Extended Data Fig. 3: Both non-canonical as well as canonical sphingolipids are upregulated upon ischaemia–reperfusion in mouse hearts overexpressing wild-type or mutant (C133W) SPTLC1 constructs.

a, Mouse hearts expressing wild-type or mutant SPTLC1 constructs for 1 week after adeno-associated viral (AAV) infection were analysed either following sham treatment or ischaemia–reperfusion. b–f, Both non-canonical (b,c) and canonical (d–g) sphingolipids are upregulated upon ischaemia–reperfusion, as can be seen for DoxSa (b), DoxDHCer (c), sphinganine (d), sphingosine (e), ceramide (f) and DHCer (g). While wild-type worms and C133W mutants are mostly similar upon ischaemia–reperfusion, C133W shows a weak increase in DoxDHCer in non-ischaemic sham (c) and lower (dihydro-)ceramide levels than WT after ischaemia–reperfusion (f,g), but surprisingly also significantly lower DoxDHCer levels than wild type after ischaemia–reperfusion (c). This shows that C133W mutation causes an increase of DoxDHCer relative to wild type only in sham conditions in which sufficient serine is available as substrate for the wild-type enzyme. Serine is not used as substrate by C133W that uses alanine instead. In ischaemic conditions, wild-type SPT readily uses alanine as a substrate, even more efficiently than C133W; n = 4 for sham and n = 8 for ischaemia–reperfusion are individual animals, bars are means with s.d. All statistical tests in b–g are unpaired two-sided Student’s t-tests. h, Plot representation of area of necrosis relative to area at risk for animals with AAV expression of wild-type and mutant (C133W) SPTLC1. Under these conditions, very little difference can be seen compared with the vehicle-treated control animals (grey line), but hearts with higher DoxDHCer levels do show a tendency of more necrosis. Vehicle linear regression from Fig. 4h; for AAV, n = 8 individual animals; coefficient of determination (r²) given for each linear regression and P as determined by ANCOVA.

Source data