Fig 2: Lack of the P2X7R Impairs Cardiac Function. Purified heart MFs and HMFs (see Materials and Methods) from WT (A) or P2X7R-KO (B) C57BL/6 mice were analyzed for P2X7R expression by Western Blot. Hsp60, TIM23, and mitochondrial calcium uptake protein 2 (MICU2) were used as markers of mitochondrial matrix, inner or outer mitochondrial membrane, respectively. Excised whole hearts from P2X7R WT (average weight g ± SEM = 0.1935 ± 0.005343, n = 6) or P2X7R-KO (average weight g ± SEM = 0.1965 ± 0.01169, n = 6) mice were weighed (C), measured by caliper to assess volume (average volume mm3 ± SEM P2X7R WT = 176.5 ± 21.20, n = 6; average volume mm3 ± SEM P2X7R-KO = 261.3 ± 9.229, n = 6) (D), and analyzed by TEM (E–G). Mitochondrial area (µm2) is expressed as average ± SEM for WT (0.1059 ± 0.002917, n = 289) versus P2X7-KO (0.08657 ± 0.003239, n = 253) (G). In vivo heart indexes were measured with the High-frequency Ultrasound imaging system (see Materials and Methods) shown in (H). Vols (I); Vold (J); SV (K); FS (L); EF (M); CO (N). Heart rate was 399±17 and 375±16 for WT and P2X7R-KO mice, respectively. Data are presented as average ± SEM of n = 16 mice for condition; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Lack of the P2X7R Reduces Physical Fitness and Surface Body Temperature. Physical fitness (A) of C57Bl/6 P2X7 WT and KO mice was investigated in a standard rotarod test by measuring time (s) spent on the wheel, as described in Materials and Methods. Data are presented as average ± SEM for WT (803.3 ± 44.74, n = 6) and P2X7R-KO mice (659.3 ± 34.45, n = 6). For body temperature measurement (B, C), WT and P2X7R-KO mice were placed on a warm surface in a 22°C heated room, and body images were acquired and measured with a Thermacam P25 Infrared Camera. Representative thermal images from one WT (right) and one P2X7R-KO (left) mouse from dorsal or ventral view (B). Each temperature measurement was performed in triplicate in a total of seven mice for condition. Data are presented as average °C ± SEM for P2X7R-KO (33.37 ± 0.2607, n = 7) and WT (34.4 ± 0.226, n = 7) mice. P-values are calculated with the two-tailed unpaired Student’s t-test. *P < 0.05.

Fig 4: Lack of the P2X7R Impairs Mitochondrial Potential, Oxygen Consumption, Calcium Level and Causes Intra-Mitochondrial NADH Accumulation. Cells, plated on 12-mm glass coverlips (A–D), were loaded with TMRM (20 nM) at 37°C for 20 min in KRB buffer. Fluorescence was measured with a Zeiss LS510 confocal microscope. Images were then analyzed with ImageJ software. Mitochondrial membrane potential (?m) was expressed as the ratio between TMRM fluorescence (in arbitrary units [AUs]) before and after FCCP addition. Exact fluorescence AU values: N13 WT (average ± SEM = 838.5 ± 55.96, n = 13) or N13 R (average ± SEM = 527.9 ± 66.60, n = 14) (A); MEFs WT (average ± SEM = 886.6 ± 59.86; n = 31) or MEFs P2X7-KO (average ± SEM = 671.5 ± 35.49, n = 43) (B); primary WT microglia (average ± SEM = 551.1 ± 81.53, n = 20) or primary P2X7-KO microglia (average ± SEM = 134.0 ± 13.69, n = 8) (C); HEK293-P2X7 (average ± SEM = 3769 ± 191.0, n =47) or HEK293 (average ± SEM = 2296 ± 187.7, n = 45) (D). Alternatively, cells were plated in XF96 96-well cell culture plate (E–H) and analyzed for oxygen consumption in a Seahorse apparatus. Exact averages ± SEM for Panels E–H are shown in Supplementary data, Table S1. OCR was normalized on cell content. Data are presented as average ± SEM of n = 3 for Panels E–G and n = 7 for Panel H. HEK293-P2X7R (average arbitrary fluorescence units ± SEM = 2717 ± 0.069, n = 57) or HEK293 (average arbitrary fluorescence units ± SEM = 0.95 ± 0.039, n = 58) cells were transfected with the mitochondrial-selective FRET-based, GCaMP fluorescent Ca2+ indicator (I), or analyzed for NADH autofluorescence with a fluorescence microscope (average arbitrary fluorescence units ± SEM, HEK293-P2X7R = 23.33 ± 2.07, n = 25; HEK293 = 31.31 ± 2.92, n = 31) (J). MEFs WT (average arbitrary fluorescence units ± SEM = 0.015 ± 0.002, n = 11) or MEFs P2X7R-KO (average arbitrary fluorescence units ± SEM = 0.026 ± 0.002, n = 23) (K), and N13 WT (average arbitrary fluorescence units ± SEM = 0.19 ± 0.01, n = 24) or N13 R (average arbitrary fluorescence units ± SEM = 0.23 ± 0.01, n = 37) (L), were also analyzed for NADH fluorescence. Fluorescence emission was acquired with an IX-81 Olympus automated epifluorescence microscope as described in Materials and Methods. Intracellular content (pmol/µL) of NADH for N13 WT (average ± SEM = 13.18 ± 0.25, n = 3) and N13 R (average ± SEM = 25.80 ± 0.39, n = 3) (i) was also measured with a NAD/NADH Assay Kit, as described in Methods. P-values are calculated with the two-tailed unpaired Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: The P2X7R Localizes to the Mitochondria. Western Blot analysis of lysates (10 µg of protein/lane) from whole cells (H), a crude MF or a HMF. Cytosolic fraction (C) was loaded as control. Cell fractionation was performed as described in Materials and Methods. IP3R3, ß-tubulin, and mitochondrial calcium uniporter (MCU) were used as markers of ER, cytosol, and mitochondria, respectively.