Neuroprotective treatment with the nitrone compound OKN-007 mitigates age-related muscle weakness in aging mice.

Despite the universal impact of sarcopenia on compromised health and quality of life in the elderly, promising pharmaceutical approaches that can effectively mitigate loss of muscle and function during aging have been limited. Our group and others have reported impairments in peripheral motor neurons and loss of muscle innervation as initiating factors in sarcopenia, contributing to mitochondrial dysfunction and elevated oxidative stress in muscle. We recently reported a reduction in α motor neuron loss in aging mice in response to the compound OKN-007, a proposed antioxidant and anti-inflammatory agent. In the current study, we asked whether OKN-007 treatment in wildtype male mice for 8-9 months beginning at 16 months of age can also protect muscle mass and function. At 25 months of age, we observed a reduction in the loss of whole-body lean mass, a reduced loss of innervation at the neuromuscular junction and well-preserved neuromuscular junction morphology in OKN-007 treated mice versus age matched wildtype untreated mice. The loss in muscle force generation in aging mice (~ 25%) is significantly improved with OKN-007 treatment. In contrast, OKN-007 treatment provided no protection in loss of muscle mass in aging mice. Mitochondrial function was improved by OKN-007 treatment, consistent with its potential antioxidative properties. Together, these exciting findings are the first to demonstrate that interventions through neuroprotection can be an effective therapy to counter aging-related muscle dysfunction.

Senolytic treatment does not mitigate oxidative stress-induced muscle atrophy but improves muscle force generation in CuZn superoxide dismutase knockout mice.

Oxidative stress is associated with tissue dysfunctions that can lead to reduced health. Prior work has shown that oxidative stress contributes to both muscle atrophy and cellular senescence, which is a hallmark of aging that may drive in muscle atrophy and muscle contractile dysfunction. The purpose of the study was to test the hypothesis that cellular senescence contributes to muscle atrophy or weakness. To increase potential senescence in skeletal muscle, we used a model of oxidative stress-induced muscle frailty, the CuZn superoxide dismutase knockout (Sod1KO) mouse. We treated 6-month-old wildtype (WT) and Sod1KO mice with either vehicle or a senolytic treatment of combined dasatinib (5 mg/kg) + quercetin (50 mg/kg) (D + Q) for 3 consecutive days every 15 days. We continued treatment for 7 months and sacrificed the mice at 13 months of age. Treatment with D + Q did not preserve muscle mass, reduce NMJ fragmentation, or alter muscle protein synthesis in Sod1KO mice when compared to the vehicle-treated group. However, we observed an improvement in muscle-specific force generation in Sod1KO mice treated with D + Q when compared to Sod1KO-vehicle mice. Overall, these data suggest that reducing cellular senescence via D + Q is not sufficient to mitigate loss of muscle mass in a mouse model of oxidative stress-induced muscle frailty but may mitigate some aspects of oxidative stress-induced muscle dysfunction.

Modulation of sarcopenia phenotypes by glutathione peroxidase 4 overexpression in mice.

Our laboratory previously showed lipid hydroperoxides and oxylipin levels are elevated in response to loss of skeletal muscle innervation and are associated with muscle pathologies. To elucidate the pathological impact of lipid hydroperoxides, we overexpressed glutathione peroxidase 4 (GPx4), an enzyme that targets reduction of lipid hydroperoxides in membranes, in adult CuZn superoxide dismutase knockout (Sod1KO) mice that show accelerated muscle atrophy associated with loss of innervation. The gastrocnemius muscle from Sod1KO mice shows reduced mitochondrial respiration and elevated oxidative stress (F2 -isoprostanes and hydroperoxides) compared to wild-type (WT) mice. Overexpression of GPx4 improved mitochondrial respiration and reduced hydroperoxide generation in Sod1KO mice, but did not attenuate the muscle loss that occurs in Sod1KO mice. In contrast, contractile force generation is reduced in EDL muscle in Sod1KO mice relative to WT mice, and overexpression of GPx4 restored force generation to WT levels in Sod1KO mice. GPx4 overexpression also prevented loss of muscle contractility at the single fibre level in fast-twitch fibres from Sod1KO mice. Muscle fibres from Sod1KO mice were less sensitive to both depolarization and calcium at the single fibre level and exhibited a reduced activation by S-glutathionylation. GPx4 overexpression in Sod1KO mice rescued the deficits in both membrane excitability and calcium sensitivity of fast-twitch muscle fibres. Overexpression of GPx4 also restored the sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO gastrocnemius muscles. These data suggest that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and in maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia. KEY POINTS: Knockout of CuZn superoxide dismutase (Sod1KO) induces elevated oxidative stress with accelerated muscle atrophy and weakness. Glutathione peroxidase 4 (GPx4) plays a fundamental role in the reduction of lipid hydroperoxides in membranes, and overexpression of GPx4 improves mitochondrial respiration and reduces hydroperoxide generation in Sod1KO mice. Muscle contractile function deficits in Sod1KO mice are alleviated by the overexpression of GPx4. GPx4 overexpression in Sod1KO mice rescues the impaired muscle membrane excitability of fast-twitch muscle fibres and improves their calcium sensitivity. Sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO muscles is decreased, and it is restored by the overexpression of GPx4. Our results confirm that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia.

