Molecular alterations in skeletal muscle in rheumatoid arthritis are related to disease activity, physical inactivity, and disability

Here, we report that in RA, skeletal muscle exhibits molecular alterations in inflammatory markers, transcriptional profiles, and metabolic signatures. Both at protein and transcriptional levels, muscle had a pro-inflammatory phenotype in RA. Additionally, differential gene expression in muscle in RA was indicative of dysregulation of muscle repair, promotion of glycolysis, and poor mitochondrial function. Upregulated glycolysis and mitochondrial inefficiency were supported by greater muscle concentrations of the glycolytic end-product pyruvate in RA. Further, disease activity and disability were related to lesser concentrations of long-chain acylcarnitines and greater concentrations of amino acid precursors for muscle fibrosis. Taken together, these alterations in proteins, gene expression, and metabolic intermediates were indicative of muscle in RA in a state of chronically activated, yet dysregulated remodeling with increased glycolysis, mitochondrial inefficiency, and fibrotic material (Fig. 2).

This represents the first report of significant markers of inflammation in muscle in RA. The clinical importance of these molecules is demonstrated by the significant association of several muscle cytokines with RA disease activity, disability, pain, and physical inactivity. The IPA-generated novel network centered on downregulation of NF-kB2, a protein that promotes non-canonical NF-kB signaling and opposes inflammatory signaling [17]. Downregulation of NF-kB2 would be predicted to favor coordinated upregulation of pro-inflammatory NF-kB signaling in muscle in RA. We were unable to determine if the muscle cytokines and pro-inflammatory transcripts in RA were derived from myocytes, inflammatory cells, or other cellular sources. Interestingly, muscle cytokine concentrations did not reflect those measured in circulation, suggesting these disease-associated inflammatory markers stem from local rather than systemic events.

Based on the strong relationships between muscle inflammatory markers and disability, pain and physical inactivity, we suspected that increased intramuscular cytokines may be indicative of a disrupted muscle remodeling process. In fact, muscle gene expression alterations in RA were consistent with promotion of satellite cell differentiation and upregulation of several facets of the normally well-coordinated process of muscle remodeling. For instance, muscle in RA was characterized by downregulation of ZFP36, the gene that encodes tristetraprolin (TTP), which reduces inflammation by destabilizing pro-inflammatory cytokine transcripts [18, 19] and prevents satellite cell activation by destabilizing myogenic regulatory factor, MyoD, mRNA [20]. Thus, the reduction in ZFP36 expression in muscle in RA would be expected to promote pro-inflammatory cytokine production and satellite cell activation.

Other gene expression changes also suggest both chronic activation and temporal dysregulation of muscle remodeling. For instance, downregulation of ZBTB16 would promote inflammation and proliferation of autoreactive T cells [21]. In contrast to the reduced ZFP36 expected to promote satellite cell activation, the increased CITED2 would be expected to reduce satellite cell activation [22, 23]. Increased expression of ABRA, RCAN1, VGLL2, MYF6 and decreased expression of RRAD would promote differentiation of satellite cells [24–30]. More descriptions of differentially expressed genes are provided in Additional file 1.

Gene expression alterations indicative of glycolysis promotion and poor mitochondrial function were supported by greater muscle concentrations of the glycolytic end-product pyruvate in RA. Further, disease activity and disability were related to lower concentrations of fatty-acid-derived long-chain acylcarnitines. One plausible explanation for this relationship is that fewer long-chain acylcarnitines indicate less oxidative metabolism and fewer mitochondria, consistent with a glycolytic phenotype. RA disease activity and disability were also related to higher concentrations of amino acid precursors for muscle fibrosis. M2-type macrophages contain arginase, which metabolizes arginine to ornithine [31]. Ornithine is converted to proline, which provides a substrate for resident fibroblasts to generate collagen. In addition to proline, collagen formation also requires glycine; glycine and proline each account for a third of the collagen amino acids. While collagen is critical for extracellular matrix production, in the setting of a chronically activated remodeling process, excess collagen production leads to fibrosis [31]. Thus, the relationships between these amino acids and disease activity and disability may indicate a fibrotic process in muscle associated with active disease that contributes to RA-associated disability.

There were several limitations to this study. RA medication regimens were not uniform among participants, and effects of these medications on skeletal muscle are unclear. Twenty-five percent of patients with RA used prednisone at low doses, which is not expected to have significant myopathic effects; despite this, they had significant alterations in muscle inflammatory markers and systemic inflammation relative to controls. Without histopathologic assessment or single cell isolations, we were unable to determine the cellular source of muscle cytokines, transcripts, or metabolites. Our findings indicate that either RA medication regimens or the RA disease process itself alters skeletal muscle inflammatory molecules, transcriptional profiles, and metabolic pathways.