Human induced pluripotent stem cell-derived myotubes to model inclusion body myositis

Abstract

Inclusion body myositis (IBM) is an inflammatory myopathy that displays proximal and distal muscle weakness. At the histopathological level, the muscles of IBM patients show inflammatory infiltrates, rimmed vacuoles and mitochondrial changes. The etiology of IBM remains unknown, and there is a lack of validated disease models, biomarkers and effective treatments. To contribute to unveil disease underpins we developed a cell model based on myotubes derived from induced pluripotent stem cells (iPSC-myotubes) from IBM patients and compared the molecular phenotype vs. age and sex-paired controls (n = 3 IBM and 4 CTL). We evaluated protein histological findings and the gene expression profile by mRNA-seq, alongside functional analysis of inflammation, degeneration and mitochondrial function. Briefly, IBM iPSC-myotubes replicated relevant muscle histopathology features of IBM, including aberrant expression of HLA, TDP-43 and COX markers. mRNA seq analysis identified 1007 differentially expressed genes (DEGs) (p-value adj < 0.01; 789 upregulated and 218 downregulated), associated with myopathy, muscle structure and developmental changes. Among these, 1 DEG was related to inflammation, 28 to autophagy and 28 to mitochondria. At the functional level, inflammation was similar between the IBM and CTL groups under basal conditions (mean cytokine expression in IBM 4.6 ± 1.4 vs. 6.7 ± 3.4 in CTL), but increased in IBM iPSC-myotubes after lipopolysaccharide treatment (72.5 ± 21.8 in IBM vs. 13.0 ± 6.7 in CTL). Additionally, autophagy was disturbed, with 40.14% reduction in autophagy mediators. Mitochondrial dysfunction was strongly manifested, showing a conserved respiratory profile and antioxidant capacity, but a 56.33% lower cytochrome c oxidase/citrate synthase ratio and a 66.59% increase in lactate secretion. Overall, these findings support patient-derived iPSC-myotubes as a relevant model for IBM, reflecting the main muscle hallmarks, including inflammation, autophagy dysfunction and mitochondrial alterations at transcriptomic, protein and functional levels.

Keywords: Autophagy; Inclusion body myositis (IBM); Inflammation; Mitochondria; Myotubes; iPSC.

Fig. 1

Flowchart: Obtaining iPSCs from fibroblasts and differentiation into muscle cells. First, a skin biopsy is taken from IBM patients and CTLs and fibroblasts grow. Fibroblasts are reprogrammed into iPSCs, which are characterized to validate their pluripotency. Afterwards, iPSCs are differentiated into muscle cells, from myogenic progenitors to myoblasts and then to myotubes. iPSC-derived myotubes are phenotyped by RNA-seq, protein and functional analysis (inflammation, autophagy and mitochondrial function) to examine if they are an appropriate disease model for IBM. Abbreviations: iPSCs: induced pluripotent stem cells; IBM: inclusion body myositis; CTL: healthy individuals. This figure was created with Biorender.com

Fig. 2

Characterization of 2 IBM and 2 CTL iPSC lines. A Phase contrast and alkaline phosphatase images. Scale bar: 200 µM. B Immunodetection of pluripotency markers OCT4 and NANOG. Scale bar: 200 mm. C Expression of the three germ layer markers by immunofluorescence analysis of in vitro embryoid body differentiation cell cultures using specific antibodies against the endodermal marker SOX17, the mesodermal marker SMA, and the ectodermal marker TUJ1. Nuclei were stained with DAPI. Scale bar: 100 mm. D Expression levels of pluripotency genes (POU5F1, SOX2, NANOG) and early lineage markers (SOX17, T, TUJ1) determined by real-time PCR in the iPSC lines and in those same lines differentiated into embryoid bodies. These tests leaded to the validation of the pluripotency in the iPSC lines. The characterization of the other IBM and CTL lines is represented in Supplementary Fig. 1

Fig. 3

Characterization of iPSC-derived myotubes in IBM vs. CTL lines. A Expression levels of muscle marker genes (MYOG, MYOD, DESMIN, and MHCI) determined by real-time PCR in the iPSC lines and in those same lines differentiated to myotubes. B Immunodetection of sarcomeric α-ACTININ. Nuclei were stained with DAPI. Scale bar: 10 μm. The positive detection of muscle markers in iPSC-myotubes confirmed iPSC lines differentiated into myotubes. IBM and CTL iPSC-myotubes displayed similar morphology and cell growth rate

Fig. 4

RNA-seq in iPSC-derived myotubes from IBM vs. CTL lines. A Principal component analysis (PCA). B Heatmap of the top 50 differentially expressed genes (DEGs) in IBM vs. CTLs. C Top 10 HPO terms. D Chord diagram of the targeted RNA-seq analysis. Each pathway is represented by a different color. The RNA seq analysis revealed myopathic, autophagy and mitochondrial DEGs in IBM vs. CTL iPSC-myotubes

Fig. 5

Protein expression of IBM muscle pathological features in IBM iPSC-myotubes. A HLA and COX IV. B TDP-43 and COX II. All inflammatory (HLA), autophagic (TDP-43) and mitochondrial markers (COX IV and COX II) were normalized by SYPRO total protein content. Abnormal levels of HLA, TDP-43, COX IV and COX II markers in IBM iPSC-myotubes matched the features in the muscle histopathology of IBM patients, suggestive of higher inflammation and dysfunctional autophagic and mitochondrial performance

Fig. 6

Secreted cytokines in the supernatant of IBM vs. CTL differentiated myotubes. A Fold change of the cytokine concentration in IBM vs. CTL in basal conditions, normalized by cell count. B Fold change of the cytokine concentration in treated vs. basal IBM vs. CTLs, normalized by cell count. The secretion of cytokines was overall preserved in basal conditions (A) but increased in IBM patients vs. CTLs after LPS exposure (B)

Fig. 7

Autophagy profile in IBM vs. CTL iPSC-derived myotubes. A Immunostaining of LC3 autophagosome marker at basal conditions vs. 6 h-bafilomycin A1-treated myotubes. Arrows pointed to the accumulated autophagosomes (green dots). Scale: 10 µM. B Autophagy protein concentration in basal vs. bafilomycin A1 treatment in IBM vs. CTLs. Autophagy was less active in IBM iPSC-myotubes than in CTLs, with less production of autophagosomes and autophagy mediators

Fig. 8

Mitochondrial profile in IBM vs. CTL iPSC-derived myotubes. A Mitochondrial respiration is represented by kinetics, basal respiration, ATP production, H + leak, coupling efficiency, maximal respiration, and spare respiratory capacity in IBM vs. CTLs. B Total antioxidant capacity (TAC) and lactate secretion in IBM vs. CTLs. C Citrate synthase (CS) activity, cytochrome c oxidase activity (COX) and COX/CS ratio. Mitochondrial respiration and TAC were conserved between cohorts, but lactate and COX/CS were reduced in IBM iPSC-myotubes displaying mitochondrial dysfunction