Early Onset of Lipofuscin Accumulation in Dystrophin-Deficient Skeletal Muscles of DMD Patients and mdx Mice

Lipofuscin, the so-called ageing pigment, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals and redox-active metal ions. Usually it accumulates in post-mitotic, long-lived cells such as neurons and cardiac muscle cells. In contrast, it is rarely seen in either normal or diseased skeletal muscle fibres. In this paper, we report that lipofuscin accumulates at an early age in both human and murine dystrophic muscles. Autofluorescent lipofuscin granules were localized, using confocal laser scanning microscopy and electron microscopy, in dystrophin-deficient skeletal muscles of X chromosome-linked young Duchenne muscular dystrophy (DMD) patients and of mdx mice at various ages after birth. Age-matched normal controls were studied similarly. Autofluorescent lipofuscin granules were observed in dystrophic biceps brachii muscles of 2-7-year-old DMD patients where degeneration and regeneration of myofibres are active, but they were rarely seen in age-matched normal controls. In normal mice, lipofuscin first appears in diaphragm muscles nearly 20 weeks after birth but in mdx muscles it occurs much earlier, 4 weeks after birth, when the primary degeneration of dystrophin-deficient myofibres is at a peak. Lipofuscin accumulation increases with age in both mdx and normal controls and is always higher in dystrophic muscles than in age-matched normal controls. At the electron microscopical level, it was confirmed that the localisation of autofluorescent granules observed by light microscopy in dystrophin-deficient skeletal muscles coincided with lipofuscin granules in myofibres and myosatellite cells, and in macrophages accumulating around myofibres and in interstitial connective tissue. Our results agree with previous biochemical and histochemical data implying increased oxidative damages in DMD and mdx muscles. They indicate that dystrophin-deficient myofibres are either more susceptible to oxidative stress, or are subjected to higher intra- or extracellular oxidative stress than normal controls, or both.     

Activity of Head Muscles During Feeding by Snakes: A Comparative Study

The adaptive radiation of colubroid snakes has involved thedevelopment of numerous prey capture specializations combinedwith conservation of a swallowing mechanism characterized byindependent movements of the right and left toothed bones ofthe skull. Synchronized electromyographic and cinematographicrecordings of swallowing in Nerodia, Elaphe, Heterodon and Agkistrodon,four diverse genera of colubroid snakes, allow a preliminaryevaluation of the relationship between prey capture and swallowing.The results indicate that the movements of the palatopterygoidbar and advance of the mandible as closing of the jaws beginsas well as patterns of muscle activity producing these movementsare similar among the four genera. Conversely, the patternsof activity of external adductors and, to some extent, the depressormandibulae differ among the four genera sampled. Analyses ofbone movements during swallowing suggest that swallowing iseffected primarily by the palatopterygoid bars. The mandiblesand their connecting soft tissues mainly press the prey againstthe palatopterygoid teeth. The mandibular teeth evidently playlittle active role in swallowing. Also, the maxilla, which displaysconsiderable morphological diversity among colubroid snakes,has little independent or direct function in swallowing, itsteeth rarely contacting the prey. The data suggest that theheads of colubroid snakes have evolved two partially separatedstructural-functional units, a medial swallowing unit and alateral prey capture unit.     

Early Detection of Myocardial Bioenergetic Deficits: A 9.4 Tesla Complete Non Invasive 31P MR Spectroscopy Study in Mice with Muscular Dystrophy

Background

Duchenne muscular dystrophy (DMD) is the most common fatal form of muscular dystrophy characterized by striated muscle wasting and dysfunction. Patients with DMD have a very high incidence of heart failure, which is increasingly the cause of death in DMD patients. We hypothesize that in the in vivo system, the dystrophic cardiac muscle displays bioenergetic deficits prior to any functional or structural deficits. To address this we developed a complete non invasive 31P magnetic resonance spectroscopy (31P MRS) approach to measure myocardial bioenergetics in the heart in vivo.