Development of skeletal muscle fibrosis in a rodent model of cancer cachexia.

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate-buffered saline or Lewis Lung Carcinoma (LLC) at 8-week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis-related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)-related genes beginning at 1wk post-LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM-related genes contrary to what was noted in female mice.

Neuronal deletion of MnSOD in mice leads to demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Neuronal oxidative stress has been implicated in aging and neurodegenerative disease. Here we investigated the impact of elevated oxidative stress induced in mouse spinal cord by deletion of Mn-Superoxide dismutase (MnSOD) using a neuron specific Cre recombinase in Sod2 floxed mice (i-mn-Sod2 KO). Sod2 deletion in spinal cord neurons was associated with mitochondrial alterations and peroxide generation. Phenotypically, i-mn-Sod2 KO mice experienced hindlimb paralysis and clasping behavior associated with extensive demyelination and reduced nerve conduction velocity, axonal degeneration, enhanced blood brain barrier permeability, elevated inflammatory cytokines, microglia activation, infiltration of neutrophils and necroptosis in spinal cord. In contrast, spinal cord motor neuron number, innervation of neuromuscular junctions, muscle mass, and contractile function were not altered. Overall, our findings show that loss of MnSOD in spinal cord promotes a phenotype of demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Lipid hydroperoxides and oxylipins are mediators of denervation induced muscle atrophy.

Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA2 and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy in vivo, we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% in vitro and by over 65% in vivo by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.

Effects of PGC-1α overexpression on the myogenic response during skeletal muscle regeneration.

The ability of skeletal muscle to regenerate from injury is crucial for locomotion, metabolic health, and quality of life. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1A) is a transcriptional coactivator required for mitochondrial biogenesis. Increased mitochondrial biogenesis is associated with improved muscle cell differentiation, however PGC1A's role in skeletal muscle regeneration following damage requires further investigation. The purpose of this study was to investigate the role of skeletal muscle-specific PGC1A overexpression during regeneration following damage. 22 C57BL/6J (WT) and 26 PGC1A muscle transgenic (A1) mice were injected with either phosphate-buffered saline (PBS, uninjured control) or Bupivacaine (MAR, injured) into their tibialis anterior (TA) muscle to induce skeletal muscle damage. TA muscles were extracted 3- or 28-days post-injury and analyzed for markers of regenerative myogenesis and protein turnover. Pgc1a mRNA was ∼10-20 fold greater in A1 mice. Markers of protein synthesis, AKT and 4EBP1, displayed decreases in A1 mice compared to WT at both timepoints indicating a decreased protein synthetic response. Myod mRNA was ∼75% lower compared to WT 3 days post-injection. WT mice exhibited decreased cross-sectional area of the TA muscle at 28 days post-injection with bupivacaine compared to all other groups. PGC1A overexpression modifies the myogenic response during regeneration.

Senolytic treatment reduces cell senescence and necroptosis in Sod1 knockout mice that is associated with reduced inflammation and hepatocellular carcinoma.

The goal of this study was to test the role cellular senescence plays in the increased inflammation, chronic liver disease, and hepatocellular carcinoma seen in mice null for Cu/Zn-Superoxide dismutase (Sod1KO). To inhibit senescence, wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib, and quercetin (D + Q) at 6 months of age when the Sod1KO mice begin exhibiting signs of accelerated aging. Seven months of D + Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels and the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1ß, CXCL-1, and GDF-15). D + Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, for example, cytokines, chemokines, macrophage levels, and Kupffer cell clusters. D + Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer and dramatically reduced the incidence of hepatocellular carcinoma. Surprisingly, D + Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin-1s reduced the markers of cellular senescence (p16, p21, and p53). Our study suggests that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age-associated pathology in the Sod1KO mice.

