Cardiac and respiratory dysfunction in Duchenne muscular dystrophy and the role of second messengers.

Duchenne muscular dystrophy (DMD) affects young boys and is characterized by the absence of dystrophin, a large cytoskeletal protein present in skeletal and cardiac muscle cells and neurons. The heart and diaphragm become necrotic in DMD patients and animal models of DMD, resulting in cardiorespiratory failure as the leading cause of death. The major consequences of the absence of dystrophin are high levels of intracellular Ca(2+) and the unbalanced production of NO that can finally trigger protein degradation and cell death. Cytoplasmic increase in Ca(2+) concentration directly and indirectly triggers different processes such as necrosis, fibrosis, and activation of macrophages. The absence of the neuronal isoform of nitric oxide synthase (nNOS) and the overproduction of NO by the inducible isoform (iNOS) further increase the intracellular Ca(2+) via a hypernitrosylation of the ryanodine receptor. NO overproduction, which further induces the expression of iNOS but decreases the expression of the endothelial isoform (eNOS), deregulates the muscle tissue blood flow creating an ischemic situation. The high levels of Ca(2+) in dystrophic muscles and the ischemic state of the muscle tissue would culminate in a positive feedback loop. While efforts continue toward optimizing cardiac and respiratory care of DMD patients, both Ca(2+) and NO in cardiac and respiratory muscle pathways have been shown to be important to the etiology of the disease. Understanding the mechanisms behind the fine regulation of Ca(2+) -NO may be important for a noninterventional and noninvasive supportive approach to treat DMD patients, improving the quality of life and natural history of DMD patients.

Expression profiling reveals novel hypoxic biomarkers in peripheral blood of adult mice exposed to chronic hypoxia.

Hypoxia induces a myriad of changes including an increase in hematocrit due to erythropoietin (EPO) mediated erythropoiesis. While hypoxia is of importance physiologically and clinically, lacunae exist in our knowledge of the systemic and temporal changes in gene expression occurring in blood during the exposure and recovery from hypoxia. To identify these changes expression profiling was conducted on blood obtained from cohorts of C57Bl-10 wild type mice that were maintained at normoxia (NX), exposed for two weeks to normobaric chronic hypoxia (CH) or two weeks of CH followed by two weeks of normoxic recovery (REC). Using stringent bioinformatic cut-offs (0% FDR, 2 fold change cut-off), 230 genes were identified and separated into four distinct temporal categories. Class I) contained 1 transcript up-regulated in both CH and REC; Class II) contained 202 transcripts up-regulated in CH but down-regulated after REC; Class III) contained 9 transcripts down-regulated both in CH and REC; Class IV) contained 18 transcripts down-regulated after CH exposure but up-regulated after REC. Profiling was independently validated and extended by analyzing expression levels of selected genes as novel biomarkers from our profile (e.g. spectrin alpha-1, ubiquitin domain family-1 and pyrroline-5-carboxylate reductase-1) by performing qPCR at 7 different time points during CH and REC. Our identification and characterization of these genes define transcriptome level changes occurring during chronic hypoxia and normoxic recovery as well as novel blood biomarkers that may be useful in monitoring a variety of physiological and pathological conditions associated with hypoxia.

Superior calcium homeostasis of extraocular muscles.

