Reactive Oxygen Species and Mitochondrial K_ATP Channels Mediate Helium-Induced Preconditioning Against Myocardial Infarction In Vivo

Abstract

Objectives

Helium produces preconditioning by activating prosurvival kinases, but the roles of reactive oxygen species (ROS) or mitochondrial K_ATP channels in this process are unknown. We tested the hypothesis that ROS and mitochondrial K_ATP channels mediate helium-induced preconditioning in vivo.

Design

Randomized, prospective study.

Setting

University research laboratory.

Participants

Male New Zealand white rabbits.

Interventions

Rabbits (n=64) were instrumented for measurement of systemic hemodynamics and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion. In separate experimental groups, rabbits (n=7 or 8 per group) were randomly assigned to receive 0.9% saline (control) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture before LAD occlusion with or without the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the mitochondrial K_ATP antagonist 5-hydroxydecanoate (5-HD; 5 mg/kg). Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni's modification of Student's t test.

Measurements and Main Results

Myocardial infarct size was determined using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium significantly (P<0.05) reduced infarct size (23±4% of the area at risk; mean±SD) compared with control (46±3%). NAC, 2-MPG, and 5-HD did not affect irreversible ischemic injury when administered alone (49±5, 45±6, and 45±3%), but these drugs blocked reductions in infarct size produced by helium (45±4, 45±2, and 44±3%).

Conclusions

The results suggest that ROS and mitochondrial K_ATP channels mediate helium-induced preconditioning in vivo.

Reperfusion after coronary artery occlusion produces large quantities of reactive oxygen species (ROS) that contribute substantially to myocardial injury^1,2. In contrast, small amounts of ROS released from mitochondria during a brief episode of ischemia before prolonged coronary occlusion and reperfusion cause preconditioning^3,4. Pretreatment with free radical scavengers abolished cardioprotection produced by ischemic preconditioning⁵. Volatile anesthetics also directly produce small quantities of ROS^6,7 (most likely from mitochondrial electron transport chain complex III⁸) through activation of mitochondrial adenosine triphosphate-regulated potassium (K_ATP) channels^9,10. As observed during ischemic preconditioning, ROS generated by this form of pharmacologic preconditioning mediated reductions in myocardial infarct size produced by the volatile agent^6,7,10. Brief, intermittent administration of helium before prolonged coronary artery occlusion and reperfusion was recently shown to protect myocardium against infarction by activating prosurvival signaling kinases [e.g., phosphotidylinositol-3-kinase (PI3K), extracellular signal-regulated kinases (Erk1/2), endothelial nitric oxide synthase (eNOS)], attenuating the detrimental actions of glycogen synthase kinase, and inhibiting mitochondrial transition in vivo^11–13. Whether oxygen-derived free radical intermediates play a role in helium-induced preconditioning is unknown. Brief exposure to the anesthetic noble gas xenon produced cardioprotection by activating mitochondrial K_ATP channels¹⁴, but the role of mitochondrial K_ATP channels in cardioprotection by nonanesthetic noble gases remains undefined. Thus, the current investigation also tested the hypothesis that activation of mitochondrial K_ATP channels mediates helium-induced preconditioning in rabbits.

Methods

All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Furthermore, all conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals.

