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John D Porter

Publications and source records attributed to John D Porter.

14 recordsLinked to original sources
PubMed2003

Temporal gene expression profiling of dystrophin-deficient (mdx) mouse diaphragm identifies conserved and muscle group-specific mechanisms in the pathogenesis of muscular dystrophy.

Mutations in dystrophin are the proximate cause of Duchenne muscular dystrophy (DMD), but pathogenic mechanisms linking the absence of dystrophin from the sarcolemma to myofiber necrosis are not fully known. The muscular dystrophies also have properties not accounted for by current disease models, including the temporal delay to disease onset, broad species differences in severity, and diversity of skeletal muscle responses. To address the mechanisms underlying the differential targeting of muscular dystrophy, we characterized temporal expression profiles of the diaphragm in dystrophin-deficient (mdx) mice between postnatal days 7 and 112 using oligonucleotide microarrays and contrasted these data with published hindlimb muscle data. Although the diaphragm and hindlimb muscle groups differ in severity of response to dystrophin deficiency, and exhibited substantial divergence in some transcript categories including inflammation and muscle-specific genes, our data show that the general mechanisms operative in muscular dystrophy are highly conserved. The two muscle groups principally differed in expression levels of differentially regulated genes, as opposed to the non-conserved induced/repressed transcripts defining fundamentally distinct mechanisms. We also identified a postnatal divergence of the two wild-type muscle group expression profiles that temporally correlated with the onset and progression of the dystrophic process. These findings support the hypothesis that conserved disease mechanisms interacting with baseline differences in muscle group-specific transcriptomes underlie their differential responses to DMD. We further suggest that muscle group-specific transcriptional profiles contribute toward the muscle targeting and sparing patterns observed for a variety of metabolic and neuromuscular diseases.

Animals↗
PubMed2003

Dissection of temporal gene expression signatures of affected and spared muscle groups in dystrophin-deficient (mdx) mice.

Although dystrophin mutations are the proximate cause of Duchenne muscular dystrophy (DMD), interactions among heterogeneous downstream mechanisms may be key phenotypic determinants. Temporal gene expression profiling was used to identify and correlate diverse transcriptional patterns to one another and to the disease course, for both affected and spared muscle groups, in postnatal day 7-112 dystrophin-deficient (mdx) mice. While 719 transcripts were differentially expressed at one or more ages in leg muscle, only 56 genes were altered in the spared extraocular muscles (EOM). Contrasting molecular signatures of affected versus spared muscles provide compelling evidence that the absence of dystrophin alone is necessary but not sufficient to cause the patterned fibrosis, inflammation and failure of muscle regeneration characteristic of dystrophinopathy. Dystrophic and adaptive changes in the microarray profiles were further quantified using an aggregate disease load index (DLI) to measure stage-dependent transcriptional impact in both muscles. DLI analysis highlighted the divergent responses of EOM and leg muscle groups. Cellular process-specific DLIs in leg muscle identified positively correlated temporal expression profiles for some gene classes, and the independence of others, that are linked to major disease components. Data also showed a previously unrecognized transient and selective developmental delay in pre-necrotic mdx skeletal muscle that was confirmed by qPCR. Taken together, validation and targeting of signaling pathways responsible for the coordination of the fibrotic, proteolytic and inflammatory mechanisms shown here for mdx muscle may yield new therapeutic means of mitigating the devastating consequences of DMD.

Animals↗
PubMed2003

Paradoxical absence of M lines and downregulation of creatine kinase in mouse extraocular muscle.

