Abstract 4335925: Clinical Level of Endothelin-1 Causes ROCK2-Mediated Constriction of Porcine Coronary Arterioles Independent of Protein Kinase C Signaling

Background: Myocardial ischemia in the absence of obstructive coronary artery disease along with diminished coronary flow reserve is diagnostic for coronary microvascular dysfunction (CMD). Studies implicate vasoconstriction by endothelin-1 (ET-1) or through protein kinase C (PKC) activation in CMD development. However, the molecular signaling in coronary microvascular constriction to ET-1 and PKC is incompletely understood. Herein, coronary arteriolar constrictions to a clinical level of ET-1 and PKC activator phorbol 12,13-dibutyrate (PDBu) were directly characterized in vitro. Aim: Determine roles of ET-1 receptors, extracellular Ca 2+ , Rho kinase isoforms, PKC, and myosin light chain phosphatase subunit MYPT1 in coronary arteriolar constrictions to ET-1 and PDBu. Methods: The vasomotor response and signaling pathway of isolated and pressurized (60 cmH 2 O) pig coronary arterioles (<100 µm maximal diameter) to vasoconstrictors were studied using videomicroscopic, pharmacologic, and molecular tools. Results: Coronary arterioles developed basal tone with resting diameters (40-50 µm) about 50% of maximal diameters. ET-1 (0.1 nM) and PDBu (0.1 µM) evoked comparable degree of constriction. Without crossover effects, ROCK inhibitor H-1152 and broad-spectrum PKC inhibitor bisindolylmaleimide XI, abolished constriction to ET-1 and PDBu, respectively. Expression of ROCK2 was greater than ROCK1 in arterioles, and ROCK2 but not ROCK1 siRNA attenuated ET-1-induced constriction. Increased pMYPT1 (Thr850) was detected in ET-1-constricted but not PDBu-constricted arterioles. In the absence of extracellular Ca 2+ , arterioles lost basal tone and did not constrict to ET-1 and PDBu. In the presence of extracellular Ca 2+ , L-type voltage-operated Ca 2+ channel (L-VOCC) blocker nifedipine abolished both basal tone and constriction to PDBu but did not alter constriction to ET-1. ET A receptor antagonist BQ123 but not ET B receptor antagonist BQ788 inhibited constriction to ET-1. Conclusions: Our findings indicate divergent mechanisms for ET-1- and PKC-induced constriction of coronary arterioles. ET-1 binds to arteriolar ET A receptors and triggers extracellular Ca 2+ entry. Subsequent activation of ROCK2 phosphorylates MYPT1 independent of PKC. By contrast, PKC activation linked to L-VOCCs independent of ROCK and MYPT1 elicits constriction. These insights suggest that ROCK2 and L-VOCCs may provide druggable targets in CMD associated with elevated ET-1 and PKC activation, respectively.