An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an enigmatic condition characterized by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”), and by fatigue whose severity and associated requirement for rest are excessive and disproportionate to the fatigue-inducing activity. There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolizing patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by a reduction of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signaling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters, and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in the resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

Cell-Based Blood Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

“…As a consequence, the gender proportions in the total cohort also differed significantly (p = 0.002) between the patient and control groups used for the lymphocyte death rates. Nevertheless, this additional cohort allowed us to verify our previous finding [32], that the lymphocyte death rates in culture were not age-or gender-dependent, and to confirm published reports that lymphocyte viability is stable for long periods in frozen storage [33,43].…”

“…We reported, in an accompanying paper, that after recovery from frozen storage, PBMCs from ME/CFS patients die markedly faster in culture than those from healthy controls [32]. A time course revealed that the percentage of dead cells, and the difference between patients and controls, increased dramatically from 24 to 72 h of culture.…”

“…For most of the work reported here, we used the same age-and gender-matched participant groups as reported in the accompanying paper [32]. This cohort included 51 ME/CFS patients (86% female, median age 51, age range 26-71) and 22 healthy controls (68% female, median age 43, age range 21-67).…”

“…This method has been described in an accompanying paper [32]. Briefly, lymphocytes were isolated by Ficoll-Paque density centrifugation and counted.…”

“…It has long been suspected that mitochondrial dysfunction might play a role in the cytopathology, but the small number of direct investigations of this had produced confusing, contradictory results. We recently reported both specific mitochondrial dysfunction and cellular signalling dysregulation in ME/CFS patient blood-derived lymphoblasts, as well as an associated reduction in the viability in culture of ex vivo, stored PBMCs from patients versus healthy controls [32]. We now examine the discriminatory utility of these differences as components of a potential, blood-based diagnostic test.…”