Fig. 6. Disrupted propionyl-CoA handling evokes changes in protein phosphorylation.
a, Representative cGMP FRET trace in NRVMs treated for 48 h with 3 mM propionate (PRO) to induce Pde9a expression, showing the effect of PDE9A-specific inhibitor (PF-9613; 100 µM), IBMX (100 µM) and SNAP/BAY-41 (50 µM and 5 µM, respectively). Double-normalised FRET signal in response to PF-9613 treatment at steady state, showing significant increase in propionate-treated (PRO) myocytes. Bar chart shows quantification of the effect of PF-9613 relative to untreated (CON) NRVMs. Hierarchical analysis of 88–117 myocytes from n = 4 isolations. Unpaired two-tailed t-test. ****P < 0.0001. Mean ± s.e.m. b, cGMP assay in murine cardiac lysates, normalized to protein content (n = 8 biologically independent samples per sex and genotype). Violin plots show distribution of measurements as a proxy of the scope for cGMP signaling. F-test was performed to compare variance in WT versus PA hearts (*P < 0.05). c, Transmembrane H+ fluxes generated by Na+/H+ exchange activity in myocytes isolated from mouse hearts. Female PA myocytes have higher NHE1 activity, which is consistent with a higher engagement of cGMP signaling triggered by natriuretic peptide signaling. Two-way ANOVA analysis of data from 20–35 myocytes from six isolations per category. Mean ± s.e.m. d, Phosphoproteomics of lysates prepared from female PA and WT hearts (n = 6 biologically independent samples per sex and genotype). Differentially abundant peptides (Padj < 0.05), color-coded by type of protein. Shape of symbol indicates the most likely kinase (phosphosite functional score >3) for the given peptide substrate. Two-sample t-test, corrected for multiple comparisons. F, female; M, male; NS, not significant.