Differential RNA Expression Profile of Skeletal Muscle Induced by Experimental Autoimmune Myasthenia Gravis in Rats

Abstract

The differential susceptibility of skeletal muscle by myasthenia gravis (MG) is not well understood. We utilized RNA expression profiling of extraocular muscle (EOM), diaphragm (DIA), and extensor digitorum (EDL) of rats with experimental autoimmune MG (EAMG) to evaluate the hypothesis that muscles respond differentially to injury produced by EAMG. EAMG was induced in female Lewis rats by immunization with acetylcholine receptor purified from the electric organ of the Torpedo. Six weeks later after rats had developed weakness and serum antibodies directed against the AChR, animals underwent euthanasia and RNA profiling performed on DIA, EDL, and EOM. Profiling results were validated by qPCR. Across the three muscles between the experiment and control groups, 359 probes (1.16%) with greater than 2-fold changes in expression in 7 of 9 series pairwise comparisons from 31,090 probes were identified with approximately two-thirds being increased. The three muscles shared 16 genes with increased expression and 6 reduced expression. Functional annotation demonstrated that these common expression changes fell predominantly into categories of metabolism, stress response, and signaling. Evaluation of specific gene function indicated that EAMG led to a change to oxidative metabolism. Genes related to muscle regeneration and suppression of immune response were activated. Evidence of a differential immune response among muscles was not evident. Each muscle had a distinct RNA profile but with commonality in gene categories expressed that are focused on muscle repair, moderation of inflammation, and oxidative metabolism.

Gene Expression Profiling of Orbital Muscles in Treatment-Resistant Ophthalmoplegic Myasthenia Gravis

“…Figure 4C depicts the representative biological categories of strongly correlated gene pairs in the OP-MG network and inverse in controls. Of the most differentially correlated gene pairs which were inverse by phenotype (n=13; p 0.001), >50% involved a gene whose transcripts were substantially altered in EAMG [17] suggesting altered MG pathways in the muscle transcriptome of OP-MG vs controls. The broad category of 'muscle atrophy' signalling was the most representative functional category accounting for two-thirds of the most differential and inversely correlated gene pairs by phenotype with oxidative metabolism the next most frequent category (p 0.001) (genes may be implicated in 2 categories).…”

“…OP-MG genes served as "seed queries" which informed the broader enquiry of additional gene candidates in related pathways (OP-MG pathways). Genes previously shown to be dysregulated by MG in human limb muscles [16], in vitro models or experimental autoimmune myasthenia gravis (EAMG) [17] were also included. As reduced contractility (as occurs in OP-MG EOMs) [1,18] potentially alters the regulation of biological pathways in muscle, we also pro led genes previously shown to be dysregulated in the EOMs of cases with ocular misalignment/strabismus due to other non-myasthenic causes.…”

“…The reference genes were selected from genes that have previously been validated in human [9,[19][20][21] and rodent EOMs [17,22] and showed stability in a patient derived muscle model [7].…”

“…CANX encodes calnexin, a chaperone protein that facilitates the assembly of AChR subunits [34], FAM92A1 has recently been recognized for its importance in mitochondrial functioning [35] and both FAM69A and PEF1 have poorly characterized biological function. PEF1 encoding Pe in, which may be involved with endoplasmic reticulum-golgi transport and/or calcium binding [36], connected with a 'module' of genes (Table 2) which were pro led because they showed altered muscle expression levels in EAMG [17], and they are known to regulate mitochondrial biogenesis and oxidative metabolism. PARK2, encoding parkin, a major regulator of mitophagy [31], appeared to form a subnode of negatively correlated genes, several of which are involved in muscle regeneration (e.g.…”

“…WIF1 encodes Wnt inhibitory factor-1, and as an inhibitor of Wnt signalling may affect muscle regeneration and endplate AChR clustering [39]. MT1A encodes metallothionein-1A and protects cells against oxidative stress [in [17]]. Increased expression of metallothionein 1 has been reported in atrophic muscle, possibly by preventing the upregulation of the insulin growth factor (IGF)-1 pathway [40], and in the EOMs of the EAMG rodent models [17].…”