Experimental Preparation

Male New Zealand white rabbits weighing between 2.5 and 3.0 kg were anesthetized with intravenous sodium pentobarbital (30 mg/kg) as previously described¹¹. Additional doses of pentobarbital were titrated as required to assure that pedal and palpebral reflexes were absent throughout the experiment. Briefly, a tracheostomy was performed through a midline incision, and each rabbit was ventilated with positive pressure using an air-oxygen mixture (fractional inspired oxygen concentration = 0.30). Arterial blood gas tensions and acid-base status were maintained within a normal physiological range by adjusting the respiratory rate or tidal volume throughout the experiment. A pulse oximeter was placed on the right hind paw of each rabbit for measurement of continuous arterial oxygen saturation. Heparin-filled catheters were positioned in the right carotid artery and the left jugular vein for measurement of arterial blood pressure and fluid or drug administration, respectively. Maintenance fluids (0.9% saline; 15 ml·kg⁻¹·min⁻¹) were continued for the duration of each experiment. A thoracotomy was performed at the left fourth intercostal space, and the heart was suspended in a pericardial cradle. A prominent branch of the left anterior descending coronary artery (LAD) was identified, and a silk ligature was placed around this vessel approximately halfway between the base and the apex for the production of coronary artery occlusion and reperfusion. Intravenous heparin (500 U) was administered immediately before LAD occlusion. Coronary artery occlusion was verified by the presence of epicardial cyanosis and regional dyskinesia in the ischemic zone, and reperfusion was confirmed by observing an epicardial hyperemic response. Hemodynamics were continuously recorded on a polygraph throughout each experiment.

Experimental Protocol

The experimental design is illustrated in Figure 1. Baseline hemodynamics, arterial blood gas tensions, and arterial oxygen saturation were recorded 30 min after instrumentation was completed. All rabbits underwent a 30 min LAD occlusion followed by 3 h of reperfusion. In eight separate groups, rabbits (n= 7 to 8 per group) were randomly assigned (Latin square design) to receive 0.9% saline (control) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of 70% nitrogen-30% oxygen before coronary artery occlusion in the presence or absence of the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the selective mitochondrial K_ATP channel antagonist 5-hydroxydeconate (5-HD; 5 mg/kg). NAC and 2-MPG were dissolved in 0.9% saline and administered as intravenous infusions over 30 and 75 min, respectively. 5-HD was dissolved in 0.9% saline and administered intravenously 30 min before LAD occlusion. The doses of NAC, 2-MPG, and 5-HD used in the current investigation did not produce hemodynamic effects nor affect infarct size when administered alone in an identical rabbit model^7,10. The doses of NAC, 2-MPG, and 5-HD also abolished isoflurane-induced production of ROS as detected by dihydroethidium staining independent of prolonged coronary artery occlusion and reperfusion in rabbits^7,10. Taken together, these previous findings suggest that these particular antagonists are effective and selective in the doses that were used in the current investigation.

Figure 1.

Schematic illustration depicting the experimental protocol used in the current investigation. Abbreviations: NAC = N-acetylcysteine; 2-MPG = N-2-mercaptoproprionyl glycine; 5-HD = 5-hydroxydecanoate

Measurement of Myocardial Infarct Size

Myocardial infarct size was measured as previously described¹⁵. Briefly, the LAD was reoccluded at the completion of each experiment and 3 ml of patent blue dye was injected intravenously. The left ventricular area at risk for infarction was separated from surrounding normal areas (stained blue), and the two regions were incubated at 37°C for 20 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer adjusted to pH 7.4. Infarcted and noninfarcted myocardium within the area at risk were carefully separated and weighed after storage overnight in 10% formaldehyde. Myocardial infarct size was expressed as a percentage of the area at risk. Rabbits that developed intractable ventricular fibrillation and those with an area at risk less than 15% of total left ventricular mass were excluded from subsequent analysis.

Statistical Analysis

Statistical analysis of data within and between groups was performed with multiple analysis of variance (ANOVA) for repeated measures followed by Bonferroni's modification of Student's t test¹⁶. Changes were considered statistically significant when P<0.05. All data are expressed as mean ± standard deviation (SD).