PGC-1α overexpression is not sufficient to mitigate cancer cachexia in either male or female mice.

Cancer cachexia (CC) accounts for 20%-40% of cancer-related deaths. Mitochondrial aberrations have been shown to precede muscle atrophy in different atrophy models, including cancer. Therefore, this study investigated potential protection from the cachectic phenotype through overexpression of peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α). First, to establish potential of mitochondria-based approaches we showed that the mitochondrial antioxidant MitoTEMPO (MitoT) attenuates myotube atrophy induced by Lewis lung carcinoma (LLC) cell conditioned media. Next, cachexia was induced in muscle-specific PGC-1α overexpressing (MCK-PCG1α) or wildtype (WT) littermate mice by LLC implantation. MCK-PCG1α did not protect LLC-induced muscle mass loss. In plantaris, Atrogin mRNA content was 6.2-fold and ∼11-fold greater in WT-LLC vs WT-phosphate-buffered saline (PBS) for males and females, respectively (p < 0.05). MitoTimer red:green ratio for male PGC was ∼65% higher than WT groups (p < 0.05), with ∼3-fold more red puncta in LLC than PBS (p < 0.05). Red:green ratio was ∼56% lower in females WT-LLC vs PGC-LLC (p < 0.05). In females, no change in red puncta was noted across conditions. Lc3 mRNA content was ∼73% and 2-fold higher in male and female LLC mice, respectively, vs PBS (p < 0.05). While MitoT could mitigate cancer-induced atrophy in vitro, PGC-1α overexpression was insufficient to protect muscle mass and mitochondrial health in vivo despite mitigation of cachexia-associated signaling pathways.

Mitochondrial aberrations during the progression of disuse atrophy differentially affect male and female mice.

BACKGROUND: Disuse decreases muscle size and is predictive of mortality across multiple pathologies. Detriments to mitochondrial function are hypothesized to underlie disuse-induced muscle atrophy. Little data exist on early mechanisms contributing to onset of these pathologies, nor is it known how they differ between sexes. The purpose of this study was to examine differential and conserved responses to mitochondrial quality control in male and female mice during the development and progression of disuse-induced atrophy. METHODS: One hundred C57BL/6J mice (50 male and 50 female) were hindlimb unloaded to induce disuse atrophy for 0 (con), 24, 48, 72, or 168 h. At designated time-points, extensor digitorum longus, gastrocnemius, and soleus muscles were collected for analysis of mitochondrial quality control markers. RESULTS: One hundred sixty-eight hours of disuse resulted in ~25% lower oxidative muscle fibre CSA in both male (P = 0.003) and female (P = 0.02) mice without any differences due to disuse in glycolytic fibres. In male mice, 48 h of unloading was sufficient to result in ~67% greater mitochondrial oxidative stress as assessed by the reporter gene pMitoTimer compared with 0 h (P = 0.002), this mitochondrial stress preceded detectable muscle loss. However in female mice, mitochondrial oxidative stress did not occur until 168 h of disuse (~40% greater mitochondrial oxidative stress in 168 h compared with 0 h of disuse, P < 0.0001). Blunted oxidative stress in female mice appeared to coincide with greater inductions of autophagy and mitophagy in female mice (~3-fold greater BNIP3 and ~6-fold greater LC3II/I ratio P < 0.0001 and P = 0.038 respectively). Male mice overall had greater reactive oxygen species (ROS) production compared with female mice. Female mice had a greater induction of ROS within 24 h of disuse (~4-fold greater compared with 0 h, P < 0.0001); whereas male mice did not have greater ROS production until 168 h of disuse (~2-fold greater, P < 0.0001). Although all muscle types exhibited some alterations to mitochondrial quality control, such as increased markers of mitophagy and fission, the soleus muscle in both male and female mice exhibited consistent alterations to various markers of mitochondrial quality. Markers of mitochondrial translation were approximately 30-50% lower within 24 h of unloading in both male and female soleus muscle (P value ranges: <0.0001-0.03). CONCLUSIONS: Disuse negatively affects mitochondria differentially between sexes during development of muscle wasting. Acutely, female mice may forgo muscle mass to maintain mitochondrial quality compared with male mice. These differences may contribute to divergent clinical manifestations of atrophy.

Tumor burden negatively impacts protein turnover as a proteostatic process in noncancerous liver, heart, and muscle, but not brain.

