Oscillatory work and the step that generates force in single myofibrils from rabbit psoas.

The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (Pi) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the Pi release step. The question asked here is whether the force generation occurs before Pi release or after release. We investigated the effect of Pi on oscillatory work production in single myofibrils and found that Pi-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of Pi in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have Pi attached. With sinusoidal analysis, we obtained the kinetic constants around the Pi release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before Pi is released, and the same force is maintained after Pi is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

Changes of mitochondrial lipid molecules, structure, cytochrome c and ROS of beef Longissimus lumborum and Psoas major during postmortem storage and their potential associations with beef quality.

This study investigated changes in mitochondrial lipid molecules and their potential associations with Longissimus lumborum (LL) and Psoas major (PM) quality during storage. A total of 1610 lipid species that matched with 36 lipid classes were identified from isolated mitochondria. PM had more key lipid molecules at storage d-1 including diacylglycerol and triacylglycerol (may play roles in membrane stability), phosphatidylethanolamine, acyl carnitine and cardiolipin (involved in energy metabolism), and cardiolipin and phosphatidylserine (important factors in apoptosis). Correspondingly, with extended storage time, mitochondrial structure, cytochrome c and reactive oxygen species (ROS) were changed, and muscle oxidation intensified. These changes have close associations with shear force and water holding capacity (WHC). Compared with LL, PM had higher content of lipid classes, more mitochondrial ROS, greater muscle oxidation, and lower shear force and WHC. These findings provided new insights into the effects of lipidome on mitochondrial structure, ROS and cytochrome c, and their potential associations with beef quality.

Effects of Low-Intensity and Long-Term Aerobic Exercise on the Psoas Muscle of mdx Mice: An Experimental Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle disease characterized by the absence of the protein dystrophin, which causes a loss of sarcolemma integrity, determining recurrent muscle injuries, decrease in muscle function, and progressive degeneration. Currently, there is a need for therapeutic treatments to improve the quality of life of DMD patients. Here, we investigated the effects of a low-intensity aerobic training (37 sessions) on satellite cells, peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α protein (PGC-1α), and different types of fibers of the psoas muscle from mdx mice (DMD experimental model). Wildtype and mdx mice were randomly divided into sedentary and trained groups (n = 24). Trained animals were subjected to 37 sessions of low-intensity running on a motorized treadmill. Subsequently, the psoas muscle was excised and analyzed by immunofluorescence for dystrophin, satellite cells, myosin heavy chain (MHC), and PGC-1α content. The minimal Feret's diameters of the fibers were measured, and light microscopy was applied to observe general morphological features of the muscles. The training (37 sessions) improved morphological features in muscles from mdx mice and caused an increase in the number of quiescent/activated satellite cells. It also increased the content of PGC-1α in the mdx group. We concluded that low-intensity aerobic exercise (37 sessions) was able to reverse deleterious changes determined by DMD.

Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.

In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.

Augmented Renal Clearance, Muscle Catabolism and Urinary Nitrogen Loss: Implications for Nutritional Support in Critically Ill Trauma Patients.

The main objective of this pilot study was to determine the association between augmented renal clearance (ARC), urinary nitrogen loss and muscle wasting in critically ill trauma patients. We conducted a retrospective analysis of a local database in 162 critically ill trauma patients without chronic renal dysfunction. Nutritional-related parameters and 24 h urinary biochemical analyses were prospectively collected and averaged over the first ten days after admission. Augmented renal clearance was defined by a mean creatinine clearance (CLCR) > 130 mL/min/1.73 m2. The main outcome was the cumulated nitrogen balance at day 10. The secondary outcome was the variation of muscle psoas cross-sectional area (ΔCSA) calculated in the subgroup of patients who underwent at least two abdominal CT scans during the ICU length of stay. Overall, there was a significant correlation between mean CLCR and mean urinary nitrogen loss (normalized coefficient: 0.47 ± 0.07, p < 0.0001). ARC was associated with a significantly higher urinary nitrogen loss (17 ± 5 vs. 14 ± 4 g/day, p < 0.0001) and a lower nitrogen balance (-6 ± 5 vs. -4 ± 5 g/day, p = 0.0002), without difference regarding the mean protein intake (0.7 ± 0.2 vs. 0.7 ± 0.3 g/kg/day, p = 0.260). In the subgroup of patients who underwent a second abdominal CT scan (N = 47), both ΔCSA and %ΔCSA were higher in ARC patients (-33 [-41; -25] vs. -15 [-29; -5] mm2/day, p = 0.010 and -3 [-3; -2] vs. -1 [-3; -1] %/day, p = 0.008). Critically ill trauma patients with ARC are thus characterized by a lower nitrogen balance and increased muscle loss over the 10 first days after ICU admission. The interest of an increased protein intake (>1.5 g/kg/day) in such patients remains a matter of controversy and must be confirmed by further randomized trials.

