Fig 2: Correction of mitochondrial dysfunction in ATM-deficient cells by C7. (A) Mitochondrial function analysis was performed using the Seahorse XF24 extracellular flux analyser. 2DG causes a major increase in oxygen consumption rate (OCR) and respiratory capacity in ATM-deficient cells which was reduced to normal levels with C7 co-treatment. (C) Significant reduction in basal OCR by C7 in untreated ATM-deficient cells. (D) Determination of ATP levels at 1 h after exposure to 2DG in the presence and absence of C7. (E) Determination of ATP levels at 5 h after exposure to 2DG in the presence and absence of C7. ATP was assayed using a colorimetric method as described in Methods. All data are plotted as mean +/- SD, n = 3, *p < 0.01, ns for p > 0.05 using two-way ANOVA (A, B and C) and unpaired two-tailed Student's t-test (D, E, and F).

Fig 3: Anaplerotic effect of heptanoate in correcting the mitochondrial defect in ataxia-telangiectasia (A–T) cells. Inhibition of glycolysis with 2DG treatment and the resulting nutrient stress activates ATM and enhances ER-mitochondrial signalling for the transfer of Ca2+ between the two organelles. This signalling is deficient in A-T cells rendering them hypersensitive to this form of metabolic stress. Co-treatment of ATM-deficient cells with heptanoate (C7, a product of triheptanoin metabolism) corrects all aspects of the signalling defect and restores sensitivity to metabolic stress to control levels. The underlying mechanism is that heptanoate is converted to acetyl CoA and propionyl CoA, both of which enter and help boost the TCA cycle and correct the mitochondrial function in ATM-deficient cells. More functional mitochondria lead to enhanced interaction with the ER which increases the transfer of Ca2+ to promote cell survival. These results suggest that heptanoate by increasing access to TCA intermediates improves mitochondrial efficiency and in turn improves ER-mitochondrial interaction and cell bioenergetics. The TCA cycle depicted here is a simpler version involving some of the key metabolites and is not comprehensive.

Fig 4: Correction of hypersensitivity to nutrient stress (glycolysis inhibition) and elevated ROS in A-T cells by heptanoate (C7). (A) Correction of sensitivity to nutrient stress in ATM-deficient cells by C7. HBEC (control), B3 (ATM-deficient cells generated by CRISPR/Cas9). 2DG, 2 deoxy glucose; C7, heptanoate. (B) Correction of sensitivity to nutrient stress by C7 in primary airway epithelial cells. Mean of two controls (C215, C218) and two A-T patient cells (AT009, AT014). (C) Correction of sensitivity in glucose-deprived olfactory neurosphere (ONS)-derived cells by C7. (D) Determination of mitochondrial ROS using MitoSOX labelling. NAC, N-acetylcysteine anti-oxidant. (E) Quantitation of the MitoSOX data was carried out by measuring fluorescence intensity for at least 4 separate determinations of cells in defined areas. (F) Correction of cell survival in control and ATM-deficient cells in response to NAC co-treatment with 2DG. C7 is included as a positive control. Scale bar, 5 µm. All data are plotted as mean +/- SEM. n = 3, *p < 0.05 and ns for p > 0.05 using unpaired two-tailed Student's t-test (E) and two-way ANOVA (A,B,C and F).

Fig 5: Effect of C7 on the IP3R1-GRP75-VDAC1 Ca2+channel between ER and mitochondria. (A) Co-staining using an antibody against Grp75 (green) and TOMM20 (red) was performed on HBEC and B3 cells in the presence or absence of 2DG and C7. An increase in the fluoresce intensity was observed in HBEC cells following exposure to 2DG and not in B3 but the signal was enhanced to control levels in these cells in response to C7. (B) Quantitation of fluorescence intensity for Grp75 shows that C7 significantly increased the intensity of GRP75 labelling in ATM-deficient cells treated with 2DG, Scale bar, 5 µm (C) Proximity ligation assay (PLA) was also carried out here to confirm increased interaction between GRP75 and IP3R1. Unlike that in HBEC cells, an increase in the number of foci in ATM-deficient cells was not observed after 2DG but in the presence of C7, the number of foci was comparable to 2 DG-treated controls. (D) Quantitation of PLA foci per cell. Scale bar, 5 µm. (E) Immunoblotting for Grp75, IP3R1, and VDAC1 in cells following exposure to 2DG and C7 treatment again confirmed ATM activation after nutrient stress but showed no differences in IP3R1-GRP75-VDAC1 protein levels after 2DG treatment or when C7 was added. p97 protein was used as a loading control. All data are plotted as mean +/- SEM. n = 3, *p < 0.05, ns for p > 0.5 using unpaired two-tailed Student's t-test.