Fig. 3: Raising and lessening propionyl-CoA in vitro with medium interventions.
a, Calibration experiment to titrate the amount of exogenous propionate needed produce a rise in propionyl-CoA in the range found in PA hearts. Forty-eight-hour treatment of NRVMs with up to 6 mM sodium propionate. Measurements of acyl-CoAs in NRVM lysates (n = 6 biologically independent samples from six isolations; left axis) were compared to measurements in PA and WT mice (right axis). Mathematical minimisation procedure best-fitted calibration constants to indicate that ∼1 mM propionate produces a rise in propionyl-CoA within the range of PA mice (red dots). Mean ± s.e.m. b, 13C tracing experiment in NRVMs with raised propionyl-CoA (exogenous propionate treatment) and for raised β-alanine (histidine in medium replaced with carnosine; yellow banding). Table shows the four treatment conditions (A/B/C/D). For conditions A/B, Ile and Val were uniformly labeled (13C on all carbons). For conditions C/D, 1 mM labeled propionate (13C at carbon-1) was added to the medium. Condition C/D produced a rise in propionate. n = 7 biologically independent samples per condition from seven isolations. Mean ± s.e.m. */** P < 0.05/<0.01. c, IC–MS identifies Krebs cycle intermediates. MeCit/Cit, 2-methylcitrate/citrate ratio. */** P < 0.05/<0.01. Mean ± s.e.m. d, 13C tracing of Ile/Val carbons (conditions A/B) or propionate carbon-1 (conditions C/D), expressed as percent molar enrichment. n = 7 per condition from seven isolations. Key shows number of 13C-labeled atoms. e, Acyl-CoA measurements expressed either as absolute quantification calibrated against standard curves or as relative AUC (without standards). n = 7 biologically independent samples per condition from seven isolations. */** P < 0.05/<0.01 for effect of propionate treatment. #/## P < 0.05/<0.01 for effect of raising β-alanine. Statistical testing by repeated-measures two-way ANOVA. Mean ± s.e.m. f, Summary of findings. Brown-filled symbols indicate carbons of acetyl-CoA or propionyl-CoA competing for entry into the Krebs cycle as citrate or 2-methylcitrate. Raising β-alanine favors the former. Propionate also enters the Krebs cycle as succinyl-CoA, but this pathway is not normally available in PA due to PCC inactivation. 2M2PE-CoA, trans-2-methyl-pentenoyl-CoA; 2M3HB-CoA, 2-methyl-3-hydroxybutyryl-CoA; BH(I)B-CoA, β-hydroxy(iso)butyryl-CoA; Ile, isoleucine; MM-CoA, methylmalonyl-CoA; Val, valine.