Prognostic value of metabolic activity of the psoas muscle evaluated by preoperative 18F-FDG PET-CT in breast cancer: a retrospective cross-sectional study.

BACKGROUND: This study aimed to evaluate the potential of metabolic activity of the psoas muscle measured by 18F-fluorodeoxyglucose positron emission tomography-computed tomography to predict treatment outcomes in patients with resectable breast cancer. METHODS: The medical records of 288 patients who had undergone surgical resection for stages I-III invasive ductal carcinoma of the breast between January 2014 and December 2014 in Pusan National University Hospital were reviewed. The standardized uptake values (SUVs) of the bilateral psoas muscle were normalized using the mean SUV of the liver. SUVRmax was calculated as the ratio of the maximum SUV of the average bilateral psoas muscle to the mean SUV of the liver. SUVRmean was calculated as the ratio of the mean SUV of the bilateral psoas muscle to the mean SUV of the liver. RESULTS: Univariate analyses identified a higher T stage, higher N stage, estrogen receptor negativity, progesterone receptor negativity, human epidermal growth factor receptor 2 positivity, triple-negative breast cancer, mastectomy (rather than breast-conserving surgery), SUVRmean > 0.464, and SUVRmax > 0.565 as significant adverse factors for disease-free survival (DFS). Multivariate Cox regression analysis revealed that N3 stage (hazard ratio [HR] = 5.347, P = 0.031) was an independent factor for recurrence. An SUVRmax > 0.565 (HR = 4.987, P = 0.050) seemed to have a correlation with shorter DFS. CONCLUSIONS: A higher SUVRmax of the psoas muscle, which could be a surrogate marker of insulin resistance, showed strong potential as an independent prognostic factor for recurrence in patients with resectable breast cancer.

L-leucine stimulation of glucose uptake and utilization involves modulation of glucose - lipid metabolic switch and improved bioenergetic homeostasis in isolated rat psoas muscle ex vivo.

The antidiabetic effect of l-leucine has been attributed to its modulatory effect on glucose uptake and lipid metabolism in muscles. However, there is a dearth on its effect on glucose metabolism in muscles. Thus, the present study investigated the effect of l-leucine - stimulated glucose uptake on glucose metabolism, dysregulated lipid metabolic pathways, redox and bioenergetic homeostasis, and proteolysis in isolated psoas muscle from Sprague Dawley male rats. Isolated psoas muscles were incubated with l-leucine (30-240 µg/mL) in the presence of 11.1 mMol glucose at 37 ˚C for 2 h. Muscles incubated in only glucose served as the control, while muscles not incubated in l-leucine and/or glucose served as the normal control. Metformin (6.04 mM) was used as the standard antidiabetic drug. Incubation with l-leucine caused a significant increase in muscle glucose uptake, with an elevation of glutathione levels, superoxide dismutase, catalase, E-NTPDase and 5'nucleotidase activities. It also led to the depletion of malondialdehyde and nitric oxide levels, ATPase, chymotrypsin, acetylcholinesterase, glycogen phosphorylase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and lipase activities. There was an alteration in lipid metabolites, with concomitant activation of glycerolipid metabolism, fatty acid metabolism, and fatty acid elongation in mitochondria in the glucose-incubated muscle (negative control). Incubation with l-leucine reversed these alterations, and concomitantly deactivated the pathways. These results indicate that l-leucine-enhanced muscle glucose uptake involves improved redox and bioenergetic homeostasis, with concomitant suppressed proteolytic, glycogenolytic and gluconeogenetic activities, while modulating glucose - lipid metabolic switch.

Low muscle mass assessed by psoas muscle area is associated with clinical adverse events in elderly patients with heart failure.

