WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome

Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by various disabling symptoms including exercise intolerance and is diagnosed in the absence of a specific cause, making its clinical management challenging. A better understanding of the molecular mechanism underlying this apparent bioenergetic deficiency state may reveal insights for developing targeted treatment strategies. We report that overexpression of Wiskott-Aldrich Syndrome Protein Family Member 3 (WASF3), here identified in a 38-y-old woman suffering from long-standing fatigue and exercise intolerance, can disrupt mitochondrial respiratory supercomplex formation and is associated with endoplasmic reticulum (ER) stress. Increased expression of WASF3 in transgenic mice markedly decreased their treadmill running capacity with concomitantly impaired respiratory supercomplex assembly and reduced complex IV levels in skeletal muscle mitochondria. WASF3 induction by ER stress using endotoxin, well known to be associated with fatigue in humans, also decreased skeletal muscle complex IV levels in mice, while decreasing WASF3 levels by pharmacologic inhibition of ER stress improved mitochondrial function in the cells of the patient with chronic fatigue. Expanding on our findings, skeletal muscle biopsy samples obtained from a cohort of patients with ME/CFS showed increased WASF3 protein levels and aberrant ER stress activation. In addition to revealing a potential mechanism for the bioenergetic deficiency in ME/CFS, our study may also provide insights into other disorders associated with fatigue such as rheumatic diseases and long COVID.

Keywords: ER stress; WASF3; fatigue; mitochondria; supercomplex.

Fig. 1.

Mitochondrial dysfunction in a patient with chronic fatigue associates with increased WASF3 expression. (A) Noninvasive assessment of in vivo mitochondrial function in a patient with lifelong fatigue (S1) compared with a control sibling (S2) using ³¹P-MRS. The phosphocreatine (PCr) recovery time constant (T_c) in their tibialis anterior muscle was calculated from PCr levels measured during the recovery phase after foot exercise. The y axis shows PCr levels relative to the preexercise baseline (mean and range of two scans are shown). The ³¹P-MRS test of patient S1 was repeated (2nd test) after control S2 testing was performed. (B) Oxygen consumption rate (OCR) of skin fibroblasts obtained from S1 and S2. Specificity of OCR is demonstrated by using complex I (rotenone, Rot) and complex V (oligomycin, Oligo) inhibitors (n = 5). (C) Immunoblots of two independently cultured fibroblast samples of siblings S1 and S2. MTCO1 and MTCO2 denote mitochondrial cytochrome c oxidase subunits 1 and 2, respectively, of respiratory complex IV (CIV). pS and pT/Y denote phosphorylated serine and threonine/tyrosine, respectively. Levels of WASF3 and MTCO1 were quantified (n = 3). (D) OCR measured in patient S1 fibroblasts after knockdown of WASF3 (n = 7 to 10). Cells were transduced with lentivirus expressing nonspecific (NS) or WASF3 shRNA. Spare respiratory capacities (calculated by subtracting basal OCR from maximal OCR after FCCP treatment) of the cells were also compared as follows: Patient S1 vs. Control S2; NS shRNA vs. WASF3 shRNA transduced S1 cells (n = 14 to 17). (E) Immunoblots of S1 fibroblasts after WASF3 knockdown (also refer to SI Appendix, Fig. S1) (one representative blot of n = 3). Values are mean $\pm$ SE. Statistical difference by unpaired, two-tailed Student’s t test. *P < 0.05.

Fig. 2.

WASF3 overexpression disrupts mitochondrial respiration and decreases exercise capacity in mice. (A) Immunoblots of C2C12 myoblasts stably transfected with empty vector or WASF3 cDNA. Mitochondria (Mito) were purified for immunoblotting MTCO2 in the C2C12 cells (one representative blot of n = 3). (B) Oxygen consumption rate (OCR) of C2C12 cells as described in panel A (n = 13). (C) Immunoblots of WASF3 and other indicated proteins in hindlimb tibialis anterior muscle (SKM) and other tissues of ~10-wk-old WASF3 Tg and wild-type mice (one representative blot of n = 3). (D) Levels of WASF3 and MTCO1/2 from immunoblots shown in panel (C) were quantified and normalized to that in SKM of wild-type mice (n = 3). (E, Left) OCR and extracellular acidification rate (ECAR) of whole soleus muscle from ~10-wk-old mice were measured simultaneously using a Seahorse XF24 Analyzer. Shown are ratios of OCR to ECAR, representing oxidative phosphorylation and glycolysis, respectively (n = 8). Middle panel: lactate levels in tail blood of ~10-wk-old mice (n = 14 to 15). Right panel: glycogen content of gastrocnemius muscles of ~16-wk-old mice (n = 5 to 9). (F) Body weights of 10-wk-old WASF3 Tg and littermate wild-type male mice (n = 7 to 19). (G) Exercise endurance of 10-wk-old WASF3 Tg compared with littermate wild-type mice measured by treadmill testing (n = 7 to 19). (H) Four-paw grip strength of indicated ~13-wk-old mice (n = 7 to 10). Male mice were used for all experiments. Values are mean $\pm$ SE. Statistical difference by unpaired, two-tailed Student’s t test. *P < 0.05; n.s. (nonsignificant).

Fig. 3.