RESULTS

Sixty-four rabbits were instrumented to obtain 59 successful infarct size experiments. Two rabbits were excluded because the left ventricular area at risk was less than 15% of the total left ventricular mass. Three rabbits were excluded because intractable ventricular fibrillation occurred during coronary artery occlusion. Arterial blood gas tensions were maintained within the physiologic range during administration of helium in all groups (data not shown). Arterial oxygen saturation remained at 100% during and after administration of helium with or without other drug interventions (data not shown). Baseline systemic hemodynamics were similar between groups (table 1), but mean arterial pressure was greater in rabbits randomized to receive 2-MPG compared with 0.9% saline. Helium did not affect hemodynamics. Mean arterial pressure and rate-pressure product were greater in rabbits receiving NAC or 2-MPG alone before LAD occlusion compared with those treated with 0.9% saline. Brief coronary artery occlusion and reperfusion significantly (P<0.05) reduced rate-pressure product in all experimental groups. There were no differences in hemodynamics between groups during LAD occlusion and reperfusion. Body weight, left ventricular mass, area at risk weight, and the ratio of area at risk to left ventricular mass were similar between groups (table 2). Left ventricular weight and area at risk weight were less in rabbits randomized to receive NAC compared with 0.9% saline, but the ration of area at risk to left ventricular mass was similar between these groups. Brief, intermittent exposure to 70% helium before LAD occlusion reduced myocardial infarct size (23±4% of the left ventricular area at risk) as compared with control rabbits (46±3%; figure 2). Administration of NAC, 2-MPG, or 5-HD alone did not affect infarct size (49±5, 45±6, and 45±3%, respectively), but these drugs abolished helium-induced cardioprotection (45±4, 44±2, and 44±3%, respectively).

Table 1.

Hemodynamics

				Reperfusion (min)
	Baseline	Intervention	Occlusion	60	120	180
HR (min−1)
CON	236±23	226±21	230±27	216±16*	211±21*	203±20*
He	255±29	238±28	228±14*	220±29*	209±25*	200±27*
5-HD	243±28	236±22	225±27	221±29	208±26*	203±27*
5-HD + He	229±24	219±22	206±19*	196±16*	189±10*	184±9*
NAC	253±28	253±22	244±31	238±33	235±37	232±39
NAC + He	246±28	234±24	223±18	214±22*	207±23*	196±29*
2-MPG	236±24	254±26	261±35	236±11	229±25	237±34
2-MPG + He	226±23	221±20	206±25	205±23	192±19*	186±18*
MAP (mmHg)
CON	71±9	65±9	61±10	62±10	63±11	62±11
He	75±8	80±17	69±7	66±10	68±6	67±16
5-HD	69±2	74±10	61±11	60±12	55±10	54±9
5-HD + He	71±7	71±9	61±11	65±10	64±10	66±7
NAC	85±9	86±6†	76±11	71±11*	72±12*	73±13*
NAC + He	75±8	74±6	60±13	61±8*	62±10*	61±6*
2-MPG	89±8†	84±6†	68±16*	68±15*	73±21	75±19
2-MPG + He	72±4	71±5	55±12*	63±10	63±10	65±9
RPP (min−1•mmHg•10−3)
CON	18.9±2.4	17.0±2.9	16.1±1.8	15.6±2.6*	15.5±3.2*	14.7±3.4*
He	21.6±3.7	21.6±6.1	17.9±2.3	16.8±3.3*	16.4±2.4*	15.3±4.1*
5-HD	19.4±2.3	19.6±2.7	15.6±1.6	15.5±3.2*	13.3±3.1*	13.2±2.7*
5-HD + He	18.5±2.7	17.7±3.1	14.7±3.2*	14.8±3.0*	14.1±2.3*	14.1±1.8*
NAC	24.5±4.5	24.4±3.0†	21.2±4.1	19.4±4.9*	19.7±5.6*	19.8±6.1
NAC + He	21.0±3.5	19.7±3.2	15.3±3.3*	15.1±2.0*	14.9±2.3*	13.8±2.0*
2-MPG	23.6±2.9	24.5±3.5†	20.2±4.2	18.3±3.5*	19.1±5.2*	20.1±4.9
2-MPG + He	18.5±2.0	17.9±2.5	13.6±4.0*	15.0±2.8*	14.0±2.5*	14.0±2.5*

Data are mean±SD

^*Significantly (P<0.05) different from baseline

^†Significantly (P<0.05) different from corresponding control

Abbreviations: HR = heart rate; MAP = mean arterial pressure; RPP = rate pressure product; CON = control; He = helium; 5-HD = 5-hydroxydecanoate; NAC = N-acetylcysteine; 2-MPG = 2-mercaptoproprionyl glycine.

