BackgroundCancer cachexia is largely irreversible, at least via nutritional means, and responsible for 20–40% of cancer‐related deaths. Therefore, preventive measures are of primary importance; however, little is known about muscle perturbations prior to onset of cachexia. Cancer cachexia is associated with mitochondrial degeneration; yet, it remains to be determined if mitochondrial degeneration precedes muscle wasting in cancer cachexia. Therefore, our purpose was to determine if mitochondrial degeneration precedes cancer‐induced muscle wasting in tumour‐bearing mice.MethodsFirst, weight‐stable (MinStable) and cachectic (MinCC) Apc Min/+ mice were compared with C57Bl6/J controls for mRNA contents of mitochondrial quality regulators in quadriceps muscle. Next, Lewis lung carcinoma (LLC) cells or PBS (control) were injected into the hind flank of C57Bl6/J mice at 8 week age, and tumour allowed to develop for 1, 2, 3, or 4 weeks to examine time course of cachectic development. Succinate dehydrogenase stain was used to measure oxidative phenotype in tibialis anterior muscle. Mitochondrial quality and function were assessed using the reporter MitoTimer by transfection to flexor digitorum brevis and mitochondrial function/ROS emission in permeabilized adult myofibres from plantaris. RT‐qPCR and immunoblot measured the expression of mitochondrial quality control and antioxidant proteins. Data were analysed by one‐way ANOVA with Student–Newman–Kuels post hoc test.ResultsMinStable mice displayed ~50% lower Pgc‐1α, Pparα, and Mfn2 compared with C57Bl6/J controls, whereas MinCC exhibited 10‐fold greater Bnip3 content compared with C57Bl6/J controls. In LLC, cachectic muscle loss was evident only at 4 weeks post‐tumour implantation. Oxidative capacity and mitochondrial content decreased by ~40% 4 weeks post‐tumour implantation. Mitochondrial function decreased by ~25% by 3 weeks after tumour implantation. Mitochondrial degeneration was evident by 2 week LLC compared with PBS control, indicated by MitoTimer red/green ratio and number of pure red puncta. Mitochondrial ROS production was elevated by ~50 to ~100% when compared with PBS at 1–3 weeks post‐tumour implantation. Mitochondrial quality control was dysregulated throughout the progression of cancer cachexia in tumour‐bearing mice. In contrast, antioxidant proteins were not altered in cachectic muscle wasting.ConclusionsFunctional mitochondrial degeneration is evident in LLC tumour‐bearing mice prior to muscle atrophy. Contents of mitochondrial quality regulators across Apc Min/+ and LLC mice suggest impaired mitochondrial quality control as a commonality among pre‐clinical models of cancer cachexia. Our data provide novel evidence for impaired mitochondrial health prior to cachectic muscle loss and provide a potential therapeutic target to prevent cancer cachexia.

BackgroundCancer cachexia occurs in approximately 80% of cancer patients and is a key contributor to cancer‐related death. The mechanisms controlling development of tumour‐induced muscle wasting are not fully elucidated. Specifically, the progression and development of cancer cachexia are underexplored. Therefore, we examined skeletal muscle protein turnover throughout the development of cancer cachexia in tumour‐bearing mice.MethodsLewis lung carcinoma (LLC) was injected into the hind flank of C57BL6/J mice at 8 weeks age with tumour allowed to develop for 1, 2, 3, or 4 weeks and compared with PBS injected control. Muscle size was measured by cross‐sectional area analysis of haematoxylin and eosin stained tibialis anterior muscle. 2H2O was used to assess protein synthesis throughout the development of cancer cachexia. Immunoblot and RT‐qPCR were used to measure regulators of protein turnover. TUNEL staining was utilized to measure apoptotic nuclei. LLC conditioned media (LCM) treatment of C2C12 myotubes was used to analyse cancer cachexia in vitro.ResultsMuscle cross‐sectional area decreased ~40% 4 weeks following tumour implantation. Myogenic signalling was suppressed in tumour‐bearing mice as soon as 1 week following tumour implantation, including lower mRNA contents of Pax7, MyoD, CyclinD1, and Myogenin, when compared with control animals. AchRδ and AchRε mRNA contents were down‐regulated by ~50% 3 weeks following tumour implantation. Mixed fractional synthesis rate protein synthesis was ~40% lower in 4 week tumour‐bearing mice when compared with PBS controls. Protein ubiquitination was elevated by ~50% 4 weeks after tumour implantation. Moreover, there was an increase in autophagy machinery after 4 weeks of tumour growth. Finally, ERK and p38 MAPK phosphorylations were fourfold and threefold greater than control muscle 4 weeks following tumour implantation, respectively. Inhibition of p38 MAPK, but not ERK MAPK, in vitro partially rescued LCM‐induced loss of myotube diameter.ConclusionsOur findings work towards understanding the pathophysiological signalling in skeletal muscle in the initial development of cancer cachexia. Shortly following the onset of the tumour‐bearing state alterations in myogenic regulatory factors are apparent, suggesting early onset alterations in the capacity for myogenic induction. Cancer cachexia presents with a combination of a loss of protein synthesis and increased markers of protein breakdown, specifically in the ubiquitin‐proteasome system. Also, p38 MAPK may be a potential therapeutic target to combat cancer cachexia via a p38‐FOX01‐atrogene‐ubiquitin‐proteasome mechanism.

