Upregulation of CAV3, CAVIN1 and CAVIN4 in iRMD skeletal muscle biopsies

To define the transcriptomic profile of iRMD skeletal muscle, RNA sequencing was performed on muscle biopsies from a cohort of eight iRMD patients (Supplemental Table 1). Findings were compared to those from a cohort of previously published non-disease control skeletal muscle biopsies [35]. Hierarchical clustering and PCA analysis showed clear separation between iRMD patient muscle biopsies and non-disease controls (Supplemental Fig. 1 and 2). When compared to non-disease controls, 8640 genes displayed significantly (padj < 0.05, FC > 1.5) altered expression in iRMD patient muscle biopsies. 4577 genes showed increased expression in iRMD muscle and 4063 were diminished in expression.

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

As previous studies have demonstrated acquired patchy loss of caveolin-3 in iRMD skeletal muscle and because hRMD is caused by mutations in CAV3 or CAVIN1, we first determined whether expression of these genes is altered in iRMD [11, 15, 20, 42]. Compared to non-disease controls, iRMD patient muscle demonstrated significantly increased mRNA expression of CAV3 and CAVIN1 (Fig. 1).

Given the recent discovery of anti-cavin-4 auto-antibodies and patchy loss of sarcolemmal cavin-4 in iRMD, we also sought to determine if CAVIN4 expression was altered in iRMD skeletal muscle [25]. Similarly to CAV3, CAVIN4 expression also was significantly increased in iRMD skeletal muscle samples compared to non-disease control (Fig. 1).

Further, when iRMD patient muscle biopsies were compared to an independent GTEX cohort of non-disease controls, CAVIN4 and CAV3 remained greater than five-fold upregulated, although CAVIN1 was not differentially expressed (Supplemental Table 2).

Pathways of Skeletal Muscle Function and Calcium Ion Signaling are altered in iRMD patient skeletal muscle

We next sought to determine whether pathways of skeletal muscle function were significantly altered in iRMD patient muscle biopsies compared to non-disease controls.

Gene Ontology pathway analysis (GO-Term) was performed on all genes with significantly altered expression between iRMD and non-disease control. Several pathways involved in skeletal muscle development or differentiation were identified as enriched (Fig. 2A). Notably, these pathways included CAV3 and CAVIN4, identifying these genes as key players of skeletal muscle alteration in iRMD (Fig. 2B, and Supplemental Table 3). Differential pathways showed relatively equal splits between genes increased and decreased in expression in iRMD patient muscle biopsies (Fig. 2B, C). Further, pathways of both T-tubule organization as well as regulation of cardiac muscle contraction by calcium ion signaling were significantly and differentially expressed in iRMD skeletal muscle compared to non-disease controls (Fig. 2A, Supplemental Table 3).

Additionally, bridging integrator 1 (BIN1), which encodes a cavin-4 interacting protein concentrated at T-tubules known to cause a centronuclear myopathy when mutated, is also dysregulated in iRMD (Supplemental Table 4) [30, 43]. Such findings support a critical role for calcium dysregulation underlying the phenomenon of rippling (Fig. 2A, C).

Channels of muscle excitation and mitochondria remain unchanged at the protein level in iRMD muscle

Given iRMD is characterized by wave-like muscle contractions and stretch induced muscle mounding, we aimed to determine whether changes in expression of genes involved in muscle excitation or relaxation occur in iRMD skeletal muscle. This was performed by utilizing the transcriptomics data set and immunofluorescence staining quantification.

The pathways of muscle excitation and relaxation have been previously well characterized. In brief, calcium initially enters the cytoplasm via membrane calcium channel Cav1.1 (CACNA1S). This influx of calcium triggers ryanodine receptor 1 (Ryr1) to release calcium from the sarcoplasmic reticulum [44, 45]. To allow relaxation, calcium is pumped back into the extracellular space by the plasma membrane Ca++ ATPase (PMCA) and into the sarcoplasmic reticulum by SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) [45]. Phospholamban (PLN) has been demonstrated to inhibit the SERCA activity allosterically, causing significant reduction in pump affinity for calcium and decreasing ATPase activity of SERCA [46, 47].

Compared to non-disease controls, iRMD muscle shows a significant increase in CACNA1S expression (5.38 fold increase, p = 7.5E−42), while RYR1 expression was not significantly different (1.22 fold increase, p = 0.178). We also assessed and compared protein levels of CACNA1S and RYR1 between iRMD skeletal muscle and non-disease controls and found no significant difference between CACNA1S and RYR1 immunofluorescence quantification (Fig. 3). Given ATP is produced by mitochondria in close proximity to T-tubules forming mitochondrial associated membranes with the structure, and that the pattern of colocalization is disrupted in other myopathic disorders, we also analyzed TOMM20 RNA expression and protein level. Though TOMM20 expression was increased, there was no change in the protein level by immunohistochemical quantification or its colocalization with RYR1 in iRMD compared to controls (Fig. 3).