The M lines are structural landmarks in striated muscles, necessary for sarcomeric stability and as anchoring sites for the M isoform of creatine kinase (CK-M). These structures, especially prominent in fast skeletal muscles, are missing in rodent extraocular muscle, a particularly fast and active muscle group. In this study, we tested the hypotheses that 1). myomesin and M protein (cytoskeletal components of the M lines) and CK-M are downregulated in mouse extraocular muscle compared with the leg muscles, gastrocnemius and soleus; and 2). the expression of other cytosolic and mitochondrial CK isoforms is correspondingly increased. As expected, mouse extraocular muscles expressed lower levels of myomesin, M protein, and CK-M mRNA than the leg muscles. Immunocytochemically, myomesin and M protein were not detected in the banding pattern typically seen in other skeletal muscles. Surprisingly, message abundance for the other known CK isoforms was also lower in the extraocular muscles. Moreover, total CK activity was significantly decreased compared with that in the leg muscles. Based on these data, we reject our second hypothesis and propose that other energy-buffering systems may be more important in the extraocular muscles. The downregulation of major structural and metabolic elements and relative overexpression of two adenylate kinase isoforms suggest that the extraocular muscle group copes with its functional requirements by using strategies not seen in typical skeletal muscles.

Adenylate Kinase↗
PubMed2003

Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice.

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin-glycoprotein complex (DGC)-competent and DGC-deficient myofibers distributed in a fiber type-specific pattern. Up-regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type-specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and alpha-syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, alpha7beta1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Adaptation, Physiological↗
PubMed2003

Persistent over-expression of specific CC class chemokines correlates with macrophage and T-cell recruitment in mdx skeletal muscle.

Prior studies and the efficacy of immunotherapies provide evidence that inflammation is mechanistic in pathogenesis of Duchenne muscular dystrophy. To identify putative pro-inflammatory mechanisms, we evaluated chemokine gene/protein expression patterns in skeletal muscle of mdx mice. By DNA microarray, reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, and immunoblotting, convergent evidence established the induction of six distinct CC class chemokine ligands in adult MDX: CCL2/MCP-1, CCL5/RANTES, CCL6/mu C10, CCL7/MCP-3, CCL8/MCP-2, and CCL9/MIP-1gamma. CCL receptors, CCR2, CCR1, and CCR5, also showed increased expression in mdx muscle. CCL2 and CCL6 were localized to both monocular cells and muscle fibers, suggesting that dystrophic muscle may contribute toward chemotaxis. Temporal patterns of CCL2 and CCL6 showed early induction and maintained expression in mdx limb muscle. These data raise the possibility that chemokine signaling pathways coordinate a spatially and temporally discrete immune response that may contribute toward muscular dystrophy. The chemokine pro-inflammatory pathways described here in mdx may represent new targets for treatment of Duchenne muscular dystrophy.

Animals↗
PubMed2003

Comprehensive expression profiling by muscle tissue class and identification of the molecular niche of extraocular muscle.

Muscle tissue is an elegant model for biologic integration of structure with function and is frequently affected by a variety of inherited diseases. Traditional muscle classes--skeletal, cardiac, and smooth--share basic aspects of contractile and energetics mechanisms but also have distinctive role-specific adaptations. We used large-scale oligonucleotide microarrays to broaden knowledge of the adaptive expression patterns underlying muscle tissue differences and to identify transcript subsets that are most likely to represent candidate disease genes. Using stringent analysis criteria, we found >or=95 transcripts, which were preferentially expressed by each muscle class and were validated by inclusion of known muscle class-specific and inherited disease-related genes. Differentially expressed transcripts not previously identified as class-specific extend understanding of muscle class transcriptomes and may represent novel muscle-specific disease genes. We also analyzed the expression profile of extraocular muscle, which is divergent from other skeletal muscles, in the broader context of all major muscle classes. Data show that the extraocular muscle phenotype results from the combination of tissue-specific transcripts, novel expression levels of skeletal muscle transcripts, and partial sharing of gene expression patterns with cardiac and smooth muscle. These, and additional proteomic data, establish that extraocular muscle does not constitute a distinctive muscle class but that it does occupy a novel niche within the skeletal muscle class.

Animals↗
PubMed2003

Molecular organization of the extraocular muscle neuromuscular junction: partial conservation of and divergence from the skeletal muscle prototype.