Skeletal muscle mitochondrial degeneration is a hallmark of insulin resistance/obesity marked by lost function, enhanced ROS emission, and altered morphology which may be ameliorated by physical activity (PA). However, no prior report has examined mitochondrial quality control regulation throughout biogenesis, fusion/fission dynamics, autophagy, and mitochondrial permeability transition pore (MPTP) in obesity. Therefore, we determined how each process is impacted by Western diet (WD)-induced obesity and whether voluntary PA may alleviate derangements in mitochondrial quality control mechanisms. Despite greater mitochondrial content following WD (COX-IV and Cytochrome C), induction of biogenesis controllers appears impaired (failed induction of PGC-1α). Mitochondrial fusion seems diminished (reduced MFN2, Opa1 proteins), with no significant changes in fission, suggesting a shift in balance of dynamics regulation favoring fission. Autophagy flux was promoted in WD (reduced p62, increased LC3II:I ratio); however, mitophagy marker BNIP3 is reduced in WD which may indicate reduced mitophagy despite enhanced total autophagy flux. MPTP regulator Ant mRNA is reduced by WD. Few processes were impacted by physical activity. Finally, mitochondrial quality control processes are partially promoted by PGC-1α, as PGC-1α transgenic mice display elevated mitochondrial biogenesis and autophagy flux. Additionally, these mice exhibit elevated Mfn1 and Opa1 mRNA, with no change in protein content suggesting these factors are transcriptionally promoted by PGC-1α overexpression. These data demonstrate dysfunctions across mitochondrial quality control in obesity and that PGC-1α is sufficient to promote multiple, but not necessarily all, aspects of mitochondrial quality control. Mitochondrial quality control may therefore be an opportune target to therapeutically treat metabolic disease.

No abstract

Obese mice have impaired inflammatory and protein synthetic signalling that may negatively influence muscle regeneration.

Skeletal muscle is capable of robust self-repair following mild trauma, yet in cases of traumatic volumetric muscle loss (VML), where more than 20% of a muscle's mass is lost, this capacity is overwhelmed. Current autogenic whole muscle transfer techniques are imperfect, which has motivated the exploration of implantable scaffolding strategies. In this study, the use of an allogeneic decellularized skeletal muscle (DSM) scaffold with and without the addition of minced muscle (MM) autograft tissue was explored as a repair strategy using a lower-limb VML injury model (n = 8/sample group). We found that the repair of VML injuries using DSM + MM scaffolds significantly increased recovery of peak contractile force (81 -3% of normal contralateral muscle) compared to unrepaired VML controls (62 -4%). Similar significant improvements were measured for restoration of muscle mass (88 -3%) in response to DSM + MM repair compared to unrepaired VML controls (79 -3%). Histological findings revealed a marked decrease in collagen dense repair tissue formation both at and away from the implant site for DSM + MM repaired muscles. The addition of MM to DSM significantly increased MyoD expression, compared to isolated DSM treatment (21-fold increase) and unrepaired VML (37-fold) controls. These findings support the further exploration of both DSM and MM as promising strategies for the repair of VML injury.

Muscle atrophy is a hallmark of cancer cachexia resulting in impaired function and quality of life and cachexia is the immediate cause of death for 20-40% of cancer patients. Multiple microRNAs (miRNAs) have been identified as being involved in muscle development and atrophy; however, less is known specifically on miRNAs in cancer cachexia. The purpose of this investigation was to examine the miRNA profile of skeletal muscle atrophy induced by cancer cachexia to uncover potential miRNAs involved with this catabolic condition. Phosphate-buffered saline (PBS) or Lewis lung carcinoma cells (LLC) were injected into C57BL/6J mice at 8 wk of age. LLC animals were allowed to develop tumors for 4 wk to induce cachexia. Tibialis anterior muscles were extracted and processed to isolate small RNAs, which were used for miRNA sequencing. Sequencing results were assembled with mature miRNAs, and functions of miRNAs were analyzed by Ingenuity Pathway Analysis. LLC animals developed tumors that contributed to significantly smaller tibialis anterior muscles (18.5%) and muscle cross-sectional area (40%) compared with PBS. We found 371 miRNAs to be present in the muscle above background levels. Of these, nine miRNAs were found to be differentially expressed. Significantly altered groups of miRNAs were categorized into primary functionalities including cancer, cell-to-cell signaling, and cellular development among others. Gene network analysis predicted specific alterations of factors contributing to muscle size including Akt, FOXO3, and others. These results create a foundation for future research into the sufficiency of targeting these genes to attenuate muscle loss in cancer cachexia.

SUMMARY During aging, there is a progressive loss of volume and function in skeletal muscle that impacts mobility and quality of life. The repair of skeletal muscle is regulated by tissue-resident stem cells called satellite cells (or muscle stem cells [MuSCs]), but in aging, MuSCs decrease in numbers and regenerative capacity. The transcriptional networks and epigenetic changes that confer diminished regenerative function in MuSCs as a result of natural aging are only partially understood. Herein, we use an integrative genomics approach to profile MuSCs from young and aged animals before and after injury. Integration of these datasets reveals aging impacts multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility, and patterns of transcription factor (TF) binding activities. Collectively, these datasets facilitate a deeper understanding of the regulation tissue-resident stem cells use during aging and healing.