Fig. 1
I prostanoid receptor (IP) activation attenuates pressure overload-induced heart failure by enhancing glucose oxidation. a Basal respiration and maximum respiration in IP KO and WT NMCMs in the presence of glucose, pyruvate and glutamine. (n = 5, two-sided t test, *P < 0.05). b The variation of 13C-glucose metabolic flux in Cay10441-treated cardiomyocytes. Fractional enrichments of glucose-6-phosphate (G6P, m + 6), pyruvate (m + 3), lactate (m + 3), and fructose-6-phosphate (F6P, m + 6), citrate (m + 1-m + 6), alpha ketoglutarate (m + 1-m + 5), succinate (m + 1-m + 4), Fumarate (m + 1-m + 4) and malate(m + 1-m + 4) as shown. Red up-arrow indicates upregulation in Cay10441-treated cardiomyocytes; green down-arrows indicate downregulation in Cay10441-treated cardiomyocytes. c Effect of Cay0441 treatment on PDH activity in Ang II-stimulated HL-1 cells (n = 5, two-sided t test, *P < 0.05). d Western blot analyses of the effect of Cay10441 on protein expression levels of PDHA1, PDHA1,p293S DLAT, and DLD in Ang II-stimulated HL-1 cells. e Western blot analyses of the effect of Cay10441 treatment on acetylation of PDHA1 in Ang II-stimulated HL-1 cells. f Effect of acetyl-deficient K → R mutant of PDHA1 at 321 lysine (K321R) on PDHA1 activity in Cay10441- and Ang II-stimulated HL-1 cells. The PDH activity in untreated HL-1 cells was normalized. (n = 6, one-way ANOVA, Tukey multiple comparisons test was used to compare the mean of each group, *P < 0.05). g Effect of acetyl-deficient K → R mutant of PDHA1 at 321 lysine (K321R) on mitochondrial Acetyl-CoA level in Cay10441- and Ang II-stimulated HL-1 cells. (n = 9, two-way ANOVA, Tukey multiple comparisons test was used to compare the mean of each group, *P < 0.05). h Western blot results of the effect of ACAT1 silencing on PDHA1 acetylation in Cay10441-treated HL-1 cells. i Effect of ACAT1 silencing on PDH activity in Cay10441-treated HL-1 cells (n = 6, two-way ANOVA, Tukey multiple comparisons test was used to compare the mean of each group, *P < 0.05). j Western blot analysis of the effect of PKA inhibitor H89 on Cicaprost-induced ACAT1 phosphorylation in Ang II-stimulated HL-1 cells. k Western blot analysis of the effect of PKA inhibitor H89 on Cicaprost-induced suppression of PDHA1 acetylation in Ang II-stimulated HL-1 cells. l MS/MS spectra showing PKA-mediated phosphorylation of ACAT1 Ser54 (top) and Ser69 (upper). The in vitro PKA kinase assay was performed using synthetic peptides ATRTPIGSFLGSLSLLPATK or SLLPTAKLGSIAIQGAIEKA, followed by LC-MS/MS analysis. m Western blot analysis of the effect of the ACAT1 S54/S69A double mutation on forskolin-induced ACAT1 phosphorylation in Ang II-stimulated HL-1 cells. n Western blot analysis of the effect of the ACAT1 S54/S69A double mutation on PDHA1 acetylation in forskolin and Ang II-stimulated HL-1 cells. o Structural basis of ACAT1 inactivation by Ser54 and Ser69 phosphorylation. The left panel shows the ___location of Ser54 and Ser69 (red spheres) in the tetrameric ACAT1 holoenzyme (PDB 2IBW). The four subunits of the enzyme are represented by different colors. The right panels show structural elements surrounding Ser54 and Ser69. Dashed lines indicate potential hydrogen bonds. p Western blot analysis of the effect of the ACAT1 S54/69A mutation on Cicaprost-induced ACAT1 phosphorylation in Ang II-stimulated HL-1 cells. q Western blot analysis of the effect of the ACAT1 S54/69A mutation on PDHA1 acetylation in Cicaprost- and Ang II-stimulated HL-1 cells. r Ejection fraction (EF) of aortic constricted-CMIP−/− and LC mice (n = 10–14, two-way ANOVA, Tukey multiple comparisons test was used to compare the mean of each group, *P < 0.05). s PDH activity in heart tissues from aortic constricted-CMIP−/− mice (n = 10, two-way ANOVA, Tukey multiple comparisons test was used to compare the mean of each group, *P < 0.05). t Mitochondrial Acetyl-CoA level in heart tissues from aortic constricted-CMIP−/− and LC mice (n = 9–10, two-way ANOVA, Tukey multiple comparisons test was used to compare the mean of each group, *P < 0.05). u ACAT1 phosphorylation and PDHA1 acetylation levels in heart tissues from aortic constricted-CMIP−/− mice. v Ejection fraction (EF) of NS304-treated AAC mice (n = 11, two-sided t test, *P < 0.05). w Effect of NS304 on PDH activity of heart tissues from AAC mice after NS304 treatment (n = 6, two-sided t test, *P < 0.05). x Mitochondrial acetyl-CoA level in heart tissues from NS304-treated AAC mice (n = 6 per group, two-sided t test, *P < 0.05). y ACAT1 phosphorylation and PDHA1 acetylation levels in heart tissues from NS304-treated mice. z Mechanistic diagram for IP receptor-mediated glucose oxidation in cardiomyocytes by ACAT1 S54/69 phosphorylation. Data are shown as mean ± SEM
