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Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice ¹

JOHN D. PORTER^*,,,2, ANITA P. MERRIAM^*, SANGEETA KHANNA^*, FRANCISCO H. ANDRADE, CHELLIAH R. RICHMONDS, PATRICK LEAHY, GEORGIANA CHENG^*, PARASKEVI KARATHANASIS^||, XIAOHUA ZHOU^*, LINDA L. KUSNER, MARVIN E. ADAMS^¶, MICHAEL WILLEM^**, ULRIKE MAYER and HENRY J. KAMINSKI^,

Departments of
^* Ophthalmology,
Neurology,
Neurosciences, and
The Comprehensive Cancer Center, Case Western Reserve University and The Research Institute of University Hospitals of Cleveland, Cleveland, Ohio, USA;
^|| Department of Toxicology, University of Kentucky, Lexington, Kentucky, USA;
^¶ Department of Physiology and Biophysics, University of Washington, Seattle, Washington, USA;
^** Max-Planck-Institute for Biochemistry, Martinsried, Germany; and
School of Biological Sciences, Wellcome Trust Centre for Cell-Matrix Research, Manchester, UK

²Correspondence: Department of Ophthalmology, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106-5068, USA. E-mail: jdp7{at}po.cwru.edu

SPECIFIC AIMS

Our aim was to test mechanisms potentially responsible for protection of the extraocular muscles in an animal model of Duchenne muscular dystrophy.

PRINCIPAL FINDINGS

Using the mdx mouse, we systematically tested putative mechanisms that could protect extraocular muscle from the cyclic degeneration and regeneration characteristic of dystrophin-based muscular dystrophy. Putative muscle-sparing mechanisms were predicted from existing models of the pathogenesis of muscular dystrophy, including functional replacement of dystrophin by utrophin, maintained calcium homeostasis in the face of sarcolemmal defects, preservation of nitric oxide (NO) myofiber-to-vasculature signaling, and compensation by an 7ß1 integrin to laminin-2 linkage that parallels the dystrophin-glycoprotein complex (DGC). We used high density oligonucleotide microarrays to uncover any other unanticipated mechanisms that may contribute to the absence of pathology in eye muscle.

1. Dystrophin-deficient extraocular muscles expresses sarcolemmal utrophin, but in a fiber type-specific fashion
Our data established that the expression of utrophin at neuromuscular junctions of wild-type extraocular muscle was augmented in mdx mice to include the entire sarcolemma of three of six extraocular muscle fiber types: the orbital singly innervated fiber type and the orbital and global multiply innervated fiber types (Fig. 1 ). Accessory extraocular muscles, which lack these three fiber types, did not exhibit sarcolemmal utrophin immunoreactivity. In contrast to other skeletal muscles, utrophin up-regulation occurred in intact, adult fiber types, as central nuclei and other signs of muscle damage or regeneration were absent from mdx extraocular muscle.

View larger version (127K): [in this window] [in a new window]   Figure 1. Immunocytochemical localization of dystrophin (dys) and utrophin (utr) in wt (C57) and mdx extraocular muscles. Dystrophin is displaced from the sarcolemma in mdx EOM (A, B). Utrophin is normally restricted to adult EOM nmjs (C), identified with -bungarotoxin staining (BT; D). In mdx, utrophin is no longer restricted to nmj sites, but also localizes to the sarcolemma of orbital layer fibers (E, F) and the global multiply innervated fiber type (G, I). Multiply innervated fibers were identified by their immunoreactivity for slow (ß-cardiac) myosin heavy chain (sMyHC) in adjacent sections (H). Bars, 25 µm (A–F in panel F; G–I in panel I).

2. The fiber type-specific expression of utrophin was accompanied by preservation of the dystrophin-glycoprotein complex
The three extraocular muscle fiber types expressing sarcolemmal utrophin in mdx also showed normal localization of the dystroglycans, sarcoglycans, syntrophins, and dystrobrevins by immunocytochemistry. Dystrophin-glycoprotein complex immunoreactivity was absent from the other three extraocular muscle fiber types and from the accessory extraocular muscles.

