Unraveling calcium dysregulation and autoimmunity in immune mediated rippling muscle disease

Abstract

Rippling Muscle Disease (RMD) is a rare skeletal myopathy characterized by abnormal muscular excitability manifesting with wave-like muscle contractions and percussion-induced muscle mounding. Hereditary RMD is associated with caveolin-3 or cavin-1 mutations. Recently, we identified cavin 4 autoantibodies as a biomarker of immune-mediated RMD (iRMD), though the underlying disease-mechanisms remain poorly understood. Transcriptomic studies were performed on muscle biopsies of 8 patients (5 males; 3 females; ages 26-to-80) with iRMD. Subsequent pathway analysis compared iRMD to human non-disease control and disease control (dermatomyositis) muscle samples. Transcriptomic studies demonstrated changes in key pathways of muscle contraction and development. All iRMD samples had significantly upregulated cavin-4 expression compared to controls, likely compensatory for autoantibody-mediated protein degradation. Proteins involved in muscle relaxation (including SERCA1, PMCA and PLN) were significantly increased in iRMD compared to controls. Comparison of iRMD to dermatomyositis transcriptomics demonstrated significant overlap in immune pathways, and the IL-6 signaling pathway was markedly increased in all iRMD patient muscle biopsies and increased in the majority of iRMD patients' serum. This study represents the first muscle transcriptomic analysis of iRMD patients and dissects underlying disease mechanisms. Increase of sarcolemmal and cellular calcium channels as well as PLN, an inhibitor of the SERCA pump for calcium into the sarcoplasm, likely alters the calcium dynamics in iRMD. These changes in crucial components of muscle relaxation may underlie rippling by altering calcium flux. Our findings provide crucial insights into the differential expression of genes regulating muscle relaxation and highlight potential disease pathomechanisms.

Keywords: Immune mediated rippling muscle disease; Interferon; Interleukin-6; Myopathy; Transcriptomics.

Fig. 1

Proteins implicated in hereditary RMD (hRMD) and immune mediated RMD (iRMD) display increased expression in iRMD. RNA was isolated from iRMD skeletal muscle biopsies and sequenced, and compared against previously published non-disease controls (Ctrl). Z-scores for CAV3, CAVIN1, and CAVIN4 were calculated from expression levels across samples. iRMD = immune mediated rippling muscle disease, Ctrl = non-disease control biopsies

Fig. 2

iRMD Skeletal Muscle shows alterations in pathways of calcium regulation, muscle contraction, muscle development, and muscle differentiation. A Bubble plot of GO Term analysis of differentially expressed genes in iRMD skeletal muscle compared to healthy controls shows pathways of muscle t-tubules, calcium regulation, muscle differentiation, and muscle development are significantly altered in iRMD. B Heatmap of differentially expressed genes in muscle organ development GO-Term. C Heatmap of differentially expressed genes in regulation of cardiac muscle contraction by calcium ion signaling GO-Term

Fig. 3

CACNA1S, RYR1 and TOM20 expression level and immunofluorescence in iRMD compared to non-disease controls. A Heatmap of CACNA1S expression in transcriptomic data set of iRMD compared to non-disease controls. B Immunofluorescence staining of CACNA1S showed no significant difference in protein level. C Significantly increased expression was found for TOMM20 but not RYR1 using RNA sequencing transcriptomic analysis on iRMD patient’s skeletal muscle biopsies compared to non-disease controls. D Immunofluorescence staining of RYR1 and TOMM20 demonstrated no significant difference in expression or colocalization in iRMD patient’s compared to non-disease controls. Scale bar = 10um., ns = not significant

Fig. 4

Skeletal muscle from iRMD patients shows altered expression and protein level of critical regulators of calcium flux and muscle relaxation. A Heatmap showing expression of PLN, PMCA, and SERCA1 in iRMD patient skeletal muscle biopsies compared to non-disease controls. B Immunofluorescent staining of iRMD skeletal muscle compared to non-disease controls. *p < 0.05, **p < 0.01. Scale bars: PLN = 50um, PMCA = 10um, SERCA1 = 50um

Fig. 5

Interferon type-1 and Interferon type-2 pathways are not induced in iRMD skeletal muscle biopsies. A Heatmap of expression z-scores of Interferon type-1 inducible genes in iRMD skeletal muscle biopsies compared to non-disease controls. B Heatmap of expression z-scores of Interferon type-2 inducible genes in iRMD skeletal muscle biopsies compared to non-disease controls

Fig. 6

The IL-6 pathway is activated in iRMD. Heatmap of IL-6 inducible genes in iRMD skeletal muscle biopsies compared to non-disease controls

Fig. 7

Schematic illustration of pathophysiologic changes in iRMD. Left: Calcium enters the skeletal muscle cytosol through Cav1.1 and RyR1. In iRMD, antibodies bind to Cavin-4 at caveolae. IL6 expression and protein level are increased. Right: SERCA1 and PMCA, proteins involved in muscle relaxation, are increased at the protein level. PLN, an inhibitor of SERCA1, is dramatically increased in expression in iRMD skeletal muscle. This may interfere with the ability of calcium to return to the sarcoplasmic reticulum, instead exiting through PMCA to the extracellular space. Blue arrows: RNA expression level. Red arrows: Protein expression level