Extraocular muscles (EOMs) are a unique group of skeletal muscles with unusual physiological properties such as being able to undergo rapid twitch contractions over extended periods and escape damage in the presence of excess intracellular calcium (Ca(2+)) in Duchenne's muscular dystrophy (DMD). Enhanced Ca(2+) buffering has been proposed as a contributory mechanism to explain these properties; however, the mechanisms are not well understood. We investigated mechanisms modulating Ca(2+) levels in EOM and tibialis anterior (TA) limb muscles. Using Fura-2 based ratiometric Ca(2+) imaging of primary myotubes we found that EOM myotubes reduced elevated Ca(2+) ˜2-fold faster than TA myotubes, demonstrating more efficient Ca(2+) buffering. Quantitative PCR (qPCR) and western blotting revealed higher expression of key components of the Ca(2+) regulation system in EOM, such as the cardiac/slow isoforms sarcoplasmic Ca(2+)-ATPase 2 (Serca2) and calsequestrin 2 (Casq2). Interestingly EOM expressed monomeric rather than multimeric forms of phospholamban (Pln), which was phosphorylated at threonine 17 (Thr17) but not at the serine 16 (Ser16) residue. EOM Pln remained monomeric and unphosphorylated at Ser16 despite protein kinase A (PKA) treatment, suggesting differential signalling and modulation cascades involving Pln-mediated Ca(2+) regulation in EOM. Increased expression of Ca(2+)/SR mRNA, proteins, differential post-translational modification of Pln and superior Ca(2+) buffering is consistent with the improved ability of EOM to handle elevated intracellular Ca(2+) levels. These characteristics provide mechanistic insight for the potential role of superior Ca(2+) buffering in the unusual physiology of EOM and their sparing in DMD.

Identification of the neuromuscular junction transcriptome of extraocular muscle by laser capture microdissection.

PURPOSE: To examine and characterize the profile of genes expressed at the synapses or neuromuscular junctions (NMJs) of extraocular muscles (EOMs) compared with those expressed at the tibialis anterior (TA). METHODS: Adult rat eyeballs with rectus EOMs attached and TAs were dissected, snap frozen, serially sectioned, and stained for acetylcholinesterase (AChE) to identify the NMJs. Approximately 6000 NMJs for rectus EOM (EOMsyn), 6000 NMJs for TA (TAsyn), equal amounts of NMJ-free fiber regions (EOMfib, TAfib), and underlying myonuclei and RNAs were captured by laser capture microdissection (LCM). RNA was processed for microarray-based expression profiling. Expression profiles and interaction lists were generated for genes differentially expressed at synaptic and nonsynaptic regions of EOM (EOMsyn versus EOMfib) and TA (TAsyn versus TAfib). Profiles were validated by using real-time quantitative polymerase chain reaction (qPCR). RESULTS: The regional transcriptomes associated with NMJs of EOMs and TAs were identified. Two hundred seventy-five genes were preferentially expressed in EOMsyn (compared with EOMfib), 230 in TAsyn (compared with TAfib), and 288 additional transcripts expressed in both synapses. Identified genes included novel genes as well as well-known, evolutionarily conserved synaptic markers (e.g., nicotinic acetylcholine receptor (AChR) alpha (Chrna) and epsilon (Chrne) subunits and nestin (Nes). CONCLUSIONS: Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. The definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype.

Distinctive patterns of microRNA expression in extraocular muscles.

The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other muscles. We and others have shown that EOMs have a unique transcriptome and proteome. Here we investigated the expression pattern of microRNAs (miRNAs), as they may play a role in generating the unique EOM allotype. We isolated RNA and screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. Seventy-four miRNAs were found to be differentially regulated (P value <0.05) of which 31 (14 upregulated, 17 downregulated) were differentially regulated at signal strength >500. Muscle-specific miRNAs miR-206 and miR-499 were upregulated and miR-1, miR-133a, and miR-133b were downregulated in EOM. Quantitative PCR (qPCR) analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using cotransfection of precursor miRNAs with reporter constructs containing the 3'-untranslated region of predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular makeup of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne muscular dystrophy and may assist in development of therapeutic strategies.

Transcriptional and functional differences in stem cell populations isolated from extraocular and limb muscles.

The extraocular muscles (EOMs) are a distinct muscle group that displays an array of unique contractile, structural, and regenerative properties. They also have differential sensitivity to certain diseases and are enigmatically spared in Duchenne muscular dystrophy (DMD). The EOMs are so distinct from other skeletal muscles that the term "allotype" has been coined to highlight EOM group-specific properties. We hypothesized that increased and distinct stem cells may underlie the continual myogenesis noted in EOM. The side population (SP) stem cells were isolated and studied. EOMs had 15x higher SP cell content compared with limb muscles. Expression profiling revealed 348 transcripts that define the EOM-SP transcriptome. Over 92% of transcripts were SP specific, because they were absent in previous whole muscle microarray studies. Cultured EOM-SP cells revealed superior in vitro proliferative capacity. Finally, assays of the committed progenitors or satellite cells performed on myofibers isolated from EOM and limb muscles independently validated the increased proliferative capacity of these muscles. We suggest a model in which unique EOM stem cells contribute to the continual myogenesis noted in EOM and consistent with a role for their sparing in DMD. We believe the greater numbers of stem cells, their unique transcriptome, the greater proliferative capacity of EOM stem cells, and the greater number of satellite cells also offer clues for novel cell-based therapeutic strategies.