Methods and Results

Six control and nine mdx mice at 5 months of age were used for the study. A standard 3D -Image Selected In vivo Spectroscopy (3D-ISIS) sequence was used to provide complete gradient controlled three-dimensional localization for heart 31P MRS. These studies demonstrated dystrophic hearts have a significant reduction in PCr/ATP ratio compare to normal (1.59±0.13 vs 2.37±0.25, p<0.05).

Conclusion

Our present study provides the direct evidence of significant cardiac bioenergetic deficits in the in vivo dystrophic mouse. These data suggest that energetic defects precede the development of significant hemodynamic or structural changes. The methods provide a clinically relevant approach to use myocardial energetics as an early marker of disease in the dystrophic heart. The new method in detecting the in vivo bioenergetics abnormality as an early non-invasive marker of emerging dystrophic cardiomyopathy is critical in management of patients with DMD, and optimized therapies aimed at slowing or reversing the cardiomyopathy.     

A Comparative Analysis and Review of lysyl Residues Affected by Posttranslational Modifications

Post-translational modification is the most common mechanism of regulating protein function. If phosphorylation is considered a key event in many signal transduction pathways, other modifications must be considered as well. In particular the side chain of lysine residues is a target of different modifications; notably acetylation, methylation, ubiquitylation, sumoylation, neddylation, etc. Mass spectrometry approaches combining highly sensitive instruments and specific enrichment strategies have enabled the identification of modified sites on a large scale. Here we make a comparative analysis of the most representative lysine modifications (ubiquitylation, acetylation, sumoylation and methylation) identified in the human proteome. This review focuses on conserved amino acids, secondary structures preference, subcellular localization of modified proteins, and signaling pathways where these modifications are implicated. We discuss specific differences and similarities between these modifications, characteristics of the crosstalk among lysine post translational modifications, and single nucleotide polymorphisms that could influence lysine post-translational modifications in humans.     

Revertant Fibers in the mdx Murine Model of Duchenne Muscular Dystrophy: An Age- and Muscle-Related Reappraisal

Muscles in Duchenne dystrophy patients are characterized by the absence of dystrophin, yet transverse sections show a small percentage of fibers (termed “revertant fibers”) positive for dystrophin expression. This phenomenon, whose biological bases have not been fully elucidated, is present also in the murine and canine models of DMD and can confound the evaluation of therapeutic approaches. We analyzed 11 different muscles in a cohort of 40 mdx mice, the most commonly model used in pre-clinical studies, belonging to four age groups; such number of animals allowed us to perform solid ANOVA statistical analysis. We assessed the average number of dystrophin-positive fibers, both absolute and normalized for muscle size, and the correlation between their formation and the ageing process. Our results indicate that various muscles develop different numbers of revertant fibers, with different time trends; besides, they suggest that the biological mechanism(s) behind dystrophin re-expression might not be limited to the early development phases but could actually continue during adulthood. Importantly, such finding was seen also in cardiac muscle, a fact that does not fit into the current hypothesis of the clonal origin of “revertant” myonuclei from satellite cells. This work represents the largest, statistically significant analysis of revertant fibers in mdx mice so far, which can now be used as a reference point for improving the evaluation of therapeutic approaches for DMD. At the same time, it provides new clues about the formation of revertant fibers/cardiomyocytes in dystrophic skeletal and cardiac muscle.     

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Background

The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.

Methodology/Principal Findings

We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.

Conclusions/Significance

Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.     

Magnetic Resonance Imaging and Spectroscopy Assessment of Lower Extremity Skeletal Muscles in Boys with Duchenne Muscular Dystrophy: A Multicenter Cross Sectional Study

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that results in functional deficits. However, these functional declines are often not able to be quantified in clinical trials for DMD until after age 7. In this study, we hypothesized that ¹H₂O T₂ derived using ¹H-MRS and MRI-T₂ will be sensitive to muscle involvement at a young age (5–7 years) consistent with increased inflammation and muscle damage in a large cohort of DMD subjects compared to controls.

