Altered TRPM7-Dependent Calcium Influx in Natural Killer Cells of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling multisystemic condition. The pathomechanism of ME/CFS remains unestablished; however, impaired natural killer (NK) cell cytotoxicity is a consistent feature of this condition. Calcium (Ca²⁺) is crucial for NK cell effector functions. Growing research recognises Ca²⁺ signalling dysregulation in ME/CFS patients and implicates transient receptor potential ion channel dysfunction. TRPM7 (melastatin) was recently considered in the pathoaetiology of ME/CFS as it participates in several Ca²⁺-dependent processes that are central to NK cell cytotoxicity which may be compromised in ME/CFS. TRPM7-dependent Ca²⁺ influx was assessed in NK cells isolated from n = 9 ME/CFS patients and n = 9 age- and sex-matched healthy controls (HCs) using live cell fluorescent imaging techniques. Slope (p < 0.05) was significantly reduced in ME/CFS patients compared with HCs following TRPM7 activation. Half-time of maximal response (p < 0.05) and amplitude (p < 0.001) were significantly reduced in the HCs compared with the ME/CFS patients following TRPM7 desensitisation. Findings from this investigation suggest that TRPM7-dependent Ca²⁺ influx is reduced with agonism and increased with antagonism in ME/CFS patients relative to the age- and sex-matched HCs. The outcomes reported here potentially reflect TRPM3 dysfunction identified in this condition suggesting that ME/CFS is a TRP ion channelopathy.

Keywords: calcium; chronic fatigue syndrome; myalgic encephalomyelitis; natural killer cell; transient receptor potential melastatin 7.

Figure 1

Ca²⁺ influx parameters from the stimulation of NK cells using 40 µM NTB. (a) Comparison of pooled slope values. (b) Comparison of pooled T1/2 values. (c) Comparison of pooled amplitude values. (d) Example Ca²⁺ influx recording with 40 µM NTB for TRPM7 activation and 1 µM ionomycin for non-specific Ca²⁺ influx in a HC. (e) Example Ca²⁺ influx recording with 40 µM NTB for TRPM7 activation and 1 µM ionomycin for non-specific Ca²⁺ influx in a ME/CFS patient. Data were collected from n = 9 HCs matched to n = 9 ME/CFS patients. The Ca²⁺ influx parameters were calculated from the smoothed (red) graph, which was generated by smoothing the raw (black) recording in OriginLabs displayed in (d,e). Values obtained from NTB stimulation for each cell were normalised to the ionomycin response curve within the same recording. The total number of cells analysed following the exclusion of definitive outliers (number of recordings excluded: HC = 14, ME/CFS = 25) are presented above the corresponding bar graph. All data are presented as mean ± SD with significance as determined using independent Mann–Whitney U tests. * p < 0.05. Abbreviations: calcium (Ca²⁺), healthy control (HC), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), naltriben (NTB), half-time of maximum response (T1/2).

Figure 2

Ca²⁺ influx parameters from the inhibition and desensitisation of NK cells using 6 µM NS8593. (a) Comparison of pooled slope values. (b) Comparison of pooled T1/2 values. (c) Comparison of pooled amplitude values. (d) Example Ca²⁺ influx recording with 6 µM NS8593 for TRPM7 activation, 6 µM NS8593 + 40 µM NTB for TRPM7 desensitisation, followed by 40 µM NTB for TRPM7 activation and 1 µM ionomycin for non-specific Ca²⁺ influx in a HC. (e) Example Ca²⁺ influx recording with 6 µM NS8593 for TRPM7 activation, 6 µM NS8593 + 40 µM NTB for TRPM7 desensitisation, followed by 40 µM NTB for TRPM7 activation and 1 µM ionomycin for non-specific Ca²⁺ influx in a ME/CFS patient. Data were collected from n = 9 HCs matched to n = 9 ME/CFS patients. The Ca²⁺ influx parameters were calculated from the smoothed (red) graph, which was generated by smoothing the raw (black) recording in OriginLabs displayed in (d,e). Values obtained from NS8593 inhibition for each cell were normalised to the ionomycin response curve within the same recording. The total number of cells analysed following the exclusion of definitive outliers (number of recordings excluded: HC = 6, ME/CFS = 7) are presented above the corresponding bar graph. All data are presented as mean ± SD with significance as determined using independent Mann–Whitney U tests. * p < 0.05, *** p < 0.001. Abbreviations: calcium (Ca²⁺), healthy control (HC), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), naltriben (NTB), half-time of maximum response (T1/2).