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

Mitochondria, essential organelles with diverse functions, require continuous quality control to maintain their integrity and function. Mitochondrial proteases, such as LONP1 and CLPP, are key players in this process, selectively targeting and removing damaged or dysfunctional proteins. Disruptions in mitochondrial proteostasis are linked to various diseases, including metabolic disorders, cancer, and neurodegenerative diseases. However, the physiological consequences of stress-induced impairment of mitochondrial protein quality control remain poorly understood. Skeletal muscle, the largest metabolically active tissue, is particularly vulnerable to mitochondrial dysfunction. Mitochondrial quality control is crucial for maintaining skeletal muscle function under various stresses. Muscle disuse, whether due to inactivity or aging, causes muscle loss (atrophy) and weakness, significantly impacting quality of life and mortality. This muscle loss results from an imbalance between protein synthesis and degradation. While ATP-dependent and -independent mitochondrial proteases degrade damaged proteins, the role of specific proteases in muscle disuse remains unclear. LONP1, an evolutionarily conserved serine peptidase, is a central component of mitochondrial protein quality control in mammals. Several proteins have been identified as LONP1 substrates, and LONP1 deficiency in mice is embryonically lethal. Mutations in LONP1 are linked to CODAS syndrome. While mitochondrial quality control is increasingly recognized as important for skeletal muscle function, the role of LONP1 in muscle stress response and regulation remains unexplored. This study aims to investigate the role of LONP1 in mediating mitochondrial protein quality control in skeletal muscle during disuse.

The existing literature strongly supports the critical role of mitochondrial proteases in maintaining cellular health and function. Studies have highlighted the involvement of disturbed mitochondrial proteostasis in various diseases, underscoring the importance of understanding the mechanisms regulating mitochondrial protein quality control. The relationship between mitochondrial health and skeletal muscle function has also been extensively studied, with research showing that impairments in mitochondrial function directly contribute to muscle atrophy and weakness. Specifically, the role of ATP-dependent proteases like LONP1 and CLPP in maintaining mitochondrial integrity and function has been emphasized. However, the specific contribution of LONP1 to skeletal muscle mass and strength in response to disuse has been less explored, creating the need for this study.

This study employed a multi-faceted approach, combining in vivo studies in mice with in vitro experiments in cultured myocytes and human patient samples. To investigate the effects of LONP1 deficiency, the researchers generated skeletal muscle-specific Lonp1-knockout (LONP1 mKO) mice by breeding Lonp1-floxed mice with mice expressing Cre recombinase in postnatal skeletal muscle. They employed denervation and hindlimb immobilization models to induce muscle disuse in mice. The researchers also analyzed human supraspinatus muscle samples from patients with and without muscle atrophy. Mitochondrial function was assessed through various techniques, including electron microscopy to examine mitochondrial ultrastructure, respirometry to measure mitochondrial respiration rates, and analysis of mitochondrial DNA content. Protein turnover was evaluated using MitoTimer reporter mice. The researchers also examined protein synthesis and degradation pathways, focusing on the ubiquitin-proteasome system and autophagy. In vitro studies involved culturing primary skeletal myocytes from Lonp1-floxed mice and inducing LONP1 deletion via adenoviral Cre expression. They also used a transgenic mouse model overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known LONP1 substrate, to study the effects of mitochondrial protein overload. Additional techniques employed included quantitative RT-PCR, Western blotting, immunofluorescence, immunoprecipitation, siRNA knockdown, and iTRAQ mass spectrometry. Statistical analyses included two-tailed unpaired Student’s t-tests, one-way ANOVAs, and Pearson's correlation tests.