PURPOSE: The phenotypically novel extraocular muscles (EOMs) exhibit fundamental differences in innervation and neuromuscular junction (NMJ) morphology from other skeletal muscles. In the current study, the morphology and molecular organization of NMJs of EOM singly innervated (SIF) and multiply innervated (MIF) fiber types were evaluated and the distribution of molecules involved in formation and maintenance of NMJs were specifically characterized. METHODS: Adult mouse EOM NMJ organization was examined by immunofluorescence and confocal microscopy. Differential cellular localization of components of two established synaptic signaling pathways, (1) neuregulin and erbB receptors 2, 3, and 4 and (2) agrin, MuSK, and rapsyn and select NMJ-associated structural proteins were studied for EOM SIF and MIF populations. Endplate topography and structure were also studied, using both confocal microscopy and transmission electron microscopy, with NMJ morphologic organization correlated with specific EOM fiber types. RESULTS: Confocal fluorescence microscopy demonstrated that, for NMJs of both EOM SIFs and MIFs, components of neuregulin and agrin pathways and the major components of the junctional dystrophin-glycoprotein complex (DGC) colocalized with acetylcholine receptor (AChR) aggregates. However, EOM exhibited novel fiber-type-specific extrasynaptic localization of two key DGC signaling-related molecules: alpha-dystrobrevin 1 (global MIFs) and syntrophin beta1 (global MIFs and orbital MIFs and SIFs). CONCLUSIONS: The data establish that the molecular organization of EOM SIF and MIF NMJs includes the same signaling and structural molecules previously characterized for other skeletal muscles. By contrast, divergence in other aspects of the synaptic and nonsynaptic sarcolemmal organization of EOM fiber types may underlie the unique responses of these muscles in a variety of neuromuscular disorders.

Animals↗
PubMed2003

Comprehensive evaluation of the extraocular muscle critical period by expression profiling in the dark-reared rat and monocularly deprived monkey.

PURPOSE: To address the consequences of visual deprivation paradigms in rat (dark rearing) and monkey (monocular deprivation) on extraocular muscle (EOM) development using genome-wide expression profiling. METHODS: Serial analysis of gene expression (SAGE) was used to determine alterations in the EOM transcriptome induced by dark rearing of rats from birth to postnatal day 45. Data were compared with previously published normal EOM SAGE library. DNA microarray similarly assessed changes in gene expression patterns of EOMs of monkeys reared from birth to 4 months of age with monocular deprivation. RESULTS: Dark rearing produced changes in expression of 280 transcripts in rat EOM. Of these, 71 were known genes representing functional categories that included energy metabolism/mitochondrial-related (21%), protein synthesis and modification (14%), lipid metabolism (13%), and muscle-related (6%) transcripts. Together, the predominant pattern reflected an energetic shift toward fatty acid beta-oxidation and integrated alterations in both myofibers and supportive tissues. The response of monkey rectus muscles to monocular deprivation was considerably less severe. CONCLUSIONS: The visual deprivation paradigms used in this study mimic alterations that are associated with the common disorders of strabismus, congenital cataract, and amblyopia. These data show that postnatal EOM maturation is broadly susceptible to changes in activity patterns that are a consequence of visuomotor maldevelopment. The data extend the concept of an EOM-critical period and establish that activity patterns in developing eye movement systems play vital determinant roles in the novel EOM phenotype.

Animals↗
PubMed2003

Postnatal suppression of myomesin, muscle creatine kinase and the M-line in rat extraocular muscle.

The M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM), is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.

Adaptation, Physiological↗
PubMed2002

A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice.