3. Sarcolemmal integrity remains intact and calcium homeostatic systems do not adapt in dystrophin-deficient extraocular muscle
The established vital dye permeability of mdx skeletal muscles was absent from all extraocular muscle fiber types. In addition to exclusion of Evans blue dye, mdx extraocular muscle contrasted with other skeletal muscles in that it did not show elevation of total calcium level, as determined by atomic absorption spectroscopy. Furthermore, there was no adaptive increase in Ca²⁺-ATPase (SERCA) content in mdx extraocular muscle.

4. Neuronal NOS (nNOS) activity and localization is partially preserved in mdx extraocular muscle, but is not mechanistic in muscle sparing
By monitoring ³H-arginine to ³H-citrulline conversion, we determined that the dystrophin-deficient reduction in nNOS was less in extraocular muscle (27%) than in other skeletal muscles (48–54%). Mdx extraocular muscle fiber types that retained the DGC also showed immunoreactivity for nNOS at the sarcolemma. Mice deficient in both dystrophin and -syntrophin (or expressing -syntrophin deficient in the nNOS binding PDZ domain), however, exhibited normal anatomy.

5. Extraocular muscles are spared in mice deficient in both dystrophin and 7 integrin
By quantitative PCR, expression of 7A integrin in mdx extraocular muscle was unchanged from wild-type whereas 7B showed only a modest increase (1.74-fold, P<0.01). Extraocular muscles in mice deficient in dystrophin and 7 integrin, however, were unchanged from wild-type, mdx, or 7 integrin-deficient mice.

6. Microarray did not identify alternative adaptive strategies in mdxextraocular muscle
Postnatal day 56 mdx extraocular muscle was compared with age-matched controls (n=5/strain) by DNA microarray using Affymetrix MG-U74Av1 arrays. Of >10,000 probes evaluated, only 7 met criteria for differential regulation (Fig. 2 ). Down-regulated genes in mdx EOM included erythroid differentiation regulator (Edr, –2.2-fold), dystrophin (Dmd, –2.5-fold), and two ESTs (AI853899, –2.0-fold; AW125272, –1.8-fold). Up-regulated genes included metallothionein 1 and 2 (Mt1, 1.6-fold; Mt2, 2.4-fold) and cardiac ankyrin-like repeat protein (Alrp, 3.1-fold).

View larger version (65K): [in this window] [in a new window]   Figure 2. Hierarchical cluster analysis of differentially expressed genes in extraocular and hindlimb muscle. Very few differentially regulated genes (n=7) were found in the mdx eye muscle (A). Genes identified from a reanalysis of our prior hindlimb muscle data (n=373) were plotted against eye values for the same genes (B). Genes induced (Leg) and down-regulated (Leg) in mdx are shown. The intersection of genes induced in the wt EOM vs. wt leg (n=191) with genes induced in the mdx leg vs. wt leg (n=311) then was examined for shared genes (n=59; C). Expression levels, averaged across replicates, for the 59 genes in the Venn diagram are shown in a hierarchical cluster analysis (D).

CONCLUSIONS

Current models do not mechanistically link dystrophin deficiency to myofiber death when explaining the complex spatial, temporal, and species specificities of the disease. The complete sparing of the extraocular muscles in muscular dystrophy is an important exception unaccounted for by existing models. We have shown here that there are both DGC-competent and -deficient myofibers in mdx extraocular muscle, yet neither type exhibits pathology. Adaptive strategies predicted from existing models of dystrophinopathy were explored to determine the mechanisms responsible for extraocular muscle sparing. We also used gene expression profiling as a large-scale and unbiased means of identifying mechanisms not predicted by current disease models. Collectively, our data show that extraocular muscle is constitutively protected in mdx (Fig. 3 ) and provide compelling support for the concept of the conditional nature of dystrophinopathy.