The study's key findings demonstrate a strong correlation between LONP1 protein levels and skeletal muscle mass and strength, both in mouse models and human patients. In mice, skeletal muscle-specific deletion of LONP1 resulted in: 1. **Decreased mitochondrial LONP1 protein:** LONP1 protein levels decreased significantly in both denervation and immobilization models of muscle disuse, coinciding with muscle mass loss. This was also observed in atrophied human supraspinatus muscles. 2. **Impaired mitochondrial protein turnover:** MitoTimer analysis revealed significantly slower mitochondrial protein turnover in LONP1 mKO mice, indicating impaired mitochondrial proteostasis. 3. **Mitochondrial dysfunction:** Electron microscopy showed abnormal mitochondrial cristae structure and the presence of electro-dense aggregates in LONP1 mKO muscles. Mitochondrial respiration rates were significantly reduced. 4. **Reduced muscle mass and strength:** LONP1 mKO mice exhibited significantly reduced muscle mass, myofiber size, and grip strength compared to controls. They also showed impaired exercise performance and altered muscle fuel utilization. 5. **Autophagy activation:** Analysis of autophagy flux revealed a significant increase in mitochondrial autophagy in LONP1 mKO mice, suggesting that the loss of LONP1 triggers a compensatory autophagy response. 6. **No effect on ubiquitin-mediated protein degradation or protein synthesis:** The study found no significant changes in ubiquitin-mediated protein degradation or protein synthesis rates in LONP1 mKO muscles, suggesting that autophagy is the primary mechanism driving muscle loss in the absence of LONP1. 7. **ΔOTC overexpression recapitulates the phenotype:** Overexpressing the mitochondrial-retained ΔOTC mutant in skeletal muscle mimicked the effects of LONP1 deletion, inducing mitochondrial dysfunction, autophagy activation, and muscle loss, further supporting the role of LONP1 in maintaining mitochondrial proteostasis and muscle health. 8. **PARK7 identified as a LONP1 substrate:** Proteomic analysis identified PARK7 as a potential LONP1 substrate. Knockdown of PARK7 in LONP1-deficient myotubes reduced autophagy flux, suggesting a mechanistic link between LONP1, PARK7, and autophagy activation.

This study establishes a critical role for the mitochondrial protease LONP1 in maintaining skeletal muscle mass and function. The findings demonstrate that LONP1 deficiency leads to impaired mitochondrial proteostasis, resulting in mitochondrial dysfunction and activation of the autophagy-lysosome system. This autophagy activation, rather than changes in ubiquitin-mediated protein degradation or protein synthesis, appears to be the primary driver of muscle atrophy in the absence of LONP1. The study's findings are supported by both genetic manipulation of LONP1 in mice and in vitro studies using cultured myocytes, as well as observations in human patients with muscle atrophy. The identification of PARK7 as a potential downstream effector of LONP1, mediating autophagy activation, provides a potential mechanistic link explaining the observed muscle phenotype. The study also highlights the importance of maintaining mitochondrial proteostasis in preventing muscle loss, suggesting potential therapeutic targets for combating muscle atrophy associated with aging and disease.

This research demonstrates a crucial role for LONP1 in maintaining mitochondrial health and preserving skeletal muscle mass and strength. LONP1 deficiency leads to impaired mitochondrial protein turnover, mitochondrial dysfunction, and compensatory autophagy activation, resulting in muscle atrophy. The identification of PARK7 as a potential LONP1 substrate mediating autophagy activation provides a mechanistic insight. These findings suggest that targeting LONP1 or the autophagy pathway could be promising therapeutic strategies for preventing or treating muscle loss associated with disuse, aging, and disease. Further research is needed to explore the detailed mechanisms regulating LONP1 expression and activity in skeletal muscle and to investigate the potential of LONP1 as a therapeutic target.

The study primarily focused on mouse models and cultured myocytes, limiting the direct translation of findings to humans. While human samples were analyzed, the sample size was relatively small, potentially limiting the generalizability of the results. The precise mechanisms regulating LONP1 expression and activity in response to muscle disuse were not fully elucidated. The study primarily focused on autophagy as the mechanism linking LONP1 deficiency to muscle atrophy; other potential mechanisms might also contribute.