Table 2.

Left Ventricular Area at Risk

	N	Body Weight (g)	LV (g)	AAR (g)	AAR/LV (%)
CON	8	2703±298	3.90±0.46	1.63±0.20	42±5
He	8	2803±243	3.84±0.21	1.26±0.08	33±2
5-HD	7	2789±231	3.49±0.39	1.36±0.34	39±6
5-HD + He	7	2487±72	3.58±0.25	1.34±0.17	37±4
NAC	8	2709±280	3.08±0.48*	1.02±0.33*	33±6
NAC + He	7	2594±82	3.74±0.45	1.35±0.37	36±7
2-MPG	7	2847±170	3.32±0.63	1.18±0.48	35±9
2-MPG + He	7	2726±112	3.58±0.44	1.47±0.25	41±4

Data are mean±SD

^*Significantly (P<0.05) different from corresponding CON value

Abbreviations: LV = left ventricle; AAR = area at risk; CON = control; He = helium; 5-HD = 5-hydroxydecanoate; NAC = N-acetylcysteine; 2-MPG = 2-mercaptoproprionyl glycine.

Figure 2.

Myocardial infarct size depicted as a percentage of left ventricular area at risk in rabbits receiving 0.9% saline (control, CON) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (F_iO₂=0.30) in the presence or absence of pretreatment with the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the selective mitochondrial K_ATP channel antagonist 5-hydroxydeconate (5-HD; 5 mg/kg) before prolonged coronary artery occlusion and reperfusion. Each point represents a single experiment. All data are mean±SD. *Significantly (P<0.05) different from CON

DISCUSSION

The current results confirm our previous findings^11–13 indicating that three cycles of 5 min 70% helium-30% oxygen preconditioning interspersed with 5 min washout periods of an air-oxygen mixture reduce myocardial necrosis after prolonged coronary artery occlusion and reperfusion. The results demonstrate for the first time that pretreatment with NAC or 2-MPG abolishes this helium-induced cardioprotection, suggesting that ROS mediate preconditioning by the nonanesthetic noble gas in vivo. The results further indicate that 5-HD pretreatment blocks reductions in myocardial infarct size produced by brief, intermittent administration of helium, implicating mitochondrial K_ATP channels in this process as well. Weber et al demonstrated that mitochondrial K_ATP channels mediated cardioprotection produced by the anesthetic inert gas xenon¹⁴, and the current results extend this observation to another noble gas that is devoid of anesthetic properties, even under extreme hyperbaric conditions (>200 atm)¹⁷. Collectively, the current and previous¹⁴ data with noble gases also lend support to the hypothesis that ROS are a ubiquitous feature in preconditioning phenomena in conjunction with mitochondrial K_ATP channels. Pretreatment with low concentrations of ROS mimicked the beneficial effects of ischemic preconditioning¹⁸, and small quantities of ROS generated by mitochondria during brief periods of ischemia^3,4 or after exposure to the mitochondrial K_ATP channel agonist diazoxide^19,20 have been shown to play a central role in ischemic and pharmacologic preconditioning, respectively. Evidence that mitochondrial K_ATP activation mediates this ROS-induced cardioprotection was also provided by the observations that diazoxide enhances oxidation of the ROS probe Mitotracker® orange²⁰ (Molecular Probes, Eugene, OR) and increases ROS production as measured using 2’,7’-dichlorofluorescein diacetate in rat ventricular myocytes or isolated hearts²¹. Pretreatment with 5-HD abolished ROS generation produced by the mitochondrial K_ATP channel openers nicorandil and cromakalim in isolated rat hearts²². Similarly, the ROS scavengers NAC, 2-MPG, and Mn(III)tetrakis(4-benzoic acid)porphyrine chloride inhibited the cardioprotective effects of isoflurane in isolated⁶ and intact rabbit hearts^7,10. Scavengers of ROS also abolished the salutary actions of sevoflurane against ischemic damage in isolated guinea pig hearts^23,24. Preconditioning by isoflurane directly increased production of superoxide anion (O₂⁻) as determined using dihydroethidium staining through a mitochondrial K_ATP channel-mediated mechanism^8,10. Thus, it has become abundantly clear that ROS and mitochondrial K_ATP channels play complimentary roles during ischemic, pharmacologic, and anesthetic preconditioning, and the current results with helium suggest that ROS and mitochondrial K_ATP channels are also essential in preconditioning by the nonanesthetic noble gas.

