Depletion of essential fatty acids in muscle is associated with shorter survival of cancer patients undergoing surgery-preliminary report.

Emerging studies are reporting associations between skeletal muscle abnormalities and survival in cancer patients. Cancer prognosis is associated with depletion of essential fatty acids in erythrocytes and plasma in humans. However the relationship between skeletal muscle membrane fatty acid composition and survival is unknown. This study investigates the relationship between fatty acid content of phospholipids in skeletal muscle and survival in cancer patients. Rectus abdominis biopsies were collected during cancer surgery from 35 patients diagnosed with cancer. Thin-layer and gas chromatography were used for quantification of phospholipid fatty acids. Cutpoints for survival were defined using optimal stratification. Median survival was between 450 and 500 days when patients had arachidonic acid (AA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in muscle phospholipid below the cut-point compared to 720-800 days for patients above. Cox regression analysis revealed that low amounts of AA, EPA and DHA are risk factors for death. The risk of death remained significant for AA [HR 3.5 (1.11-10.87), p = 0.03], EPA [HR 3.92 (1.1-14.0), p = 0.04] and DHA [HR 4.08 (1.1-14.6), p = 0.03] when adjusted for sex. Lower amounts of essential fatty acids in skeletal muscle membrane is a predictor of survival in cancer patients. These results warrant investigation to restore bioactive fatty acids in people with cancer.

Lipid is heterogeneously distributed in muscle and associates with low radiodensity in cancer patients.

BACKGROUND: Low muscle radiodensity is associated with mortality in a variety of cancer types. Biochemical and morphological correlates are unknown. We aimed to evaluate triglyceride (TG) content and location as a function of computed tomography (CT)-derived measures of skeletal muscle radiodensity in cancer patients. METHODS: Rectus abdominis (RA) biopsies were collected during cancer surgery from 75 patients diagnosed with cancer. Thin-layer chromatography and gas chromatography were used for quantification of TG content of the muscle. Axial CT images of lumbar vertebra were used to measure muscle radiodensity. Oil Red O staining was used to determine the location of neutral lipids in frozen muscle sections. RESULTS: There was wide variation in RA radiodensity in repeated measures (CV% ranged from 3 to 55% based on 10 serial images) as well as within one slice (CV% ranged from 6 to 61% based on 10 subregions). RA radiodensity and total lumbar muscle radiodensity were inversely associated with TG content of RA (r = -0.396, P < 0.001, and r = -0.355, P = 0.002, respectively). Of the total percentage area of muscle staining positive for neutral lipid, 54 ± 17% was present as extramyocellular lipids (range 23.5-77.8%) and 46 ± 17% (range 22.2-76.5%) present as intramyocellular lipid droplets. CONCLUSIONS: Repeated measures revealed wide variation in radiodensity of RA muscle, both vertically and horizontally. Low muscle radiodensity reflects high level of TG in patients with cancer. Non-uniform distribution of intramyocellular and extramyocellular lipids was evident using light microscopy. These results warrant investigation of mechanisms resulting in lipid deposition in muscles of cancer patients.

Immunohistochemical phenotyping of T cells, granulocytes, and phagocytes in the muscle of cancer patients: association with radiologically defined muscle mass and gene expression.

BACKGROUND: Inflammation is a recognized contributor to muscle wasting. Research in injury and myopathy suggests that interactions between the skeletal muscle and immune cells confer a pro-inflammatory environment that influences muscle loss through several mechanisms; however, this has not been explored in the cancer setting. This study investigated the local immune environment of the muscle by identifying the phenotype of immune cell populations in the muscle and their relationship to muscle mass in cancer patients. METHODS: Intraoperative muscle biopsies were collected from cancer patients (n = 30, 91% gastrointestinal malignancies). Muscle mass was assessed histologically (muscle fiber cross-sectional area, CSA; µm2) and radiologically (lumbar skeletal muscle index, SMI; cm2/m2 by computed tomography, CT). T cells (CD4 and CD8) and granulocytes/phagocytes (CD11b, CD14, and CD15) were assessed by immunohistochemistry. Microarray analysis was conducted in the muscle of a second cancer patient cohort. RESULTS: T cells (CD3+), granulocytes/phagocytes (CD11b+), and CD3-CD4+ cells were identified. Muscle fiber CSA (µm2) was positively correlated (Spearman's r = > 0.45; p = < 0.05) with the total number of T cells, CD4, and CD8 T cells and granulocytes/phagocytes. In addition, patients with the smallest SMI exhibited fewer CD8 T cells within their muscle. Consistent with this, further exploration with gene correlation analyses suggests that the presence of CD8 T cells is negatively associated (Pearson's r = ≥ 0.5; p = <0.0001) with key genes within muscle catabolic pathways for signaling (ACVR2B), ubiquitin proteasome (FOXO4, TRIM63, FBXO32, MUL1, UBC, UBB, UBE2L3), and apoptosis/autophagy (CASP8, BECN1, ATG13, SIVA1). CONCLUSION: The skeletal muscle immune environment of cancer patients is comprised of immune cell populations from the adaptive and innate immunity. Correlations of T cells, granulocyte/phagocytes, and CD3-CD4+ cells with muscle mass measurements indicate a positive relationship between immune cell numbers and muscle mass status in cancer patients. Further exploration with gene correlation analyses suggests that the presence of CD8 T cells is negatively correlated with components of muscle catabolism.