BACKGROUND: Acute decompensated heart failure (ADHF) is a growing healthcare burden with increasing prevalence and comorbidities due to progressive aging society. Accumulating evidence suggest that low skeletal muscle mass has a negative impact on clinical outcome in elderly adult population. We sought to determine the significance of psoas muscle area as a novel index of low skeletal muscle mass in elderly patients with ADHF. METHODS: In this single-center retrospective observational study, we reviewed consecutive 865 elderly participants (65 years or older) who were hospitalized for ADHF and 392 were available for analysis (79 years [74-85], 56% male). Cross-sectional areas of psoas muscle at the level of fourth lumbar vertebra were measured by computed tomography and normalized by the square of height to calculate psoas muscle index (PMI, cm2/m2). RESULTS: Dividing the patients by the gender-specific quartile value (2.47 cm2/m2 for male and 1.68 cm2/m2 for female), we defined low PMI as the lowest gender-based quartile of PMI. Multiple linear regression analysis revealed female sex, body mass index (BMI), and E/e', but not left ventricular ejection fraction, were independently associated with PMI. Kaplan-Meier analysis showed low PMI was associated with higher rate of composite endpoint of all-cause death and ADHF re-hospitalization (P = 0.033). Cox proportional hazard model analysis identified low PMI, but not BMI, was an independent predictor of the composite endpoint (Hazard ratio: 1.52 [1.06-2.16], P = 0.024). CONCLUSIONS: PMI predicted future clinical adverse events in elderly patients with ADHF. Further studies are needed to assess whether low skeletal muscle mass can be a potential therapeutic target to improve the outcome of ADHF.

Integrated lipidomics and targeted metabolomics analyses reveal changes in flavor precursors in psoas major muscle of castrated lambs.

Castration may decrease off-odors and improve meat flavor. Meat flavor is generated through complex chemical reactions that involve hydrophilic and hydrophobic flavor precursors. In this study, we investigated the flavor precursors in psoas major muscles of castrated and intact sheep using lipidomics and targeted metabolomics. Castration decreased testosterone levels and increased intramuscular fat content. Six hundred fourteen lipid molecules confirmed showed a separation between castrated and intact sheep based on principal component analysis. Fourteen lipid species and 224 lipid molecules increased in castrated sheep. Targeted metabolomics analysis showed that 18 hydrophilic metabolites were affected by castration; however, only hypoxanthine significantly increased in the castration group. Among 45 volatiles identified, 1-octen-3-ol and hexanal were significantly higher in castrated sheep. These results revealed that lipids, hydrophilic metabolites, and volatile compounds in lamb were affected by castration, which might be beneficial in lamb quality.

Long-term retrospective observation study to evaluate effects of adiponectin on skeletal muscle in renal transplant recipients.

Although it has been reported that chronic kidney disease exacerbates sarcopenia progression, the mechanisms of the process remain unclear. Fifty-one patients who underwent renal transplantation at our hospital since 1998 (31 males and 20 females; aged 29-52 years at the time of transplantation) were retrospectively examined for the relationships among the psoas muscle index (PMI), intramuscular adipose tissue content (IMAC), serum adiponectin fractions (high-/low-molecular-weight) and new-onset diabetes after transplantation (NODAT). Before transplantation, age at kidney transplantation negatively correlated with PMI and positively correlated with IMAC (rS = - 0.427, p < 0.01; rS = 0.464, p < 0.01, respectively). Both at 1 and 5 years after transplantation, PMI was higher than before transplantation (p < 0.01). IMAC transiently decreased to - 0.39 at 1 year after kidney transplantation but subsequently increased to - 0.36 at 5 years after kidney transplantation. Multivariate analyses revealed that the mean increase in high-molecular weight adiponectin concentrations was an exacerbating factor for the mean change in PMI (p = 0.003). Moreover, the mean increases in IMAC were exacerbating factors for NODAT. In conclusion, the increase in the PMI is associated with high-molecular weight adiponectin levels after renal transplantation.

The load dependence and the force-velocity relation in intact myosin filaments from skeletal and smooth muscles.