Methods

MR data were acquired from 123 boys with DMD (ages 5–14 years; mean 8.6 SD 2.2 years) and 31 healthy controls (age 9.7 SD 2.3 years) using 3-Tesla MRI instruments at three institutions (University of Florida, Oregon Health & Science University, and Children’s Hospital of Philadelphia). T₂-weighted multi-slice spin echo (SE) axial images and single voxel ¹H-MRS were acquired from the lower leg and thigh to measure lipid fraction and ¹H₂O T₂.

Results

MRI-T₂, ¹H₂O T₂, and lipid fraction were greater (p<0.05) in DMD compared to controls. In the youngest age group, DMD values were different (p<0.05) than controls for the soleus MRI-T₂, ¹H₂O T₂ and lipid fraction and vastus lateralis MRI-T₂ and ¹H₂O T₂. In the boys with DMD, MRI-T₂ and lipid fraction were greater (p<0.05) in the oldest age group (11–14 years) than the youngest age group (5–6.9 years), while ¹H₂O T₂ was lower in the oldest age group compared to the young age group.

Discussion

Overall, MR measures of T₂ and lipid fraction revealed differences between DMD and Controls. Furthermore, MRI-T₂ was greater in the older age group compared to the young age group, which was associated with higher lipid fractions. Overall, MR measures of T₂ and lipid fraction show excellent sensitivity to DMD disease pathologies and potential therapeutic interventions in DMD, even in the younger boys.     

Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.     

Quantitative Proteomic Analysis of Serum from Pregnant Women Carrying a Fetus with Conotruncal Heart Defect Using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Labeling

Objective

To identify differentially expressed proteins from serum of pregnant women carrying a conotruncal heart defects (CTD) fetus, using proteomic analysis.

Methods

The study was conducted using a nested case-control design. The 5473 maternal serum samples were collected at 14–18 weeks of gestation. The serum from 9 pregnant women carrying a CTD fetus, 10 with another CHD (ACHD) fetus, and 11 with a normal fetus were selected from the above samples, and analyzed by using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS). The differentially expressed proteins identified by iTRAQ were further validated with Western blot.

Results

A total of 105 unique proteins present in the three groups were identified, and relative expression data were obtained for 92 of them with high confidence by employing the iTRAQ-based experiments. The downregulation of gelsolin in maternal serum of fetus with CTD was further verified by Western blot.

Conclusions

The identification of differentially expressed protein gelsolin in the serum of the pregnant women carrying a CTD fetus by using proteomic technology may be able to serve as a foundation to further explore the biomarker for detection of CTD fetus from the maternal serum.     

Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx,Largemyd and mdx/Largemyd

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Large^myd mouse and the recently generated and worst double mutant mdx/Large^myd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Large^myd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Large^myd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Large^myd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Large^myd showed similar values than both the mild mdx and worst mdx/Large^myd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.     

Translational Activity of mRNA Coding for Cytoskeletal Brain Proteins in Newborn and Adult Mice: A Comparative Study

Translational activity of mRNA coding for cytoskeletal brain proteins was used to determine the relative abundance of the mRNA in the brains of newborn and adult mice. mRNA was translated in a cell-free system containing rabbit reticulocyte factors. The products of translation were analyzed by two-dimensional gel electrophoresis and characterized by peptide map analysis. Comparison of the products of translation from newborn and from adult brain mRNA shows a 50% decrease in actin and tubulin from newborn to the adult stage. In contrast, the 70 kd neurofilament protein and glial fibrillary acidic protein show a twofold increase in the adult stage. The heat-shock protein HSP70 increases slightly (30%) whereas the brain isozyme of creatine kinase and the heat-shock protein HSP90 are three times as high in adult subjects as in newborns.     

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.