Cancer survivors are more susceptible to pathologies such as hypertension, liver disease, depression, and coronary artery disease when compared with individuals who have never been diagnosed with cancer. Therefore, it is important to understand how tumor burden negatively impacts nontumor-bearing tissues that may impact future disease susceptibility. We hypothesized that the energetic costs of a tumor would compromise proteostatic maintenance in other tissues. Therefore, the purpose of this study was to determine if tumor burden changes protein synthesis and proliferation rates in heart, brain, and liver. One million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (PBS, sham) were injected into the hind flank of female mice at ∼4.5 mo of age, and the tumor developed for 3 wk. Rates of proliferation and protein synthesis were measured in heart, brain, liver, and tumor tissue. Compared with sham, rates of protein synthesis (structural/nuclear, cytosolic, mitochondrial, and collagen) relative to proliferation were lower in the heart and liver of LLC mice, but higher in the brain of LLC mice. In the tumor tissue, the ratio of protein synthesis to DNA synthesis was approximately 1.0 showing that protein synthesis in the tumor was used for proliferation with little proteostatic maintenance. We further provide evidence that the differences in tissue responses may be due to energetic stress. We concluded that the decrease in proteostatic maintenance in liver, heart, and muscle might contribute to the increased risk of disease in cancer survivors.NEW & NOTEWORTHY We present data showing that simultaneously measuring protein synthesis and cell proliferation can help in the understanding of protein turnover as a proteostatic process in response to tumor burden. In some tissues, like hepatic, cardiac, and skeletal muscle, there was a decrease in the protein to DNA synthesis ratio indicating less proteostatic maintenance. In contrast, the brain maintained or even increased this protein to DNA synthesis ratio indicating more proteostatic maintenance.

Female mice may have exacerbated catabolic signalling response compared to male mice during development and progression of disuse atrophy.

BACKGROUND: Muscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well-established model of hindlimb unloading (HU). METHODS: One hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism). RESULTS: Both males and females lost ~30% of tibialis anterior cross-sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross-sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24-48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4-fold greater content in females compared with ~2-fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes. CONCLUSIONS: These results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.

Cancer-induced Cardiac Atrophy Adversely Affects Myocardial Redox State and Mitochondrial Oxidative Characteristics.

Cachexia presents in 80% of advanced cancer patients; however, cardiac atrophy in cachectic patients receives little attention. This cardiomyopathy contributes to increased occurrence of adverse cardiac events compared to age-matched population norms. Research on cardiac atrophy has focused on remodeling; however, alterations in metabolic properties may be a primary contributor. PURPOSE: Determine how cancer-induced cardiac atrophy alters mitochondrial turnover, mitochondrial mRNA translation machinery and in-vitro oxidative characteristics. METHODS: Lewis lung carcinoma (LLC) tumors were implanted in C57BL6/J mice and grown for 28days to induce cardiac atrophy. Endogenous metabolic species, and markers of mitochondrial function were assessed. H9c2 cardiomyocytes were cultured in LLC-conditioned media with(out) the antioxidant MitoTempo. Cells were analyzed for ROS, oxidative capacity, and hypoxic resistance. RESULTS: LLC heart weights were ~10% lower than controls. LLC hearts demonstrated ~15% lower optical redox ratio (FAD/FAD+NADH) compared to PBS controls. When compared to PBS, LLC hearts showed ~50% greater COX-IV and VDAC, attributed to ~50% lower mitophagy markers. mt-mRNA translation machinery was elevated similarly to markers of mitochondrial content. mitochondrial DNA-encoded Cytb was ~30% lower in LLC hearts. ROS scavengers GPx-3 and GPx-7 were ~50% lower in LLC hearts. Treatment of cardiomyocytes with LLC-conditioned media resulted in higher ROS (25%), lower oxygen consumption rates (10% at basal, 75% at maximal), and greater susceptibility to hypoxia (~25%) -- which was reversed by MitoTempo. CONCLUSION: These results substantiate metabolic cardiotoxic effects attributable to tumor-associated factors and provide insight into interactions between mitochondrial mRNA translation, ROS mitigation, oxidative capacity and hypoxia resistance.

Moderators of skeletal muscle maintenance are compromised in sarcopenic obese mice.

