Calcium Sensitivity, but Not Its Level, Determines Hypoxic Constriction of Porcine Coronary Arteries

Abstract

AIM: Acute hypoxia can induce transient contraction of coronary arteries, leading to myocardial ischemia and even cardiac dysfunction. However, the precise regulatory mechanisms remain unclear. In this study, we applied various interventions to isolated porcine coronary arteries by modulating cytoplasmic calcium concentrations mediated by calcium channels on the plasma membrane and sarcoplasmic reticulum, aiming to investigate the relationship between hypoxic contraction and intracellular calcium levels as well as calcium sensitization effects. METHODS: Isolated rings of the porcine left anterior descending coronary artery served as the experimental model. Based on distinct intervention targets, four core experimental groups were established. The specific grouping, sample size (n) for each group, and treatments were as follows: (1) nitric oxide (NO)-soluble guanylyl cyclase (sGC) pathway and energy metabolism intervention groups including control (n=5), nitric oxide synthase inhibitor nitro-L-arginine (NLA, 10^-4 mol/L; n=4 to 5), soluble guanylyl cyclase (sGC) antagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 3×10^-5 mol/L; n=5), endothelium-denuded (n=5), normal glucose incubation (n=3), and glucose-free incubation (n=3) groups; (2) calcium source intervention groups including normal calcium control (n=4), calcium-free incubation with 5×10^-3 mol/L ethylene glycol tetraacetic acid (EGTA; n=4), L-type calcium channel antagonist nifedipine (10^-6 mol/L; n=5 to 7), non-selective cation channel inhibitor NiCl2 (5×10^-5 mol/L; n=5 to 7), sarcoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (2×10^-6 mol/L; n=5 to 7), and inositol trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB, 10^-4 mol/L; n=5 to 7) groups; (3) myosin light chain kinase (MLCK) pathway intervention groups including control (n=4) and MLCK-specific inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7, 10^-5 mol/L; n=6) groups; (4) myosin light chain phosphatase (MLCP) activity and endothelium-dependence intervention groups including endothelium-intact (n=4) and mechanically endothelium-denuded (n=6) groups. All arterial rings were pre-contracted with either U46619 (3×10^-7 mol/L) or KCl (6×10^-2 mol/L) and then subjected to 10 minutes of hypoxia (95% N2+5% CO2). Changes in vascular tension were continuously monitored and recorded using a multi-channel physiological signal acquisition system. Furthermore, combined with Western blotting, the phosphorylation level of myosin light chain (MLC) and the activity of MLCP were determined; the phosphorylation levels of MLC and MLCP were also compared between endothelium-intact and endothelium-denuded coronary arteries under hypoxic conditions. RESULTS: (1) Hypoxic constriction of porcine coronary arteries is dependent on the activation of endothelium-derived nitric oxide (NO) and sGC in vascular smooth muscle cells. (2) Hypoxic contraction in porcine coronary arteries is independent of extracellular Ca²⁺ influx. (3) Hypoxic contraction in porcine coronary arteries does not rely on intracellular Ca²⁺ release from the sarcoplasmic reticulum. (4) Hypoxic contraction in porcine coronary arteries leads to inhibition of myosin light chain phosphatase activity, suggesting increased calcium sensitization in coronary artery smooth muscle. CONCLUSION: The mechanism underlying acute hypoxia-induced vasoconstriction exhibits distinct characteristics: it does not rely on extracellular calcium influx mediated by plasma membrane calcium channels, nor is it associated with intracellular calcium mobilization from sarcoplasmic reticulum stores. Instead, it is mediated by a significant enhancement in calcium sensitivity regulated by myosin light chain phosphatase, a process referred to as calcium sensitization.

1. Introduction

Hypoxic constriction of blood vessels was first discovered in 1976 in the context of acute hypoxia eliciting rapid contraction responses of isolated veins in dogs [1]. Subsequently, this phenomenon was reported in a number of blood vessel types including rat and mouse aorta, mesenteric and pulmonary arteries [2], canine femoral artery and coronary artery [3-7], and porcine coronary artery [8-10]. It was found more potently in constricted vessels [5]. Despite the fact that this phenomenon has been studied extensively, the detailed mechanisms that mediate this hypoxic response remain elusive.

Hypoxic constriction of blood vessels is independent of types of vasoconstrictors; norepinephrine [3-4], phenylephrine [2], prostaglandin F2α (PGF2α) [5-6] and the TP receptor agonist U-46619 [9-10] all induce substantial responses. Interestingly, endothelial released nitric oxide (NO) used to be considered as a vasodilator is indispensable for this hypoxic response. The removal of endothelium substantially blocks hypoxic constriction while supplementation with NO restores this reaction [7-8, 10]. The hypoxic response also relies on the activation of soluble guanylyl cyclase (sGC), the downstream signal of NO [10-11]. Normally, NO activates sGC to induce vasodilation [11]. The activation of sGC entails two independent pathways to perform its functions; one is to reduce intracellular Ca²⁺ ([Ca²⁺]i) levels and the other is to lower the sensitivity towards Ca²⁺ at a given concentration [12-14]. The level of [Ca²⁺]i regulates myosin light chain kinase (MLCK) activity to control phosphorylation levels of myosin light chain (MLC) and vessel tension. By contrast, MLC activity and vessel tension determined by Ca²⁺ sensitivity are achieved through the regulation of myosin light chain phosphatase (MLCP) by dephosphorylating MLC [15-19]. Nevertheless, it remains unclear as to whether these two mechanisms are involved in hypoxic constriction.