We measured protein levels of P-Cx43 after 30 minutes of LAD ligation to determine whether one of the antiarrhythmic effects of nitrite is caused by preventing Cx43 dephosphorylation. A polyclonal anti-Cx43 antibody detected one band each at 44 to 46 and at 41 kDa on Western blots. The bands with a lower and higher molecular mass represent NP-Cx43 and P-Cx43, respectively. The loading control was β-actin. Figure 4A shows a representative immunoblot using polyclonal anti-Cx43 and anti-actin antibodies. Figure 4B shows the optical density of the Cx43/actin ratio. The amount of P-Cx43 was significantly decreased after 30 minutes of LAD ligation in the control compared with the sham group (P = 0.001). Some dephosphorylation had occurred at 30 minutes after MI in the group given 0.15 mg/kg nitrite, but the amount of P-Cx43 in the 0.15 mg/kg nitrite group was significantly higher than that in the control group at 30 minutes after the onset of ischemia (P = 0.007). There was no difference in the amount of P-Cx43 among the cPTIO (P = 0.92 versus control), allopurinol (P = 0.96 versus control), and control groups.
In this study, moderate-dose nitrite may have attenuated ventricular arrhythmias induced by acute ischemia in rats via an NO-dependent mechanism, and XOR acted as a functional nitrite reductase. This antiarrhythmic effect of nitrite might, in part, be mediated via the modulation of Cx43, which is a principal ventricular gap junction protein.
The administration of 0.15 mg/kg nitrite before 30 minutes of LAD ligation alleviated the severity of ventricular arrhythmias. In contrast, 0.015 and 1.5 mg/kg nitrite did not suppress ischemia-induced ventricular arrhythmias. The curve of the dose-response relationship between nitrite and its antiarrhythmic effects was not linear but rather U-shaped. These results are similar to previous findings of hepatic,20,21 kidney,22 brain,23 and pulmonary24 injuries using comparable dosages. Although several studies20,23,24 have found that various nitrite doses confer beneficial effects in experimental animals and targeted organs or pathology, almost all of them found that nanomolar increases in the circulating nitrite concentration conferred such effects. Increased nitrite concentrations can cause nitrite-derived NO to become an essential source of harmful peroxynitrite, causing oxidant damage.29 Calvert and Lefer41 reported that a supraphysiological concentration of nitrite might cause cellular necrosis and apoptosis. Hence, nitrite exerts antiarrhythmic effects at the prevailing physiologic concentration, whereas higher concentrations might negate these therapeutic effects.
We administered allopurinol with nitrite to determine the effect of XOR on a model of ischemia-induced ventricular arrhythmias and found that allopurinol blocked the antiarrhythmic effects of nitrite. Others have also shown that nitrite-derived protection is partly mediated by XOR acting as a nitrite reductase in rat models of kidney I/R injury22 or ventilator-induced lung injury.24 Webb et al.42 have shown that XOR can pH and concentration dependently catalyze the formation of NO from nitrite and that this NO could protect the heart against I/R injury. Thus, nitrite might have been reduced to NO by XOR during ischemia in the present study, and bioactive NO might have regulated ischemia-induced arrhythmias via Cx43 modulation.
The NO donors nitroglycerin and nitroprusside also have hypotensive effects that prevent their routine application, particularly under critical conditions, even though they have advantages against ischemia-induced ventricular arrhythmias.39,43 In the experimental setting, morphine can exert significant cardiovascular effects,44 including antiarrhythmia, via activation of some opioids receptors.45 Nevertheless, it is rarely applied in the clinical setting because the required doses are extremely high. We found that the antiarrhythmic concentration of nitrite had little effect on hemodynamics. Others also have shown that the doses of nitrite required for cytoprotection have little effect on general blood pressure.24 Therefore, nitrite can be a novel cytoprotective and antiarrhythmic agent not only during the perioperative period but also when patients are in critical care units.30
The present study has some limitations. First, we focused on NO and Cx43 as pivotal mediators of the antiarrhythmic effects of nitrite. However, this was only one of the possible pathways of nitrite protection in this setting. For example, calcium overload after ischemia is also involved in the development of ischemia-induced ventricular arrhythmias.35 Although Pavlovic et al.46 reported that NO activates Na/K-ATPase and thereby limits calcium overload and arrhythmias, further study is required to elucidate detailed interactions among the nitrite-NO pathway, Cx43, and calcium overload. Second, the detailed mechanisms through which nitrite and/or NO attenuate ischemia-induced ventricular arrhythmias and preserve Cx43 from dephosphorylation remain unclear. As one possibility, the antioxidative stress effects of nitrite23,47 might prevent dysregulation of the cardiac connexon connection induced by oxidant stress.48 Third, we did not assess levels of deoxyhemoglobin, which is another possible nitrite reductase in red blood cells. Hemoglobin is an essential factor in physiologically regulating NO bioactivity in mammals, and it might be important in the pathophysiology of I/R injury.49 We cannot exclude the possible contribution of this mechanism to nitrite-derived antiarrhythmic effects. Fourth, it should be considered that there are specific differences in the electrophysiologic properties between rats and human patients. In particular, rat cardiomyocytes are known to have a much briefer action potential. Despite species differences, knowledge obtained from the study of antiarrhythmic agents in mouse, rat, dog, or other laboratory animals, and their underlying electrophysiologic and metabolic mechanisms of action, has been available in the development of strategies for attenuating ischemia-induced ventricular arrhythmias in humans. Finally, although we performed experiments in a blinded and randomized fashion to avoid type I and II errors, these errors might have arisen in our sample size.
In summary, the present findings showed that nitrite might have attenuated the severity of ischemia-induced ventricular arrhythmias via an NO-dependent mechanism. In addition, NO from nitrite via the XOR pathway reduced the extent of Cx43 dephosphorylation during MI. These results suggest that preserving the function of ventricular gap junctions, presumably via NO-mediated Cx43 phosphorylation, is one mechanism through which nitrite can alleviate ventricular arrhythmias induced by ischemia.
We thank Ryo Miyashita, MD, Yusuke Yoshikawa, MD, and Shunsuke Hayashi, Research Technologist, for valuable technical assistance (Department of Anesthesiology, Sapporo Medical University School of Medicine).
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