Figure 7.

Decreasing nitrated RhoA levels reverses mitochondrial fission and reduces cellular glycolysis in pulmonary arterial endothelial cells (PAECs) isolated from lambs with pulmonary hypertension. PAECs were treated or not with the RhoA nitration shielding peptide, NipR1 (100 ng/ml, 24 h), then the mitochondria were labeled with MitoTracker, and effects on mitochondrial fission were analyzed. NipR1 exposure does not change the aspect ratio (AR), branches, and junctions counts in Control PAEC (A) but increases the AR and the number of mitochondrial branches and junctions in Shunt PAEC (B), indicative of increased mitochondrial fusion. Western blot analysis confirmed NipR1 decreased nitrated RhoA levels (ni-RhoA, C) and decreased RhoA activity (D) in Shunt PAEC. Western blot analysis also demonstrated that NipR1 decreased the pS⁶¹⁶Drp1:total Drp1 ratio (E). NipR1 increased the mitochondrial membrane potential (F) and decreased mt-ROS levels (G) in Shunt PAEC. Western blot analysis showed this correlated with decreased HIF1α levels (H). Representative Western blot images are shown. β-Actin was used to normalize protein loading. The total ATP production rate in Shunt PAEC cells was increased by NipR1 treatment (I). This was associated with a decrease in ATP derived from cellular glycolysis (J) and an increased ATP generated by oxidative phosphorylation (OXPHOS, K). The scale bar represents 10 μm. Data are mean ± SE. ∗p < 0.05 versus untreated Shunt PAEC.