The purpose of this study was to determine whether sarcopenic obesity accelerates impairments in muscle maintenance through the investigation of cell cycle progression and myogenic, inflammatory, catabolic and protein synthetic signaling in mouse gastrocnemius muscles. At 4 weeks old, 24 male C57BL/6 mice were fed either a high fat diet (HFD, 60 % fat) or normal chow (NC, 17 % fat) for either 8-12 weeks or 21-23 months. At 3-4 months or 22-24 months the gastrocnemius muscles were excised. In addition, plasma was taken for C2C12 differentiation experiments. Mean cross-sectional area (CSA) was reduced by 29 % in aged HFD fed mice compared to the aged NC mice. MyoD was roughly 50 % greater in the aged mice compared to young mice, whereas TNF-α and IGF-1 gene expression in aged HFD fed mice were reduced by 52 % and 65 % in comparison to aged NC fed mice, respectively. Myotubes pretreated with plasma from aged NC fed mice had 14 % smaller myotube diameter than their aged HFD counterparts. Aged obese mice had greater impairments to mediators of muscle maintenance as evident by reductions in muscle mass, CSA, along with alterations in cell cycle regulation and inflammatory and insulin signaling.

Mitochondrial Function and Protein Turnover in the Diaphragm are Altered in LLC Tumor Model of Cancer Cachexia.

It is established that cancer cachexia causes limb muscle atrophy and is strongly associated with morbidity and mortality; less is known about how the development of cachexia impacts the diaphragm. The purpose of this study was to investigate cellular signaling mechanisms related to mitochondrial function, reactive oxygen species (ROS) production, and protein synthesis during the development of cancer cachexia. C57BL/J6 mice developed Lewis Lung Carcinoma for either 0 weeks (Control), 1 week, 2 weeks, 3 weeks, or 4 weeks. At designated time points, diaphragms were harvested and analyzed. Mitochondrial respiratory control ratio was ~50% lower in experimental groups, which was significant by 2 weeks of cancer development, with no difference in mitochondrial content markers COXIV or VDAC. Compared to the controls, ROS was 4-fold elevated in 2-week animals but then was not different at later time points. Only one antioxidant protein, GPX3, was altered by cancer development (~70% lower in experimental groups). Protein synthesis, measured by a fractional synthesis rate, appeared to become progressively lower with the cancer duration, but the mean difference was not significant. The development and progression of cancer cachexia induces marked alterations to mitochondrial function and ROS production in the diaphragm and may contribute to increased cachexia-associated morbidity and mortality.

Neuron-specific deletion of CuZnSOD leads to an advanced sarcopenic phenotype in older mice.

Age-associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1-/- mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2- to 4-month-old) Sod1flox/SlickHCre mice with tamoxifen to generate i-mn-Sod1KO mice. CuZnSOD protein was 40-50% lower in neuronal tissue in i-mn-Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18- to 22-month-old i-mn-Sod1KO mice. By 24 months, 22% of NMJs in i-mn-Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal-specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit.

Cancer cachexia in a mouse model of oxidative stress.

BACKGROUND: Cancer is associated with muscle atrophy (cancer cachexia) that is linked to up to 40% of cancer-related deaths. Oxidative stress is a critical player in the induction and progression of age-related loss of muscle mass and weakness (sarcopenia); however, the role of oxidative stress in cancer cachexia has not been defined. The purpose of this study was to examine if elevated oxidative stress exacerbates cancer cachexia. METHODS: Cu/Zn superoxide dismutase knockout (Sod1KO) mice were used as an established mouse model of elevated oxidative stress. Cancer cachexia was induced by injection of one million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (saline) into the hind flank of female wild-type mice or Sod1KO mice at approximately 4 months of age. The tumour developed for 3 weeks. Muscle mass, contractile function, neuromuscular junction (NMJ) fragmentation, metabolic proteins, mitochondrial function, and motor neuron function were measured in wild-type and Sod1KO saline and tumour-bearing mice. Data were analysed by two-way ANOVA with Tukey-Kramer post hoc test when significant F ratios were determined and α was set at 0.05. Unless otherwise noted, results in abstract are mean ±SEM. RESULTS: Muscle mass and cross-sectional area were significantly reduced, in tumour-bearing mice. Metabolic enzymes were dysregulated in Sod1KO mice and cancer exacerbated this phenotype. NMJ fragmentation was exacerbated in tumour-bearing Sod1KO mice. Myofibrillar protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.00847 ± 0.00205; wildtype LLC, 0.0211 ± 0.00184) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0180 ± 0.00118; Sod1KO LLC, 0.0490 ± 0.00132). Muscle mitochondrial oxygen consumption was reduced in tumour-bearing mice compared with saline-injected wild-type mice. Mitochondrial protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.0204 ± 0.00159; wild-type LLC, 0.167 ± 0.00157) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0231 ± 0.00108; Sod1 KO LLC, 0.0645 ± 0.000631). Sciatic nerve conduction velocity was decreased in tumour-bearing wild-type mice (wild-type saline, 38.2 ± 0.861; wild-type LLC, 28.8 ± 0.772). Three out of eleven of the tumour-bearing Sod1KO mice did not survive the 3-week period following tumour implantation. CONCLUSIONS: Oxidative stress does not exacerbate cancer-induced muscle loss; however, cancer cachexia may accelerate NMJ disruption.

