Tag: mitochondrial function

Altered TRPM3‐Dependent Cytosolic and Mitochondrial Calcium Influx in Natural Killer Cells of Post‐COVID‐19 Condition Patients

Abstract:

According to the World Health Organization (WHO), approximately 6% of COVID-19 cases develop serious long-term sequelae referred to as post-COVID-19 condition (PCC). Immunological disturbances such as persistent activation of immune cells and reduced cytotoxicity by natural killer (NK) cells are reported as key aspects in PCC.

Recently, electrophysiological studies by our group demonstrated impairment of transient receptor potential melastatin 3 (TRPM3) ion channels in NK cells from PCC patients. The significant reduction in TRPM3 channel function and reduced functional activity by NK cells warrants further investigation. Hence, using live cell calcium (Ca2+) imaging ex vivo, we examined the downstream impact of TRPM3 ion channel dysfunction on intracellular and mitochondrial Ca2+ mobilization in NK cells from N = 8 PCC patients, age and sex matched to N = 8 PCC healthy controls (HC).

Our findings provide new evidence of altered passive and TRPM3-mediated Ca2+ influx, significantly impacting cytoplasmic and mitochondrial Ca2+ mobilization in PCC. Passive cytosolic Ca2+ influx amplitude (p < 0.0001) was significantly reduced in PCC; however, passive mitochondrial Ca2+ mobilization (p < 0.0001) was significantly increased. Importantly, cytoplasmic and mitochondrial response rates (slope, p < 0.001) to pregnenolone sulphate stimulation were significantly reduced in PCC.

Consequently, TRPM3-dependent cytosolic (p < 0.001) and mitochondrial (p < 0.0005) Ca2+ mobilization were significantly reduced in PCC compared with HC. Altered ion channel Ca2+ signalling can severely impact both the immune system and bioenergetic processes, potentially leading to broader systemic dysregulations underpinning the pathomechanism of the PCC condition, and warrants further investigations.

Source: Magawa CT, Eaton-Fitch N, Muraki K, Marshall-Gradisnik S. Altered TRPM3-Dependent Cytosolic and Mitochondrial Calcium Influx in Natural Killer Cells of Post-COVID-19 Condition Patients. Eur J Immunol. 2026 Jul;56(7):e70240. doi: 10.1002/eji.70240. PMID: 42484498; PMCID: PMC13390657.  https://pmc.ncbi.nlm.nih.gov/articles/PMC13390657/ (Full text)

Real-Time Measurement of Mitochondrial Function and Glycolysis in Lymphoblastoid Cell Lines

Abstract:

Cells require energy in the form of ATP to function. The two main ways in which cells generate energy in mammalian cells is through glycolysis and oxidative phosphorylation (OXPHOS). Glycolysis takes place in the cytosol and involves the breakdown of glucose molecules, generating ATP and pyruvate, while OXPHOS takes place in the mitochondria and is responsible for producing the majority of ATP for the cell. A dysregulation of these cellular processes has been reported in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In order to understand the mechanisms of the disease, it is imperative to understand how the bioenergetic pathways are altered in ME/CFS.

Here we describe a method for measuring mitochondrial function and glycolytic function using the Agilent Seahorse Extracellular Flux Analyzer. We have optimized these assays for use in actively proliferating lymphoblastoid cell lines that are generated from blood cells. This assay measures oxygen consumption rate and extracellular acidification rates providing an overview of mitochondrial function and efficiency and glycolytic rate and capacity, respectively. These assays are performed on live, intact cells, and enable us to view different components and measurements of energy metabolism through the injection of different compounds that stimulate or inhibit various sections of these pathways. The below method details an optimized glycolysis and mitochondrial assay for 96-well plates with modifications noted for use in 24-well plates.

Source: Katsaros T, Missailidis D, Annesley SJ. Real-Time Measurement of Mitochondrial Function and Glycolysis in Lymphoblastoid Cell Lines. Methods Mol Biol. 2025;2920:173-202. doi: 10.1007/978-1-0716-4498-0_11. PMID: 40372684. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_11