Zacopride

What is the central question of this study?What is the mechanism of cardiac inflammation induced by α1‐adrenoceptor stimulation by NLRP3 inflammasome activation?What is the main finding and its importance?In the mechanism of cardiac inflammation induced by α1‐adrenoceptor overactivation, Kir2.1 exerts cardioprotective and anti‐inflammatory effects by inhibiting the activation of the NLRP3 inflammasome.

Overstimulation of sympathetic nerves in cardiovascular diseases can lead to impaired cardiomyocyte function and potential heart failure, which activates not only the β‐adrenoceptors but also the α1‐adrenoceptors (α1‐AR). A previous report indicated that NLRP3 inflammasome activation is involved in cardiac inflammation induced by the α1‐AR agonist phenylephrine (PE), but the mechanism is still unknown. Here, we aimed to study whether Kir2.1 is involved in cardiac inflammation caused by PE. The results from in vitro experiments showed that PE upregulated the expression levels of NLRP3, caspase‐1, interleukin (IL)‐18 and IL‐1β and downregulated the expression level of Kir2.1 in H9C2 cells. The Kir2.1 agonist zacopride downregulated the expression of NLRP3, caspase‐1, IL‐1β and IL‐18, and the Kir2.1 inhibitor ML133 upregulated their expression. To further explore the mechanism, we found that zacopride downregulated the protein expression level of p‐p65 and that ML133 upregulated it. Moreover, the nuclear factor‐κB (NF‐κB) signalling pathway inhibitor curcumenol reversed the expression of NLRP3 inflammasomes caused by PE in H9C2 cells. In in vivo experiments, the protein expression level of Kir2.1 in the PE group was significantly decreased, and the activation of Kir2.1 by zacopride reduced cardiac inflammation. In short, Kir2.1 is involved in α1‐AR overactivation, which induces cardiac inflammation, through the NF‐κB signalling pathway, and activating Kir2.1 can downregulate NLRP3 inflammation and exert cardioprotective effects induced by zacopride.

Inward rectifier potassium channels (I_K1, Kir) are known to play critical roles in arrhythmogenesis. Thus, how I_K1 agonist affects reperfusion arrhythmias needs to be clarified, and its underlying mechanisms should be determined. Reperfusion arrhythmias were modeled by coronary ligation (ischemia, 15 minutes) and release (reperfusion, 15 minutes). Zacopride (1.5-50 μg/kg in vivo, or 0.1-10 μmol/Lex vivo) was applied in the settings of pretreatment (3 minutes before coronary ligation) and posttreatment (5 minutes after coronary ligation). Hypoxia (45 minutes) /reoxygenation (30 minutes) model was established in cultured H9c2 (2-1) cardiomyocytes. Zacopride or KN93 was applied before hypoxia (pretreatment). In the setting of pre- or posttreatment, zacopride at 15 μg/kg in vivo or 1 μmol/Lin vitro exhibited superlative protections on reperfusion arrhythmias or intracellular calcium overload. Western blot data from ex vivo hearts or H9c2 (2-1) cardiomyocytes showed that I/R (H/R) induced the inhibition of Kir2.1 (the dominant subunit of I_K1 channel in ventricle), phosphorylation and oxidation of CaMKII, downregulation of SERCA2, phosphorylation of phospholamban (at Thr17), and activation of caspase-3. Zacopride treatment (1 μmol/L) was noted to strikingly restore the expression of Kir2.1 and SERCA2 and decrease the activity of CaMKII, phospholamban, and caspase-3. These effects were largely eliminated by co-application of I_K1 blocker BaCl₂. CaMKII inhibitor KN93 attenuated calcium overload and p-PLB (Thr17) in an I_K1-independent manner. I_K1-depedent inhibition of CaMKII activity is found to be a key cardiac salvage signaling under Ca²⁺ dyshomeostasis and reactive oxygen species (ROS) stress. I_K1 might be a novel target for pharmacological conditioning of reperfusion arrhythmia, especially for the application after unpredictable ischemia.