In the present study we evaluated the load dependence of force produced by isolated muscle myosin filaments interacting with fluorescently labeled actin filaments, using for the first time whole native myosin filaments. We used a newly developed approach that allowed the use of physiological levels of ATP. Single filaments composed of either skeletal or smooth muscle myosin and single filaments of actin were attached between pairs of nano-fabricated cantilevers of known stiffness. The filaments were brought into contact to produce force, which caused sliding of the actin filaments over the myosin filaments. We applied load to the system by either pushing or pulling the filaments during interactions and observed that increasing the load increased the force produced by myosin and decreasing the load decreased the force. We also performed additional experiments in which we clamped the filaments at predetermined levels of force, which caused the filaments to slide to adjust the different loads, allowing us to measure the velocity of length changes to construct a force-velocity relation. Force values were in the range observed previously with myosin filaments and molecules. The force-velocity curves for skeletal and smooth muscle myosins resembled the relations observed for muscle fibers. The technique can be used to investigate many issues of interest and debate in the field of muscle biophysics.

Circulating miR-203 derived from metastatic tissues promotes myopenia in colorectal cancer patients.

BACKGROUND: Sarcopenia frequently occurs in metastatic cancer patients. Emerging evidence has revealed that various secretory products from metastatic tumours can influence host organs and promote sarcopenia in patients with malignancies. Furthermore, the biological functions of microRNAs in cell-to-cell communication by incorporating into neighbouring or distal cells, which have been gradually elucidated in various diseases, including sarcopenia, have been elucidated. METHODS: We evaluated psoas muscle mass index (PMI) and intramuscular adipose tissue content (IMAC) using pre-operative computed tomography imaging in 183 colorectal cancer (CRC) patients. miR-203 expression levels in CRC tissues and pre-operative serum were evaluated using quantitative polymerase chain reaction. Functional analysis of miR-203 overexpression was investigated in human skeletal muscle cells (SkMCs), and cells were analysed for proliferation and apoptosis. Expressions of several putative miR-203 target genes (CASP3, CASP10, BIRC5, BMI1, BIRC2, and BIRC3) in SKMCs were validated. RESULTS: A total of 183 patients (108 men and 75 women) were included. The median age of enrolled patients at diagnosis was 68.0 years (range 35-89 years). High IMAC status significantly correlated with female gender (P = 0.004) and older age (P = 0.0003); however, no other clinicopathological factors correlated with IMAC status in CRC patients. In contrast, decreased PMI significantly correlated with female gender (P = 0.006) and all well-established disease development factors, including advanced T stage (P = 0.035), presence of venous invasion (P = 0.034), lymphovascular invasion (P = 0.012), lymph node (P = 0.001), distant metastasis (P = 0.002), and advanced Union for International Cancer Control tumour-node-metastasis stage classification (P = 0.0004). Although both high IMAC status and low PMI status significantly correlated with poor overall survival (IMAC: P = 0.0002; PMI: P < 0.0001; log-rank test) and disease-free survival (IMAC: P = 0.0003; PMI: P = 0.0002; log-rank test), multivariate Cox's regression analysis revealed that low PMI was an independent prognostic factor for both overall survival (hazard ratio: 4.69, 95% confidence interval (CI): 2.19-10, P = 0.0001) and disease-free survival (hazard ratio: 2.33, 95% CI: 1.14-4.77, P = 0.021) in CRC patients. Serum miR-203 expression negatively correlated with pre-operative PMI level (P = 0.0001, ρ = -0.25), and multivariate logistic regression analysis revealed that elevated serum miR-203 was an independent risk factor for myopenia (low PMI) in CRC patients (odds ratio: 5.16, 95% CI: 1.8-14.8, P = 0.002). Overexpression of miR-203 inhibited cell proliferation and induced apoptosis via down-regulation of BIRC5 (survivin) expression in human SkMC line. CONCLUSIONS: Assessment of serum miR-203 expression could be used for risk assessment of myopenia, and miR-203 might be a novel therapeutic target for inhibition of myopenia in CRC.

Clinical Impact of Muscle Quantity and Quality in Colorectal Cancer Patients: A Propensity Score Matching Analysis.