WASF3 localizes to mitochondria and disrupts respiratory supercomplex formation. (A) Immunoblots of the subcellular fractions (10-µg protein per lane) isolated from C2C12 myoblasts expressing control empty (−) or WASF3 cDNA-containing vector (one representative blot of n = 3). Alkaline phosphatase (AP). (B) Mitochondria isolated from the C2C12 cells as described in panel A were solubilized in 1% digitonin, resolved by BN-PAGE, and then immunoblotted with antibodies against WASF3 and CIII Rieske FeS protein (UQCRFS1). The corresponding positions of respiratory complexes CI and CIV were visualized by in-gel enzymatic assays using nitroblue tetrazolium (NBT) and diaminobenzidine (DAB), respectively (one representative blot or in-gel activity assay of n = 3). (C) Mitochondria from wild-type (−) or WASF3 Tg mouse hindlimb gastrocnemius muscle were resolved by BN-PAGE and immunoblotted using the indicated antibodies. In-gel activities of CI and CIV were visualized to confirm the supercomplexes detected by immunoblotting. Immunoblot of CV subunit ATP6 serves as control. Protein MW markers in kDa (one representative blot or in-gel activity assay of n = 3). (D) In-gel activity bands of CIV (DAB-stained, 720 kDa) and CI (NBT-stained, 1,048 kDa) were quantified (n = 3). Values are mean $\pm$ SE. Statistical difference by unpaired, two-tailed Student’s t test.

Fig. 4.

WASF3 interacts with mitochondrial CIII and prevents supercomplex CIII₂+IV formation. (A) Mitochondrial lysates of C2C12 myoblasts with or without WASF3 cDNA expression were incubated with control IgG or WASF3 antibody, resolved by BN-PAGE, and immunoblotted with CIII Rieske FeS protein antibody. The upper band at ~1,048 kDa serves as an internal control for the decreased SC III₂+IV band. CI in-gel activity was also visualized by NBT (one representative blot or in-gel activity assay of n = 3). (B) Transient expression (48 h) of empty-vector control (CTL), full-length WASF3 (amino acid residues 1-499), or C terminus truncated WASF3 (WASF3Δ, amino acid residues 1-332) in C2C12 cells and its effect on mitochondrial OCR (n = 6 to 7, two experimental repeats). (C) Plasmid vector containing HA-tagged biotin ligase (BirA), or the BirA-fused to the C terminus of full-length or C terminus truncated (WASF3Δ) WASF3 was transiently transfected into C2C12 myoblasts for 24 h. Total cell lysates were immunoblotted for the HA-tagged BirA or its chimeric proteins and MTCO1. The right panel shows biotinylated proteins bound to streptavidin beads immunoblotted with the indicated antibodies. Actin, known to interact with the C terminus of WASF3, and tubulin serve as specificity controls for the interaction assay. Protein MW markers in kDa. Values are mean $\pm$ SE. Statistical difference by one-way ANOVA.

Fig. 5.

WASF3 and ER stress are increased in skeletal muscle of ME/CFS patients. (A) Immunoblots of skeletal muscle biopsy samples from control healthy volunteers (CTL) and ME/CFS patients using the indicated antibodies. Note the reciprocal relationship between the protein levels of the CIV subunits and WASF3 or PERK. GAPDH and mitochondrial Lon peptidase 1 (LONP1) serve as protein loading controls (n = 10 to 14 from two immunoblots, see SI Appendix, Fig. S11). (B) Quantification of the indicated proteins on the immunoblots in Panel A and SI Appendix, Fig. S11. CTL (n = 10); ME/CFS (n = 14). (C) Correlation analyses between the protein levels of CIV subunits and WASF3 or PERK. MTCO1-WASF3, r = −0.55, P = 0.005; COX17-WASF3, r = −0.59, P = 0.003; MTCO1-PERK, r = −0.49, P = 0.01; COX17-PERK: r = −0.47, P = 0.02 (n = 24). (D) Immunoblot (Left) of human myoblasts treated with ER stress inducers tharpsigarin (Tg, 100 nM) or tunicamycin (Tun, 1 µg/mL) for 24 h. Immunoblot (Right) of hindlimb skeletal muscle (SKM) samples harvested from mice after treatment with another ER stress inducer lipopolysaccharide (LPS, 5 mg/kg body weight i.p.). Values are mean $\pm$ SE. Statistical difference by unpaired, two-tailed Student’s t test. *P < 0.05.

Fig. 6.

Pharmacologic inhibition of ER stress improves mitochondrial respiration. (A) Immunoblots of patient S1 fibroblasts treated with ER stress inhibitors tauroursodeoxycholic acid (TUDCA, 100 µM) or salubrinal (Salub, 10 µM) for the indicated times. (B) Mitochondria purified from sibling S2 control fibroblasts and patient S1 fibroblasts with or without 10 µM salubrinal treatment for 40 h were resolved by BN-PAGE and immunoblotted with CIII Rieske FeS protein antibody. Immunoblot of ATP6 subunit of CV serves as control. (C) Mitochondrial oxygen consumption rate (OCR) of S1 fibroblasts treated with salubrinal for 24 h (n = 6 to 7, three experimental repeats). (D) A proposed model of how WASF3 regulates mitochondrial function. ER stress–induced WASF3 interacts with CIII subunits and its physical presence prevents the assembly of supercomplex III₂+IV, as demonstrated by a similar effect observed with WASF3 antibody. The disruption of mitochondrial supercomplex by WASF3 reduces oxidative phosphorylation, which could result in symptoms such as exercise intolerance and fatigue. Values are mean $\pm$ SE. Statistical difference by unpaired, two-tailed Student’s t test. *P < 0.05.