 9–13 mice per group. (l) GSEA of genes upregulated in LONP1-deficient muscle in relation to normal aging in WT mice. m Representative lateral X-ray images of WT and LONP1 mKO mice at indicated age. n = 3–9 mice per group. Values represent mean ± SEM; for (b) and (e), *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding WT controls, ^‡P < 0.05, ^‡‡P < 0.01, ^‡‡‡P < 0.001 versus Basal, determined by one-way ANOVA coupled to a Fisher’s LSD post-hoc test; for (c) and (f–k), *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding WT controls determined by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 3. Loss of LONP1 leads to severe mitochondrial structural and functional abnormalities in skeletal muscle.

a, b Representative electron micrographs of soleus muscle showing subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in sections from WT and LONP1 mKO mice at the age of 2 weeks (a) and at 6 weeks (b). The scale bar represents 1 μm. n = 4–6 mice per group. Note, dilated, frequently lost, or appeared vesiculated cristae and electro-dense aggregates were observed in LONP1 mKO mitochondria. No abnormal mitochondrial ultrastructures were observed in the WT controls. c Results of quantitative PCR to determine mitochondrial DNA levels in GC muscle of indicated mice at 6 weeks of age using primers for NADH dehydrogenase (Nd1, mitochondria-encoded) and lipoprotein lipase (Lpl, nuclear-encoded). Nd1 levels were normalized to Lpl DNA content and expressed relative to WT (=1.0) muscle. n = 6 mice per group. d, e Mitochondrial respiration rates were determined on mitochondria isolated from WV muscles of indicated mice using pyruvate (d) or succinate (e) as substrates. Pyruvate/malate (Py/M) or succinate/rotenone (Suc/Rot)-stimulated, ADP-dependent respiration, and oligomycin (oligo)-induced are shown. n = 5 mice per group. f ATP levels were detected and normalized to the percentage of WT muscles. n = 5 mice per group. Values represent mean ± SEM; for (c–f), ***P < 0.001 versus corresponding WT controls determined by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 4. Muscle LONP1 deficiency does not affect protein synthesis and ubiquitin-mediated protein degradation but activates autophagy.

a Expression of atrophy-related genes (RT-qPCR) in GC muscle from indicated mice at the age of 2 weeks and at 6 weeks. n = 5–6 mice per group. b (Left) Western blot analysis of WT and LONP1 mKO WV muscles using indicated antibodies. (Right) Quantification of the p-Foxo3A/Foxo3A signal ratios. n = 5 mice per group. c Quantification of the ubiquitinated proteins of muscle total protein extracts from 2-week-old indicated mice. n = 5 mice per group. d Quantification of puromycin incorporation in muscles from indicated mice at the age of 2 weeks. n = 3 mice per group. e (Left) Western blot analysis of WV muscles from 2-week-old mice using indicated antibodies. (Right) Quantification of the p-AKT/AKT, p-S6K/S6K and p-4EBP1/4EBP1 signal ratios. n = 5 mice per group. f, g (Left) Confocal images of TA (f) and EDL (g) muscles in 6-week-old mt-Keima-TG mice and LONP1 mKO/mt-Keima mice. The scale bar represents 10 μm. 458-nm laser, green, 561-nm laser, red. (Right) Quantification of the red/green mt-Keima signal ratios. n = 3 mice per group. h, i Autophagy flux is increased in LONP1 mKO muscles. h (Left) Western blot analysis of mitochondrial-associated LC3-II in WV muscle from indicated mice. Mice were treated for 24 h with colchicine (Col) as indicated. (Right) Quantification of the mitochondrial-associated LC3-II/Tim23 signal ratios. n = 3–4 mice per group. i (Left) Western blot analysis of WV muscle total protein extracts prepared from indicated mice treated for 24 h with or without Col. (Right) Quantification of the LC3-II/LC3-I and P62/Tubulin signal ratios. n = 3–4 mice per group. Values represent mean ± SEM; for (a–g), *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding controls determined by two-tailed unpaired Student’s t test; for (h) and (i), *P < 0.05, ***P < 0.001 versus corresponding controls, ^‡‡P < 0.01, ^‡‡‡P < 0.001 versus vehicle controls, determined by one-way ANOVA coupled to a Fisher’s LSD post-hoc test; Source data are provided as a Source Data file.