The current results must be interpreted within the constraints of several potential limitations. Based on our previous experiments conducted with volatile anesthetics^7,8,10, it appears highly likely that O₂⁻ generated from the electron transport chain though mitochondrial K_ATP channel opening may also responsible for the observed results with helium. The sulfhydryl-containing glutathione precursor NAC produces antioxidant effects by enhancing glutathione synthesis, acting as a substrate for glutathione peroxydase, and facilitating metabolism of hydrogen peroxide (H₂O₂) by univalent reduction of O₂⁻ via preservation of intracellular reduced glutathione concentration²⁵. Large amounts of superoxide dismutase are contained within mitochondria, and this enzyme is primarily responsible for the chemical conversion of O₂⁻ to H₂O₂ and water. The subsequent reduction of H₂O₂ is catalyzed by glutathione peroxydase to which reduced glutathione serves as an electron donor during the reaction. Thus, the current observation that NAC abolishes helium-induced preconditioning indirectly infers that O₂⁻ or one of its immediate derivatives is responsible for this cardioprotective effect. 2-MPG also donates sulfhydryl groups to glutathione peroxydase and may be more mitochondria-specific than NAC^26–28. These observations also indirectly suggest that O₂⁻ derived from mitochondria or another oxygen-derived free radical intermediate produced by O₂⁻ metabolism is involved in cardioprotection by helium. Nevertheless, such conclusions must be qualified because we did not specifically determine the identity or define the source of the ROS involved in helium-induced preconditioning in the current investigation. It is also unclear based on the current results whether mitochondrial K_ATP opening acts as a trigger or end-effector for preconditioning by helium through ROS generation. Our laboratory is currently examining this hypothesis.

In addition to previously described limitations, the current results must be interpreted within the constraints of several other potential shortcomings. The duration of administration of NAC, 2-MPG, and 5-HD were heterogeneous, and these pharmacokinetic factors may have influenced the results. Plasma concentrations of NAC, 2-MPG, and 5-HD were also not determined nor were dose-response relationships to these drugs performed. Myocardial infarct size is determined primarily by the size of the area at risk and the extent of coronary collateral perfusion. The area at risk expressed as a percentage of total left ventricular mass was similar between groups in the current investigation, and coronary collateral blood flow has been shown to be minimal in rabbits²⁹. Thus, differences in collateral perfusion between groups probably did not account for the observed results, but coronary collateral blood flow was not specifically quantified. The reductions in myocardial necrosis produced by helium in the absence or presence of other drug interventions occurred independent of changes in major determinants of myocardial oxygen consumption. Nevertheless, coronary venous oxygen tension was not directly measured nor was myocardial oxygen consumption calculated. Notably, no significant differences in hemodynamics were observed amongst groups before and during coronary artery occlusion that may account for differences in infarct size observed between groups. Finally, the current results implicating a role for ROS and mitochondrial K_ATP channels in helium-induced cardioprotection were obtained in barbiturate-anesthetized, acutely instrumented rabbits. Whether similar results occur in other animal species or humans is unknown. Helium preconditioning has yet to be established in humans, but administration of this noble gas before a defined of period of myocardial ischemia be beneficial in a clinical setting in which an anesthetic is not required (e.g., inflation of an angioplasty balloon during cardiac catheterization). However, additional investigation will be required to test this intriguing hypothesis.

In summary, the current results confirm that brief, intermittent administration of helium before prolonged coronary artery occlusion and reperfusion protects myocardium against infarction. The findings further suggest that ROS and mitochondrial K_ATP channels mediate this helium-induced preconditioning in vivo.