BACKGROUND: Sarcopenia is defined as the loss of skeletal muscle mass, accompanied by decreased muscle strength, and consists of myopenia and myosteatosis. Recent evidence has suggested the predictive value of sarcopenia for the risk of perioperative and oncological outcomes in various malignancies. The aim of this study was to clarify the clinical impact of myopenia and myosteatosis in colorectal cancer (CRC) patients. METHODS: We analyzed the preoperative psoas muscle mass index and intramuscular adipose tissue content using preoperative computed tomography images from 308 CRC patients using statistical methods. RESULTS: Despite no significant correlation between myosteatosis and prognosis, preoperative myopenia significantly correlated with clinicopathological factors for disease development, including advanced tumor depth (P = 0.009), presence of lymphatic vessel invasion (P = 0.006), distant metastasis (P = 0.0007), and advanced stage classification (P = 0.013). Presence of preoperative myopenia was an independent prognostic factor for both cancer-specific survival (hazard ratio [HR]: 2.75, 95% confidence interval [CI]: 1.5-5.05, P = 0.001) and disease-free survival (HR: 3.15, 95% CI: 1.8-5.51, P = 0.0001), and was an independent risk factor for postoperative infectious complications in CRC patients (odds ratio: 2.03, 95% CI:1.17-3.55, P = 0.013). Furthermore, these findings were successfully validated using propensity score matching analysis. CONCLUSIONS: Preoperative myopenia could be useful for perioperative management, and quantification of preoperative skeletal muscle mass could identify patients as a high risk for perioperative and oncological outcomes in CRC patients.

Reduced protein diets increase intramuscular fat of psoas major, a red muscle, in lean and fatty pig genotypes.

The present study aims to assess the effects of pig's genotype (lean v. fatty) and dietary protein level (control v. reduced) on intramuscular fat (IMF) content, fatty acid composition and fibre profile of psoas major, a representative red muscle in pig's carcass scarcely studied relative to white longissimus lumborum. The experiment was conducted on 40 intact male pigs (20 Alentejana purebred and 20 Large White×Landrace×Pietrain crossbred) from 60 to 93 kg of live weight. Pigs were divided and allocated to four dietary groups: control protein diet equilibrated for lysine (17.5% of CP and 0.7% of lysine) and reduced protein diet (RPD) not equilibrated for lysine (13.1% of crude protein and 0.4% of lysine) within a 2×2 factorial arrangement (two genotypes and two diets). Alentejana purebred had higher IMF content (15.7%) and monounsaturated fatty acids (MUFA) (8.9%), whereas crossbred pigs had higher PM weight (46.3%) and polyunsaturated fatty acids (PUFA) (20.1%). The genotype also affected colour with higher lightness (15.1%) and yellowness (33.8%) and lower redness (9.9%) scores in crossbred pigs. In line with this, fatty pigs displayed more oxidative fibres (29.5%), whilst lean pigs had more glycolytic (54.4%). Relative to fatty acids, RPD increased MUFA (5.2%) and SFA (3.2%) but decreased PUFA (14.8%). Ultimately, RPD increased IMF content (15.7%) in the red muscle under study, with no impact on glycolytic to oxidative fibre type transformation.

Distribution of Synthetic Cannabinoids JWH-210, RCS-4 and Δ 9-Tetrahydrocannabinol After Intravenous Administration to Pigs.

BACKGROUND: Synthetic cannabinoids (SCs) have become an increasing issue in forensic toxicology. Controlled human studies evaluating pharmacokinetic data of SCs are lacking and only few animal studies have been published. Thus, an interpretation of analytical results found in intoxicated or poisoned individuals is difficult. Therefore, the distribution of two selected SCs, namely 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1- pentyl-indol-3-yl)methanone (RCS-4) as well as Δ9-tetrahydrocannabinol (THC) as reference were examined in pigs. METHODS: Pigs (n = 6 per drug) received a single intravenous 200 µg/kg BW dose of JWH-210, RCS- 4, or THC. Six hours after administration, the animals were exsanguinated and relevant organs, important body fluids such as bile, and tissues such as muscle and adipose tissue, as well as the bradytrophic specimens dura and vitreous humor were collected. After hydrolysis and solid phase extraction, analysis was performed by LC-MS/MS. To overcome matrix effects of the LC-MS/MS analysis, a standard addition method was applied for quantification. RESULTS: The parent compounds could be detected in every analyzed specimen with the exception of THC that was not present in dura and vitreous humor. Moderate concentrations were present in brain, the site of biological effect. Metabolite concentrations were highest in tissues involved in metabolism and/or elimination Conclusions: Besides kidneys and lungs routinely analyzed in postmortem toxicology, brain, adipose, and muscle tissue could serve as alternative sources, particularly if other specimens are not available. Bile fluid is the most appropriate specimen for SCs and THC metabolites detection.