Fig. 5. Acute deletion of LONP1 in mature muscle leads to autophagy activation and causes muscle loss and weakness.

a–g Mice were sacrificed 8 weeks after the injection of AAV-Cre or AAV-Control in 4-week-old Lonp1^f/f mice. a Western blot analysis of muscles for Lonp1^f/f mice transduced with indicated AAV. n = 8 mice per group. b TA muscles from indicated mice. c TA muscle weight from indicated mice. n = 8 mice per group. d Isometric tetanic contractions measurement. n = 3 mice per group. e, f (Left) Confocal images of TA (e) and EDL (f) muscles for Lonp1^f/f/mt-Keima mice transduced with indicated AAV. Scale bar: 10 μm. (Right) Quantification of the red/green mt-Keima signal ratios. n = 3 mice per group. g (Top) Western blot analysis of TA muscles from indicated mice. (Bottom) Quantification of the LC3-II/Tubulin signal ratios. n = 5 mice per group. h–i Primary myoblasts isolated from Lonp1^f/f mice were infected with an adenovirus overexpressing Cre or control virus, followed by differentiation into myotubes. h (Left) Images of myotubes. Scale bar: 200 μm. (Right) Measurements of myotube diameter. n = 376–418 myotubes per condition. i Total cellular protein content relative to genomic DNA in myotubes. n = 3. j Oxygen consumption rates (OCR) in myotubes. n = 5. k (Top) Confocal images of primary myoblasts expressing GFP-LC3. Scale bar: 10 μm. (Bottom) Quantification of GFP puncta per cell. n = 28–41 cells. l, m Autophagy flux is increased in LONP1 KO myocytes. (Top) Western blot analysis of myoblasts (l) and myotubes (m) subjected to adenovirus-based overexpression of Cre compared with control. Myocytes were treated for 4 h with or without chloroquine (CQ) as indicated. (Bottom) Quantification of the LC3-II/LC3-I and P62/Tubulin signal ratios. n = 3. Values represent mean ± SEM; for (c–k), *P < 0.05, ***P < 0.001 versus corresponding controls determined by two-tailed unpaired Student’s t test; for (l) and (m), *P < 0.05 versus corresponding controls, ^‡P < 0.05 versus vehicle controls, determined by one-way ANOVA coupled to a Fisher’s LSD post-hoc test; Source data are provided as a Source Data file.

Fig. 6. LONP1 controls mitochondrial proteostasis that regulates muscle cell autophagy.

a, b (Left) Confocal images of TA (a) and EDL (b) muscles from LONP1 mKO/MitoTimer and Lonp1^f/f/MitoTimer mice. The scale bar represents 10 μm. (Right) Quantification of MitoTimer Red:Green ratio. n = 3 mice per group. c, d Proteomes of crude mitochondria isolated from 2-week-old WT and LONP1 mKO muscles were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. n = 2 independent samples per group. c Venn diagram representing the proteomics results compared with MitoCarta2.0 database. d Heat-maps analysis of mass spectrometry proteomics data. Individual proteins are shown to be regulated in the LONP1 mKO muscles as denoted by the color scheme. e (Left) Western blot analysis of myotubes subjected to adenovirus-based overexpression of Cre compared with control. (Right) Quantification of the PARK7/Tubulin and PINK1/Tubulin signal ratios. n = 3. f RT-qPCR analysis of Park7 in myotubes. n = 3. g (Left) Cycloheximide (CHX) treatment of myotubes for the indicated time. (Right) Quantification of the PARK7 protein levels is shown. n = 3. h Co-IP experiments were performed by cotransfecting LONP1-GFP and PARK7-Flag in HEK293 cells as indicated at the top. Antibody against the Flag epitope was used for co-IP. The extracts (Input) from the HEK293 cells and proteins from the IP were analyzed by immunoblotting (IB). Representative results for co-IP are shown. n = 3. i Western blot analysis of LC3-II/LC3-I ratios in myotubes transfected with control siRNA or Park7 siRNA and subjected to adenovirus-based overexpression of Cre compared with control. Myotubes were treated for 4 h with or without chloroquine (CQ) as indicated. j Quantification of the LC3-II/LC3-I signal ratios in (i). n = 3. Values represent mean ± SEM; for (a), (b), (e), (f) and (g), *P < 0.05, **P < 0.01 versus corresponding controls determined by two-tailed unpaired Student’s t test; for (j), **P < 0.01 versus corresponding controls, ^‡‡P < 0.01 versus vehicle controls, determined by one-way ANOVA coupled to a Fisher’s LSD post-hoc test; Source data are provided as a Source Data file.