Targeting cPLA2 derived lipid hydroperoxides as a potential intervention for sarcopenia.

Defects in neuromuscular innervation contribute significantly to the age-related decline in muscle mass and function (sarcopenia). Our previous studies demonstrated that denervation induces muscle mitochondrial hydroperoxide production (H2O2 and lipid hydroperoxides (LOOHs)). Here we define the relative contribution of mitochondrial electron transport chain (ETC) derived H2O2 versus cytosolic phospholipase A2 (cPLA2) derived LOOHs in neurogenic muscle atrophy. We show that denervation increases muscle cPLA2 protein content, activity, and metabolites downstream of cPLA2 including LOOHs. Increased scavenging of mitochondrial H2O2 does not protect against denervation atrophy, suggesting ETC generated H2O2 is not a critical player. In contrast, inhibition of cPLA2 in vivo mitigates LOOH production and muscle atrophy and maintains individual muscle fiber size while decreasing oxidative damage. Overall, we show that loss of innervation in several muscle atrophy models including aging induces generation of LOOHs produced by arachidonic acid metabolism in the cPLA2 pathway contributing to loss of muscle mass.

Molecular changes in transcription and metabolic pathways underlying muscle atrophy in the CuZnSOD null mouse model of sarcopenia.

Mice lacking the superoxide anion scavenger CuZn superoxide dismutase (Sod1-/- mice) develop a number of age-related phenotypes, including an early progression of muscle atrophy and weakness (sarcopenia) associated with loss of innervation. The purpose of this study was to delineate the early development of sarcopenia in the Sod1-/- mice and to measure changes in the muscle transcriptome, proteome, and eicosanoid profile at the stage when sarcopenia is markedly induced in this model (7-9 months of age). We found a strong correlation between muscle atrophy and mitochondrial state 1 hydroperoxide production, which was 40% higher in isolated mitochondria from Sod1-/- mouse gastrocnemius muscle by 2 months of age. The primary pathways showing altered gene expression in Sod1-/- mice identified by RNA-seq transcriptomic analysis are protein ubiquitination, synaptic long-term potentiation, calcium signaling, phospholipase C signaling, AMPK, and TWEAK signaling. Targeted proteomics shows elevated expression of mitochondrial proteins, fatty acid metabolism enzymes, tricarboxylic acid (TCA) cycle enzymes, and antioxidants, while enzymes involved in carbohydrate metabolism are downregulated in Sod1-/- mice. LC-MS analysis of lipids in gastrocnemius muscle detected 78 eicosanoids, of which 31 are significantly elevated in muscle from Sod1-/- mice. These data suggest that mitochondrial hydroperoxide generation is elevated prior to muscle atrophy and may be a potential driving factor of changes in the transcriptome, proteome, and eicosanoid profile of the Sod1-/- mice. Together, these analyses revealed important molecular events that occur during muscle atrophy, which will pave the way for future studies using new approaches to treat sarcopenia.

Development and progression of cancer cachexia: Perspectives from bench to bedside.

Cancer cachexia (CC) is a devastating syndrome characterized by weight loss, reduced fat mass and muscle mass that affects approximately 80% of cancer patients and is responsible for 22%-30% of cancer-associated deaths. Understanding underlying mechanisms for the development of CC are crucial to advance therapies to treat CC and improve cancer outcomes. CC is a multi-organ syndrome that results in extensive skeletal muscle and adipose tissue wasting; however, CC can impair other organs such as the liver, heart, brain, and bone as well. A considerable amount of CC research focuses on changes that occur within the muscle, but cancer-related impairments in other organ systems are understudied. Furthermore, metabolic changes in organ systems other than muscle may contribute to CC. Therefore, the purpose of this review is to address degenerative mechanisms which occur during CC from a whole-body perspective. Outlining the information known about metabolic changes that occur in response to cancer is necessary to develop and enhance therapies to treat CC. As much of the current evidences in CC are from pre-clinical models we should note the majority of the data reviewed here are from preclinical models.