Myo-inositol inhibits intestinal glucose absorption and promotes muscle glucose uptake: a dual approach study.

The present study investigated the effects of myo-inositol on muscle glucose uptake and intestinal glucose absorption ex vivo as well as in normal and type 2 diabetes model of rats. In ex vivo study, both intestinal glucose absorption and muscle glucose uptake were studied in isolated rat jejunum and psoas muscle respectively in the presence of increasing concentrations (2.5 % to 20 %) of myo-inositol. In the in vivo study, the effect of a single bolus dose (1 g/kg bw) of oral myo-inositol on intestinal glucose absorption, blood glucose, gastric emptying and digesta transit was investigated in normal and type 2 diabetic rats after 1 h of co-administration with 2 g/kg bw glucose, when phenol red was used as a recovery marker. Myo-inositol inhibited intestinal glucose absorption (IC50 = 28.23 ± 6.01 %) and increased muscle glucose uptake, with (GU50 = 2.68 ± 0.75 %) or without (GU50 = 8.61 ± 0.55 %) insulin. Additionally, oral myo-inositol not only inhibited duodenal glucose absorption and reduced blood glucose increase, but also delayed gastric emptying and accelerated digesta transit in both normal and diabetic animals. Results of this study suggest that dietary myo-inositol inhibits intestinal glucose absorption both in ex vivo and in normal or diabetic rats and also promotes muscle glucose uptake in ex vivo condition. Hence, myo-inositol may be further investigated as a possible anti-hyperglycaemic dietary supplement for diabetic foods and food products.

Degradation of Kidney and Psoas Muscle Proteins as Indicators of Post-Mortem Interval in a Rat Model, with Use of Lateral Flow Technology.

We investigated potential protein markers of post-mortem interval (PMI) using rat kidney and psoas muscle. Tissue samples were taken at 12 h intervals for up to 96 h after death by suffocation. Expression levels of eight soluble proteins were analyzed by Western blotting. Degradation patterns of selected proteins were clearly divided into three groups: short-term, mid-term, and long-term PMI markers based on the half maximum intensity of intact protein expression. In kidney, glycogen synthase (GS) and glycogen synthase kinase-3ß were degraded completely within 48 h making them short-term PMI markers. AMP-activated protein kinase α, caspase 3 and GS were short-term PMI markers in psoas muscle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a mid-term PMI marker in both tissues. Expression levels of the typical long-term PMI markers, p53 and ß-catenin, were constant for at least 96 h post-mortem in both tissues. The degradation patterns of GS and caspase-3 were verified by immunohistochemistry in both tissues. GAPDH was chosen as a test PMI protein to perform a lateral flow assay (LFA). The presence of recombinant GAPDH was clearly detected in LFA and quantified in a concentration-dependent manner. These results suggest that LFA might be used to estimate PMI at a crime scene.

Thrombospondin-1 levels correlate with macrophage activity and disease progression in dysferlin deficient mice.

Dysferlinopathy is associated with accumulation of thrombospondin (TSP)-1 and macrophages, both of which may contribute to the pathogenesis of the disease. The purpose of this study was to determine whether TSP-1 levels can predict macrophage activity and disease progression in dysferlin deficient BlaJ mice, focusing on the early disease process. In 3 month-old BlaJ mice, muscle TSP-1 levels exhibited strong positive correlations with both accumulation of F4/80hi macrophages and with their in vivo phagocytic activity in psoas muscles as measured by magnetic resonance imaging and flow cytometry. Muscle TSP-1 levels also exhibited a strong negative correlation with muscle mass and strong positive correlations with histological measurements of muscle fiber infiltration and regeneration. Over the course of disease progression from 3 to 12 months of age, muscle TSP-1 levels showed more complicated relationships with macrophage activity and an inverse relationship with muscle mass. Importantly, blood TSP-1 levels showed strong correlations with macrophage activity and muscle degeneration, particularly early in disease progression in BlaJ mice. These data indicate that TSP-1 may contribute to a destructive macrophage response in dysferlinopathy and pose the intriguing possibility that TSP-1 levels may serve as a biomarker for disease progression.