Mutations in dystrophin cause Duchenne muscular dystrophy (DMD), but absent dystrophin does not invariably cause necrosis in all muscles, life stages and species. Using DNA microarray, we established a molecular signature of dystrophinopathy in the mdx mouse, with evidence that secondary mechanisms are key contributors to pathogenesis. We used variability controls, adequate replicates and stringent analytic tools, including significance analysis of microarrays to estimate and manage false positive rates. In leg muscle, we identified 242 differentially expressed genes, >75% of which have not been previously reported as altered in human or animal dystrophies. Data provide evidence for coordinated activity of numerous components of a chronic inflammatory response, including cytokine and chemokine signaling, leukocyte adhesion and diapedesis, invasive cell type-specific markers, and complement system activation. Selective chemokine upregulation was confirmed by RT-PCR and immunoblot, and may be a key determinant of the nature of the inflammatory response in dystrophic muscle. Up-regulation of secreted phosphoprotein 1 (minopontin, osteopontin) mRNA and protein in dystrophic muscle identified a novel linkage between inflammatory cells and repair processes. Extracellular matrix genes were up-regulated in mdx to levels similar to those in DMD. Since, unlike DMD, mdx exhibits little fibrosis, data suggest that collagen regulation at post-transcriptional stages mediates extensive fibrosis in DMD. Taken together, these data identify a relatively neglected aspect of DMD, suggest new treatment avenues, and highlight the value of genome-wide profiling in study of complex disease processes.

Animals↗
PubMed2002

Extraocular muscle: cellular adaptations for a diverse functional repertoire.

Oculomotor control systems are considerably more complex and diverse than are spinal skeletomotor systems. Moreover, individual skeletal muscles are frequently functional role-specific, while all extraocular muscles operate across a very wide dynamic range. We contend that the novel phenotype of the extraocular muscles is a direct consequence of the functional demands imposed upon this muscle group by the central eye movement controllers. This review highlights five basic themes of extraocular muscle biology that set them apart from more typical skeletal muscles, specifically, the (a) novel innervation pattern, (b) heterogeneity in contractile proteins, (c) structural and functional compartmentalization of the rectus and oblique muscles, (d) diversity of extraocular muscle fiber types, and (e) relationship between the novel muscle phenotype and the differential response of these muscles in neuromuscular and endocrine disease. Finally, new data from broad genome-wide profiling studies are reviewed, with global gene expression patterns lending substantial support to the notion that the extraocular muscles are fundamentally different from traditional skeletal muscle. This novel eye muscle phenotype represents an adaptation that exploits the full range of variability in skeletal muscle to meet the needs of visuomotor systems.

Animals↗
PubMed2002

Transcriptional profile of rat extraocular muscle by serial analysis of gene expression.

PURPOSE: Although extraocular muscle (EOM) is skeletal muscle, aspects of its biology are unlike other striated muscle. In this study, the broad molecular genetics profile underlying the novel EOM phenotype was examined. METHODS: Serial analysis of gene expression (SAGE) was used to quantify adult rat EOM gene transcripts. SAGE isolates and sequences 10-bp tags from defined locations in mRNA-derived cDNA. Tag sequence-location was used to extract transcript identity from a curated SAGE database, and detection frequencies reflected abundance of corresponding mRNAs. RESULTS: The 54,764 expressed sequence tags generated and sequenced from EOM included 17,602 unique tags. Of the unique tags, 7.8% were detected at high to intermediate levels (>or=5 copies), 19.3% at lower levels (2-4 copies), and 72.9% as single copies; 40% of the tags matched known expressed sequence tags (ESTs), most of which (85.7%) represented a unique EST. Tags without matches in the SAGE database and those expressed as single copies only were not considered further. SAGE tags expressed at more than 0.1% of total transcripts reflected several aspects of muscle biology, including sarcomeric structure, energy metabolism, and ribosomal protein expression. Genes highly expressed in EOM were compared with other existing muscle expression databases to identify conserved and novel patterns in EOM. CONCLUSIONS: The data provide a normative gene expression database and a novel molecular signature that will facilitate study of EOM development and function and of the mechanisms behind its preferential targeting or sparing in neuromuscular disease.

Animals↗