Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity after chronic hypoxia

Chronic hypoxia (CH) is known to exacerbate both basal and endothelin-1 (ET-1)-induced pulmonary vasoconstriction, a process mediated through the generation of reactive oxygen species (ROS) and the activation of RhoA/Rho kinase (ROCK)-dependent pathways that sensitize myofilaments to calcium. Given that ROCK facilitates actin polymerization and that the actin cytoskeleton plays a crucial role in regulating smooth muscle tone, we hypothesized that actin polymerization might be essential for the heightened basal and ET-1-induced vasoconstriction observed following CH. To explore this hypothesis, we conducted experiments on pressurized pulmonary arteries (fourth and fifth order) from both control and CH rats, where the endothelium had been disrupted. The rats were exposed to 4 weeks of hypoxia at 0.5 atm to induce CH conditions.

We employed two actin polymerization inhibitors—cytochalasin and latrunculin—and observed their effects on both basal and ET-1-induced vasoconstriction. These inhibitors significantly reduced vasoconstriction in CH vessels but had no such effect on control vessels, supporting our hypothesis that actin polymerization is involved in the enhanced vasoconstrictive response in CH conditions. To examine whether CH directly alters the actin profile in these arteries, we measured the ratios of filamentous actin (F-actin) to globular actin (G-actin) by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries, along with actin sedimentation assays on homogenized pulmonary artery lysates. Interestingly, while no differences were observed in actin polymerization between the two groups under baseline conditions, ET-1 stimulation led to increased actin polymerization in the pulmonary arteries from CH rats. This response was notably attenuated by the ROS scavenger tiron, the ROCK inhibitor fasudil, and a small-molecule inhibitor targeting mDia (a key effector of RhoA).

Further analysis through immunoblotting revealed that CH conditions increased both the phosphorylated (inactive) and total levels of cofilin, an actin disassembly factor. However, the ratio of phosphorylated cofilin to total cofilin remained unchanged, suggesting that cofilin’s role in actin dynamics during CH is not altered in the same manner. Taken together, these findings indicate that actin polymerization plays a significant role in the increased basal pulmonary arterial constriction and the enhanced ET-1-induced vasoconstrictor reactivity seen in chronic hypoxia. This effect is mediated through ROS and ROCK signaling, with mDia being an important mediator of ET-1-induced actin polymerization. Importantly, the response appears independent of changes in cofilin phosphorylation status, further indicating the complexity of the mechanisms involved.