Fig. 7. Overexpression of mitochondrial-retained ΔOTC protein in skeletal muscle activates autophagy and causes muscle loss and weakness.

a Western blot analysis of mitochondrial ΔOTC overexpression in WV muscle total protein extracts (Top) or mitochondrial fraction (Bottom) prepared from indicated mice. n = 3 mice per group. b, c Mitochondrial respiration rates were determined on mitochondria isolated from WV muscles of indicated mice using pyruvate (b) or succinate (c) as substrates. n = 3–5 mice per group. d Picture of hindlimbs and GC muscles from 8-week-old male mice. e GC and TA muscle weight from indicated mice. n = 3–5 mice per group. f (Top) Representative WGA (green) staining of GC muscle from 8 week old indicated mice. Scale bar represents 50 μm. (Bottom) Cross-sectional areas of GC myofibers were measured by ImageJ. n = 5 mice per group. g Isometric tetanic contraction measurements for isolated EDL muscle from indicated mice. n = 3–5 mice per group. h Expression of muscle atrophy-related genes (RT-qPCR) in GC muscle from indicated mice. n = 3–5 mice per group. i, j Representative immunoblot analysis of WV muscle total protein extracts from indicated mice. i (Right) Quantification of the p-AKT/AKT and p-S6K/S6K signal ratios were normalized (=1.0) to NTG controls. n = 3 mice per group. j Quantification of the LC3-II/LC3-I signal ratios were normalized (=1.0) to NTG controls and presented below the corresponding bands. k, l (Left) Representative confocal images of TA (k) and EDL (l) muscles in mt-Keima-TG mice and MCK-ΔOTC/mt-Keima mice. The scale bar represents 10 μm. 458-nm laser, green, 561-nm laser, red. (Right) Quantification of the red/green mt-Keima signal ratios normalized (=1.0) to NTG controls is shown. n = 3 mice per group. Values represent mean ± SEM; for (b), (c), (e), (g), (h), (i), (k) and (l), *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding NTG controls determined by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 8. LONP1 preserves muscle function through maintaining mitochondrial proteostasis.

a–f MCK-ΔOTC mice were injected intraperitoneally on postnatal day 3 (P3) and day 5 (P5) with or without AAV expressing LONP1 (AAV-LONP1). Terminal studies were conducted 4 weeks after the injection of AAV. b, c Representative immunoblot analysis of muscle total protein extracts from indicated mice. b (Right) Quantification of the FLAG-ΔOTC/Tubulin signal ratios normalized (=1.0) to MCK-ΔOTC mice. Ud, undetectable. n = 3 mice per group. c (Left) Representative Western blot analysis of LC3-II protein in muscle total protein extracts from indicated mice. (Right) Quantification of the LC3-II/Tubulin signal ratios were normalized (= 1.0) to NTG controls. n = 5 mice per group. d Mitochondrial respiration rates were determined on mitochondria isolated from WV muscles of indicated mice using pyruvate as substrates. Pyruvate/malate (Py/M), ADP-dependent respiration, and oligomycin (oligo)-induced are shown. n = 3–5 mice per group. e Representative WGA (green) staining of GC muscle from indicated mice. Scale bar represents 50 μm. n = 5–6 mice per group. f Quantification of fiber size distribution in GC muscles from indicated mice. n = 5–6 mice per group. g Model of LONP1-mediated mitochondrial protein quality control in the regulation of skeletal muscle mass and strength. The schematic depicts a proposed model for disuse-associated loss of muscle LONP1 that controls mitochondrial function and skeletal muscle mass. Values represent mean ± SEM; for (b), ^‡P < 0.05 versus MCK-ΔOTC determined by two-tailed unpaired Student’s t test; for (c) and (d), **P < 0.01, ***P < 0.001 versus corresponding controls, ^‡P < 0.05 versus MCK-ΔOTC, determined by one-way ANOVA coupled to a Fisher’s LSD post-hoc test; Source data are provided as a Source Data file.