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

Changes in key muscle relaxation gene expression occur at the RNA and protein level in iRMD muscle

Next, we investigated whether expression of genes involved in muscle relaxation was altered in iRMD. SERCA1 (1.39 fold, p = 0.022), PMCA (2.12 fold, p = 1.12E−10), and PLN (33.96 fold, p = 5.5E−146) all showed a statistically significant increase in expression. SERCA1 did not meet 1.5 fold change criteria (Fig. 4A), though it did attain a statistically significant padj (Fig. 4A). Immunofluorescence staining further demonstrated that protein level changes mirror expression level changes of PLN, PMCA, and SERCA1 in iRMD muscle (Fig. 4B and Supplemental Fig. 3). Though PLN RNA expression has been shown to be a marker of type 1 fibers, several other markers of type 1 fibers were not increased in expression in our RNA-seq dataset in iRMD muscle biopsies (Supplemental Table 5), suggesting that induction of PLN is not due to changes in predominant fiber type or fiber type shift [48].

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

Interferon pathways are not significantly induced in iRMD compared to healthy controls

Our group has recently demonstrated that auto-antibodies against cavin-4 are a specific biomarker of iRMD [25]. Hence, we next examined whether activation of pathways of autoimmunity observed in autoimmune myopathies are activated in iRMD. Interferon alpha has been suggested to play a key role in dermatomyositis (DM) where dendritic cells produce an abundance of interferon alpha and the interferon pathway is expressed at high levels in muscle biopsies [49, 50]. Utilizing previously described upregulated markers of type 1 interferon activation in dermatomyositis, we evaluated whether type 1 interferon-inducible gene overexpression represented a significant hallmark of iRMD [49]. In iRMD compared to non-disease controls, only one of these genes showed significantly increased expression, RSAD2, with many genes showing significantly decreased expression, including ISG15, IFI6, MX1, MX2, OAS1, IRF9, and IFI35 (Fig. 5A).

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

Given the lack of robust interferon type-1 induction, we next evaluated whether type-2 interferon signaling was activated in iRMD. Previous literature identified increased expression of interferon type-2-induceable genes in dermatomyositis, inclusion body myositis, anti-synthetase syndrome, and more recently in immune checkpoint inhibitor-dermatomyositis and myositis with associated myocarditis [49, 51]. In these disorders, specific markers of IFN2 induction included IFI30, GBP2, GBP1, PSMB8 [49, 51]. We evaluated expression of these genes in iRMD compared to non-disease controls and found that only GBP2 showed significantly increased expression, while IFI 30, GBP1, and PSMB8 were unchanged, suggesting that iRMD lacks induction of the type-2 interferon pathway as well (Fig. 5B).

The IL-6 pathway is induced in iRMD muscle

Induction of the IL-6 pathway was recently shown in patients with various subtypes of immune checkpoint inhibitor myositis [51]. IL-6 also serves as a myokine with multiple pro- and anti-inflammatory properties [52]. To evaluate whether the IL-6 pathway is induced in iRMD, we initially compiled a list of genes known to be part of the IL-6 mediated signaling pathway by gene ontology analysis (GO: 0070102) [41, 51]. When filtered by genes detected in our data set, 14 genes comprised the list of IL-6 inducible genes (IL-6ST, IL-6R, FER, CTR9, SRC, SPI1, SMAD4, STAT3, CEBPA, YAP1, JAK1, GAB1, JAK2, and ZCCHC11). All of these genes were significantly differentially expressed in iRMD patients’ muscle biopsies compared to non-disease controls except for SMAD4 and ZCCHC11 (p = 0.038 by hypergeometric test), demonstrating significant induction of the IL-6 pathway (Fig. 6). Further, when patient serum was analyzed, four out of six iRMD patients showed increased IL-6 protein levels, with half of patients showing IL-6 levels above the 99th percentile of the normal range (Supplemental Table 6).

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

Given the identification of IL-6 pathway activation through transcriptomic analysis of iRMD muscle as well as the recent finding of cavin-4 antibodies in iRMD, we next sought to compare changes in gene expression in iRMD to an autoimmune inflammatory myopathy. To accomplish this, a previously published transcriptomics dataset of dermatomyositis was reanalyzed along the same pipeline and to the same control group to compare expression level changes in dermatomyositis to iRMD [35]. Genes which were differentially expressed in both iRMD and dermatomyositis were analyzed with GO-Term analysis. Pathways involved in immune response, immune activation, or signaling pathways of immune activity were significantly enriched amongst overlapping genes between iRMD and DM (Supplemental Fig. 4). These findings likely represent overlapping nonspecific pathways of autoimmune myopathies supporting the role of immune activation in iRMD.