Mechanisms of Vascular Acetylcholine Reactivity in Cardiac Hypertrophy: The Role of Calcium Channels and Endothelial PI3K/AKT/eNOS Signaling in Rats

Cardiac hypertrophy alters vascular function and promotes myocardial damage via oxidative stress and inflammation, with calcium channels and the PI3K/AKT/eNOS signalling pathway being pivotal in vascular reactivity. Nonetheless, their precise role in acetylcholine (ACh)-mediated vascular responses during hypertrophy remains little elucidated. The study aims to investigate calcium channel functions and endothelial PI3K/AKT/eNOS signalling in modulating vascular reactivity in a rat model of cardiac hypertrophy. Aortic rings from control and ISO-treated rats were mounted in organ baths. Cumulative ACh dose–response curves (DRC) were obtained at baseline and after preincubation with Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) blocker, PI3K inhibitor, AKT inhibitor, Mas receptor antagonist (Mas blocker), and amlodipine (L-type Ca²⁺ blocker). Potency (pD₂), maximal relaxation (Emax), and area under the curve (AUC) were compared. In control vessels, Mas receptor blockade, AKT inhibition, and L-NAME each reduced ACh-induced relaxation—confirming that PI3K/AKT/eNOS-dependent nitric oxide (NO) signalling drives vasodilation. Amlodipine slightly lowered relaxation parameters, suggesting that Ca²⁺ entry supports maximal NO production. After ISO treatment, these effects were reversed: Mas blockade and amlodipine enhanced relaxation, while AKT inhibition increased pD₂ and AUC without reducing Emax. L-NAME caused weaker inhibition, reflecting impaired NO signalling. PI3K inhibition had no effect in either condition, and overall AUC changes were minimal. Isoproterenol disrupts the balance between Ca²-dependent and phosphorylation-dependent activation of eNOS, reducing NO bioavailability and rewiring Mas/AKT signalling. Reducing pathological Ca²⁺ influx and dampening AKT activity reverse endothelial responsiveness; however, Mas signalling shifts from endothelial vasoprotective in physiology to maladaptive in hypertrophy. Consequently, the L-type Ca²⁺ channels, AKT, and the Mas axis appear to be reasonable targets for rebalancing endothelial sensitivity in hypertrophic conditions.