Suppression of Superoxide-Hydrogen Peroxide Production at Site IQ of Mitochondrial Complex I Attenuates Myocardial Stunning and Improves Postcardiac Arrest Outcomes

Abstract

Objectives: Cardiogenic shock following cardiopulmonary resuscitation for sudden cardiac arrest is common, occurring even in the absence of acute coronary artery occlusion, and contributes to high rates of postcardiopulmonary resuscitation mortality. The pathophysiology of this shock is unclear, and effective therapies for improving clinical outcomes are lacking.

Design: Laboratory investigation.

Setting: University laboratory.

Subjects: C57BL/6 adult female mice.

Interventions: Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 4, 8, 12, or 16-minute potassium chloride-induced cardiac arrest followed by 90 seconds of cardiopulmonary resuscitation. Mice were then blindly randomized to a single IV injection of vehicle (phosphate-buffered saline) or suppressor of site IQ electron leak, an inhibitor of superoxide production by complex I of the mitochondrial electron transport chain. Suppressor of site IQ electron leak and vehicle were administered during cardiopulmonary resuscitation.

Measurements and main results: Using a murine model of asystolic cardiac arrest, we discovered that duration of cardiac arrest prior to cardiopulmonary resuscitation determined postresuscitation success rates, degree of neurologic injury, and severity of myocardial dysfunction. Post-cardiopulmonary resuscitation cardiac dysfunction was not associated with myocardial necrosis, apoptosis, inflammation, or mitochondrial permeability transition pore opening. Furthermore, left ventricular function recovered within 72 hours of cardiopulmonary resuscitation, indicative of myocardial stunning. Postcardiopulmonary resuscitation, the myocardium exhibited increased reactive oxygen species and evidence of mitochondrial injury, specifically reperfusion-induced reactive oxygen species generation at electron transport chain complex I. Suppressor of site IQ electron leak, which inhibits complex I-dependent reactive oxygen species generation by suppression of site IQ electron leak, decreased myocardial reactive oxygen species generation and improved postcardiopulmonary resuscitation myocardial function, neurologic outcomes, and survival.

Conclusions: The severity of cardiogenic shock following asystolic cardiac arrest is dependent on the length of cardiac arrest prior to cardiopulmonary resuscitation and is mediated by myocardial stunning resulting from mitochondrial electron transport chain complex I dysfunction. A novel pharmacologic agent targeting this mechanism, suppressor of site IQ electron leak, represents a potential, practical therapy for improving sudden cardiac arrest resuscitation outcomes.

Figure 1.

Duration of cardiac arrest determines postcardiopulmonary resuscitation (CPR) outcomes. A, Return of spontaneous circulation (ROSC) rates following 4, 8, 12, and 16 min of cardiac arrest (CA). B, Time of CPR to achieve ROSC. n = 12, 15, 14, and 9, respectively. *p < 0.05; **p < 0.01; ***p < 0.001 versus 4-min group. C, Percent left ventricular fractional shortening 15 min after achieving ROSC for different durations of CA. n = 17, 12, 12, 12, and 1, respectively. D, Kaplan-Meyer Curve demonstrating survival following different durations of CA. n = 22, 28, 34, and 9, respectively. E, Neurologic scores following CA of increasing duration. n = 12, respectively. F, Percent left ventricular fractional shortening recovery over time following CA. n = 7, 7, and 9, respectively. *p < 0.05; **p < 0.01; ***p < 0.001 versus sham. #p < 0.05.

Figure 2.

Postcardiopulmonary resuscitation myocardial dysfunction occurs in the absence of myocardium necrosis. A, Tetrazolium staining of hearts 2 hr following a 12-min cardiac arrest (CA). Hematoxylin and eosin staining (B), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (C), and cluster of differentiation 31 (CD31) staining (D) of left ventricle sections 2 hr following CA compared with sham. AU = arbitrary units, DAPI = 4′,6-diamidino-2-phenylindole, ROSC = return to spontaneous circulation.

Figure 3.

Increased reactive oxygen species (ROS) production and decreased complex I activity postcardiopulmonary resuscitation (CPR) resuscitation. A, Calcium-induced mitochondrial swelling from sham and post-CPR heart. n = 2, 3, 3, and 3, respectively. B, MitoSox staining (ThermoFisher, Waltham, MA) from cardiac arrest (CA) and sham mice heart. Fluorescence quantification is demonstrated in left graphic. n = 4, respectively. C, Fluorescence quantification of MitoSox staining on mitochondria isolated from CA and sham mice with the present of 10-mM pyruvate + 2-mM malate. n = 4, respectively. D, Complex I activity measurement directly from cardiac mitochondria. n = 4, respectively. 12 min CA (CA₁₂) + reperfusion 15 min (R₁₅) = 12-min CA + 15-min resuscitation; *p < 0.05; ***p < 0.001 versus sham. AU = arbitrary units, mPTP = mitochondrial permeability transition pore.

Figure 4.

Postcardiopulmonary resuscitation mitochondrial complex I injury. Oxygen consumption rate (OCR) measurements of cardiac mitochondria from cardiac arrest (CA) and sham. A, The sequential injection of mitochondrial inhibitors is indicated by arrows. B, Adenosine diphosphate (ADP)-stimulated OCR. C, State 3/state 4 respiration. D, Carbonilcyanide p-triflouromethoxyphenylhydrazone-stimulated OCR. E, Calculated proton leak. n = 7, respectively. 12 min CA (CA₁₂) + reperfusion 15 min (R₁₅) = 12-min CA + 15-min resuscitation; *p < 0.05; **p < 0.01; ***p < 0.001 versus sham.

Figure 5.

Suppressor of site I_Q (the ubiquinone-binding site of complex I, the active site during reverse electron transport) electron leak (S1QEL) reduces postcardiopulmonary resuscitation (CPR) myocardial stunning and improves post-CPR resuscitation outcomes. A, Effects of S1QEL (0.1, 1, and 10 μM) on succinate-induced H₂O₂ production at site I_Q of complex I post cardiac arrest (CA). n = 16, 16, 22, and 16, respectively. ***p < 0.001 versus CA group. B, Images and bar graph show that MitoSox staining in the heart tissue following CPR with and without S1QEL. n = 9, 10, and 8, respectively. *p < 0.05; ***p < 0.001 versus sham. ##p < 0.01 versus CA group. C, Return to spontaneous circulation (ROSC) following 12 min of CA and CPR time to ROSC with S1QEL and controls. n = 53, 39, respectively. D, Left ventricular fractional shortening following 12-min CA with S1QEL and controls. n = 10, 8, respectively. E, Neurologic scores in mice following CA with S1QEL and controls. n = 14, 17, respectively. F, Survival curve following CA with S1QEL and controls. S, S1QEL; n = 53, 39, respectively. *p < 0.05; **p < 0.01; ***p < 0.001 versus CA group. ROS = reactive oxygen species.