iNOS inhibition reduced nitrative stress and Akt S-nitrosylation in HFpEF heart. (A-B) The interactions of iNOS with other proteins were identified using the STRING database. iNOS (Nos2) directly interacted with Akt (A). The confidence score of the interaction of iNOS and other proteins is shown in B. (C) Urinary nitrite/nitrate concentration in mice from different experimental groups. n = 4 per group. (D-E) Representative immunostaining and semi-quantification of S-nitrosylated Akt (SNO-Akt) levels in heart tissue samples. n = 6 per group. (F-H) Representative immunostaining and semi-quantification of S-nitrosylated Akt and iNOS expression levels in neonatal rat cardiomyocytes transfected with plasmids. n = 6 per group for the detection of S-nitrosylated Akt. n = 3 per group for the detection of iNOS expression. (I-J) Representative immunostaining and semi-quantification of S-nitrosylated Akt in AC16 human cardiomyocyte cells transfected with mutated Akt plasmids. The data are shown as mean ± SEM and were analyzed using one-way ANOVA followed by Tukey’s post hoc test (C-E), or Student’s t-tests (F-H) ,or two-way ANOVA with Bonferroni post hoc test (I-J). *, P < 0.05. **, P < 0.01. ***, P < 0.0005. ****, P < 0.0001. ns, no significant.