Comparison of the myoplasmic calcium transient elicited by an action potential in intact fibres of mdx and normal mice.

The myoplasmic free [Ca2+] transient elicited by an action potential (Delta[Ca2+]) was compared in fast-twitch fibres of mdx (dystrophin null) and normal mice. Methods were used that maximized the likelihood that any detected differences apply in vivo. Small bundles of fibres were manually dissected from extensor digitorum longus muscles of 7- to 14-week-old mice. One fibre within a bundle was microinjected with furaptra, a low-affinity rapidly responding fluorescent calcium indicator. A fibre was accepted for study if it gave a stable, all-or-nothing fluorescence response to an external shock. In 18 normal fibres, the peak amplitude and the full-duration at half-maximum (FDHM) of Delta[Ca2+] were 18.4 +/- 0.5 microm and 4.9 +/- 0.2 ms, respectively (mean +/- s.e.m.; 16 degrees C). In 13 mdx fibres, the corresponding values were 14.5 +/- 0.6 microm and 4.7 +/- 0.2 ms. The difference in amplitude is statistically highly significant (P = 0.0001; two-tailed t test), whereas the difference in FDHM is not (P = 0.3). A multi-compartment computer model was used to estimate the amplitude and time course of the sarcoplasmic reticulum (SR) calcium release flux underlying Delta[Ca2+]. Estimates were made based on several differing assumptions: (i) that the resting myoplasmic free Ca2+ concentration ([Ca2+]R) and the total concentration of parvalbumin ([Parv(T)]) are the same in mdx and normal fibres, (ii) that [Ca2+](R) is larger in mdx fibres, (iii) that [Parv(T)] is smaller in mdx fibres, and (iv) that [Ca2+]R is larger and [Parv(T)] is smaller in mdx fibres. According to the simulations, the 21% smaller amplitude of Delta[Ca2+] in mdx fibres in combination with the unchanged FDHM of Delta[Ca2+] is consistent with mdx fibres having a approximately 25% smaller flux amplitude, a 6-23% larger FDHM of the flux, and a 9-20% smaller total amount of released Ca2+ than normal fibres. The changes in flux are probably due to a change in the gating of the SR Ca2+-release channels and/or in their single channel flux. The link between these changes and the absence of dystrophin remains to be elucidated.

Combination of peptide OFFGEL fractionation and label-free quantitation facilitated proteomics profiling of extraocular muscle.

Several label-free quantitation strategies have been introduced that obliterate the need for expensive isotopically labeled molecules. However label-free approaches have considerably higher demands in respect of repeatability of sample preparation and fractionation than multiplexing isotope labeling-based strategies. OFFGEL fractionation promises the necessary separation efficiency and repeatability. To test this platform, 12-fraction peptide OFFGEL electrophoresis and online reversed-phase LC connected to a quadrupole TOF mass spectrometer were used to determine differences of the physiological, pathological and biochemical distinct extraocular muscle allotype in comparison to hind-limb muscle. Close to 70% of the peptides separated by OFFGEL electrophoresis were detected only in a single fraction. To determine the separation repeatability of four samples, we compared the ion volumes of multiple peptides deriving from the thick filament-associated protein titin over several fractions and determined a coefficient of variation below 20%. Of the 474 proteins identified, 61 proteins were differently expressed between the two muscle allotypes and were involved in metabolism, muscle contraction, stress response, or gene expression. Several expression differences were validated using immunohistochemistry and Western blot analysis. We therefore consider peptide OFFGEL fractionation an effective and efficient addition to our label-free quantitative proteomics workflow.

Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes.

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.