Clinical and biological characterization of skeletal muscle tissue biopsies of surgical cancer patients.

BACKGROUND: Researchers increasingly use intraoperative muscle biopsy to investigate mechanisms of skeletal muscle atrophy in patients with cancer. Muscles have been assessed for morphological, cellular, and biochemical features. The aim of this study was to conduct a state-of-the-science review of this literature and, secondly, to evaluate clinical and biological variation in biopsies of rectus abdominis (RA) muscle from a cohort of patients with malignancies. METHODS: Literature was searched for reports on muscle biopsies from patients with a cancer diagnosis. Quality of reports and risk of bias were assessed. Data abstracted included patient characteristics and diagnoses, sample size, tissue collection and biobanking procedures, and results. A cohort of cancer patients (n = 190, 88% gastrointestinal malignancies), who underwent open abdominal surgery as part of their clinical care, consented to RA biopsy from the site of incision. Computed tomography (CT) scans were used to quantify total abdominal muscle and RA cross-sectional areas and radiodensity. Biopsies were assessed for muscle fibre area (µm2 ), fibre types, myosin heavy chain isoforms, and expression of genes selected for their involvement in catabolic pathways of muscle. RESULTS: Muscle biopsy occurred in 59 studies (total N = 1585 participants). RA was biopsied intraoperatively in 40 studies (67%), followed by quadriceps (26%; percutaneous biopsy) and other muscles (7%). Cancer site and stage, % of male participants, and age were highly variable between studies. Details regarding patient medical history and biopsy procedures were frequently absent. Lack of description of the population(s) sampled and low sample size contributed to low quality and risk of bias. Weight-losing cases were compared with weight stable cancer or healthy controls without considering a measure of muscle mass in 21 out of 44 studies. In the cohort of patients providing biopsy for this study, 78% of patients had preoperative CT scans and a high proportion (64%) met published criteria for sarcopenia. Fibre type distribution in RA was type I (46% ± 13), hybrid type I/IIA (1% ± 1), type IIA (36% ± 10), hybrid type IIA/D (15% ± 14), and type IID (2% ± 5). Sexual dimorphism was prominent in RA CT cross-sectional area, mean fibre cross-sectional area, and in expression of genes associated with muscle growth, apoptosis, and inflammation (P < 0.05). Medical history revealed multiple co-morbid conditions and medications. CONCLUSIONS: Continued collaboration between researchers and cancer surgeons enables a more complete understanding of mechanisms of cancer-associated muscle atrophy. Standardization of biobanking practices, tissue manipulation, patient characterization, and classification will enhance the consistency, reliability, and comparability of future studies.

Multitargeted kinase inhibitors imatinib, sorafenib and sunitinib perturb energy metabolism and cause cytotoxicity to cultured C2C12 skeletal muscle derived myotubes.

Tyrosine kinase inhibitors (TKIs) have advanced cancer treatment and prognosis but have also resulted in adverse effects such as fatigue, diarrhea, hypothyroidism, and other toxicities. We investigated TKI effects on skeletal muscle as a possible explanation of TKI induced fatigue. Changes in mitochondrial function due to inhibition of oxidative phosphorylation complexes, generation of superoxides, and inhibition of key transporters involved in uptake of glucose and/or nucleosides may result in alteration of energy metabolism and/or mitochondrial function. We investigated effects of imatinib, sorafenib and sunitinib on these processes in cultured C2C12 murine skeletal muscle cells. Imatinib, sorafenib and sunitinib were cytotoxic to C2C12 cells with IC50 values of 20, 8 and 8 µM, respectively. Imatinib stimulated glucose uptake and inhibited complex V activity by 35% at 50 µM. Sorafenib inhibited complex II/III and V with IC50 values of 32 and 28 µM, respectively. Sorafenib caused activation of caspase 3/7 and depolarization of mitochondrial membranes occurred very rapidly with complete loss at 5-10 µM. Sunitinib inhibited Complex I with an IC50 value of 38 µM and caused ATP depletion, caspase 3/7 activation, an increase in reactive oxygen species (ROS), and decreased nucleoside and glucose uptake. In conclusion, imatinib, sunitinib and sorafenib caused changes in mitochondrial complex activities, glucose and nucleoside uptake leading to decreased energy production and mitochondrial function in a skeletal muscle cell model, suggesting that these changes may play a role in fatigue, one of the most common adverse effects of TKIs.

