Fig. 7: 12,13-diHOME attenuates CaMKII activity.

a Phosphorylated CaMKII (pCaMKII) levels are significantly reduced in aged mouse hearts following TNT-Ephx1/2 treatment compared to sham controls (white bar, n = 6; TNT, light blue bar, n = 6). b In WT mice on a high-fat diet, TNT-Ephx1/2 lowers pCaMKII expression, an effect absent in nNOS−/− mice. Bar colors: white (WT Sham; n = 5), gray (WT TNT; n = 5), open red (nNOS⁻/⁻ Sham; n = 5), red (nNOS−/− TNT; n = 5). c In vitro CaMKII activity assay after 5-min incubation with 12,13-diHOME (0–25 μM) and [³²P] (2.5 μCi) in 100 μL reaction buffer (~800 μg CaMKII). Bar colors: white (0 μM), light to dark gray (5–25 μM). d, e MST analysis shows direct binding of 12,13-diHOME to CaMKIIδ with Kd = 19.1 μM (no ATP) and 23.7 μM (with ATP). f Acute 12,13-diHOME treatment (blue bar, n = 8) raises cardiac levels vs. 9,10-diHOME (white bar, n = 8), g and reduces pCaMKII levels. h, i Basal and maximal oxygen consumption rates (OCR) in human fibroblasts. Bar colors: white (PBS, n = 9), gray (12,13-diHOME, n = 10), red (AT2 + OA, n = 12), purple (12,13-diHOME + AT2 + OA, n = 10). *P < 0.001 vs. PBS; ^†P < 0.001 vs. 12,13-diHOME; ^#P < 0.001 vs. AT2 + OA. j usXBP1/sXBP1 ratio in murine fibroblasts after treatment with OA, AT2, and 12,13-diHOME. *P < 0.001 vs. PBS; ^†P < 0.001 vs. 12,13-diHOME; ^#P < 0.001 vs. AT2 + OA. k, l Basal and maximal OCR in human fibroblasts. Bar colors: white (PBS, n = 6), gray (12,13-diHOME, n = 6), red (AT2 + OA, n = 8), blue (12,13-diHOME + AT2 + OA, n = 6). Data are expressed as mean ± SEM. Two-way ANOVA with Tukey’s test c, h–l; unpaired two-tailed t test (a, b, f, g). Imaging data in (f) and (g) were obtained from three independent cohorts.