View larger version (29K): [in this window] [in a new window]   Figure 3. Scheme depicting molecular mechanisms tested here establishing that extraocular muscle adaptations are not responsible for sparing in dystrophin deficiency. DNA microarray suggests that alternate ECM (ecm) -to-cytoskeleton organization is a constitutive difference between extraocular and other skeletal muscles that may protect eye muscle in mdx.

It is generally accepted that the structural linkage between cytoskeleton and ECM and the signaling functions associated with the dystrophin-glycoprotein scaffold is required for myofiber viability. Yet we show here that half of the extraocular muscle fiber types in mdx are DGC deficient but remain structurally and functionally intact. Our data establish that extraocular muscle accomplishes what other skeletal muscles do not: the natural retention of utrophin and corresponding maintenance of the DGC at the sarcolemma in adult mdx myofibers. Moreover, we identified a novel fiber type specificity in extraocular muscle utrophin expression and corresponding DGC retention in these same fiber types. The DGC-deficient extraocular muscle fibers nonetheless retain sarcolemmal integrity, as evidenced by Evans blue dye and Ca²⁺ exclusion.

Partial DGC preservation in extraocular muscle, in conjunction with recent data revitalizing the ischemia hypothesis for muscular dystrophy, suggested that nNOS-mediated anti-ischemia mechanisms could be responsible for muscle sparing. Partial preservation of extraocular nNOS activity and fiber type-specific sarcolemmal nNOS localization reported here is consistent with this hypothesis. However, the absence of extraocular muscle myofiber necrosis in dystrophin/-syntrophin double mutants strongly suggests that nNOS retention is not viable as a rescue mechanism in mdx extraocular muscle.

7ß1 deficiency itself is thought to be responsible for a form of muscular dystrophy, and 7 integrin up-regulation in dystrophic muscle is a potential muscle rescue mechanism. If integrin serves a sparing role, then mice deficient in dystrophin and 7 integrin should exhibit dystrophic muscles. However, double mutant extraocular muscle could not be differentiated from wild-type or mdx. Thus, despite evidence that 7ß1 integrin can substitute for the DGC in other muscles, it is not implicated in extraocular muscle sparing.

We used expression profiling to determine whether transcriptional adaptations might spare extraocular muscle in ways not predicted by current models. In contrast to broad transcript changes in mdx limb representing pathology and adaptation, very few genes were differentially expressed in extraocular muscle. No genes consistent with predictions of Duchenne models (e.g., transmembrane, calcium binding, or antioxidant) were induced in mdx extraocular muscle. We concluded that extraocular muscle does not actively adapt at the transcriptional level to manage the absence of dystrophin.

We used a bioinformatics strategy to narrow the transcript profile that might be responsible for extraocular muscle sparing in muscular dystrophy. By intersecting the list of genes that are more highly expressed in wild-type extraocular vs. wild-type leg muscle with the list of genes induced in mdx leg muscle, we identified 59 genes that may play either adaptive or constitutively protective roles in the two muscle groups in dystrophin deficiency. Differential expression of ECM and cytoskeleton was a major theme (20 of 59 genes). A specialized cytoskeletal to ECM organization could provide considerable elasticity and resilience that is vital to extraocular muscle integrity. The elasticity/tensile strength associated with ECM organization may support myofibers during the constant high-level activity experienced in normal extraocular muscle, then fortuitously guard this muscle group against eccentric contraction damage in dystrophin deficiency.

In summary, convergent evidence strongly suggests that constitutive properties, not adaptation, determine the fate of mdx EOM.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0810fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0810fje

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This Article Full Text (PDF) All Versions of this Article: 17/8/893 02-0810fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by PORTER, J. D. Articles by KAMINSKI, H. J. Search for Related Content PubMed PubMed Citation Articles by PORTER, J. D. Articles by KAMINSKI, H. J.