Fish oil mitigates myosteatosis and improves chemotherapy efficacy in a preclinical model of colon cancer.

BACKGROUND: This study aimed to assess whether feeding a diet containing fish oil was efficacious in reducing tumor- and subsequent chemotherapy-associated myosteatosis, and improving tumor response to treatment. METHODS: Female Fischer 344 rats were fed either a control diet for the entire study (control), or switched to a diet containing fish oil (2.0 g /100 g of diet) one week prior to tumor implantation (long term fish oil) or at the start of chemotherapy (adjuvant fish oil). Chemotherapy (irinotecan plus 5-fluorouracil) was initiated 2 weeks after tumor implantation (cycle-1) and 1 week thereafter (cycle-2). Reference animals received no tumor or treatment and only consumed the control diet. All skeletal muscle measures were conducted in the gastrocnemius. To assess myosteatosis, lipids were assessed histologically by Oil Red O staining and total triglyceride content was quantified by gas chromatography. Expression of adipogenic transcription factors were assessed at the mRNA level by real-time RT-PCR. RESULTS: Feeding a diet containing fish oil significantly reduced tumor- and subsequent chemotherapy-associated increases in skeletal muscle neutral lipid (p<0.001) and total triglyceride content (p<0.03), and expression of adipogenic transcription factors (p<0.01) compared with control diet fed animals. The adjuvant fish oil diet was as effective as the long term fish oil diet in mitigating chemotherapy-associated skeletal muscle fat content, and in reducing tumor volume during chemotherapy compared with control fed animals (p<0.01). CONCLUSION: Long term and adjuvant fish oil diets are equally efficacious in reducing chemotherapy-associated myosteatosis that may be occurring by reducing expression of transcription factors involved in adipogenesis/lipogenesis, and improving tumor-response to chemotherapy in a neoplastic model.

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle.

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.

Effects of long-term creatine feeding and running on isometric functional measures and myosin heavy chain content of rat skeletal muscles.

The purpose of this study was to investigate whether creatine (Cr) supplementation during 12 weeks of phasic high-frequency voluntary wheel running would result in a faster myosin heavy chain (MHC) isoform profile in the rat mixed fast-twitch plantaris and alter its corresponding isometric contractile properties. The fast-twitch extensor digitorum longus and medial gastrocnemius and slow-twitch soleus were also studied. Forty weanling Sprague-Dawley male rats were assigned to one of four groups: creatine-sedentary (Cre-Sed); creatine-voluntary running (Cre-Run); control-sedentary (Con-Sed); control-voluntary running (Con-Run). Daily running distance was similar between Cre-Run and Con-Run. Average daily Cr ingestion was also similar being 2.4+/-0.17 and 3.0+/-0.14 g/kg in Cre-Sed and Cre-Run, respectively. Total creatine (TCr) content was elevated (P<0.03) in the plantaris of Cre-Run [211.4+/-16.9 mmol/kg dry weight (dw)], compared with Con-Run (175.1+/-5.69). In the plantaris, MHCIIb was 13% greater (P<0.00001) in Cre-Run compared with Con-Run, while MHCIId/x and MHCIIa were lower in Cre-Run by 7 and 6% (P<0.0002), respectively. No differences were observed in twitch force, time-to-peak tension, half-rise time or half-fall time. Greater tetanic force production (P<0.05) in Cre-Sed compared with Con-Sed corresponded to a 12% increase in MHCIId/x (P<0.0001) and a 12% decrease in MHCIIb (P<0.0006). The fatigue index of the plantaris at 10 s (FI(10s)) was reduced only after running (Cre-Run vs Con-Run), while in all other muscles the FI(10s) was lower only in the Cre-Sed group. In conclusion, Cr supplementation had differential effects on MHC isoform content and fatigability that depended on the level of contractile activity. Cr feeding combined with running exercise resulted in a faster MHC-based phenotype in the rat plantaris but the impact on associated isometric contractile properties was minimal.