Isoflurane postconditioning protects against reperfusion injury by preventing mitochondrial permeability transition by an endothelial nitric oxide synthase-dependent mechanism

Abstract

Background: The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. The authors addressed this issue using eNOS mice.

Methods: In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before and 3 min after reperfusion. Ca+-induced mitochondrial permeability transition (MPT) pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function.

Results: Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 +/- 10% (n = 10) in control to 48 +/- 10%, 41 +/- 8% (n = 8, P < 0.05), and 38 +/- 10% (n = 8, P < 0.05), respectively, and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by N-nitro-L-arginine methyl ester (a nonselective nitric oxide synthase inhibitor) administered either before ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca+ loading than did controls (78 +/- 29 microm vs. 40 +/- 25 microm CaCl2 per milligram of protein, n = 10, P < 0.05) to open the MPT pore. Hearts from eNOS mice displayed no marked differences in infarct size, cardiac function, and sensitivity of MPT pore to Ca+, compared with wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function, or the amount of Ca+ necessary to open the MPT pore in mitochondria isolated from the eNOS hearts compared with control hearts.

Conclusions: IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.

Figure 1

Schematic diagram depicting the experimental protocols. A: effects of different concentrations of isoflurane on myocardial infarct size in WT mice following I/R; B: effects of disruption of the eNOS gene on infarct size; C: effects of different concentrations of isoflurane on cardiac function in Langendorff-perfused WT hearts following I/R; D: role of nitric oxide in the cardioprotective effect produced by isoflurane; E: effects of isoflurane on MPT and cardiac function in WT and eNOS^-/- mice. eNOS^-/- = endothelial nitric oxide synthase gene knockout; I/R = ischemia/reperfusion; ISO_0.5 = 0.5 MAC isoflurane; ISO_1.0 = 1.0 MAC isoflurane; ISO_1.5 = 1.5 MAC isoflurane; L-NAME = N^G-nitro-L-arginine methyl ester; MAC = minimum alveolar concentration; MPT = mitochondrial permeability transition; WT = wild-type.

Figure 2

Concentration-dependent decreases in myocardial infarct size by isoflurane postconditioning (IsoPC) in wild-type mice subjected to 30 min of coronary occlusion followed by 2 h of reperfusion. A: area at risk expressed as a percentage of left ventricle area; B: myocardial infarct size expressed as a percentage of area at risk. IsoPC was produced by 0.5, 1.0, or 1.5 minimum alveolar concentration of isoflurane (ISO_0.5, ISO_1.0, or ISO_1.5) administered during the last 5 min of ischemia and first 3 min of reperfusion. *P < 0.05 versus control (n = 8-10 mice/group).

Figure 3

Myocardial infarct size was decreased by isoflurane postconditioning (IsoPC) in wild-type mice but not in endothelial nitric oxide synthase knockout (eNOS^-/-) mice subjected to 30 min of coronary occlusion followed by 2 h of reperfusion. IsoPC was produced by 1.0 minimum alveolar concentration of isoflurane (ISO_1.0) administered during the last 5 min of ischemia and first 3 min of reperfusion. A: area at risk; B: infarct size; C: representative heart slices from a wild-type control mouse; D: heart slices from a wild-type ISO_1.0 mouse; E: heart slices from an eNOS^-/- control mouse; F: heart slices from an eNOS^-/- ISO_1.0 mouse. The hearts were stained with 2,3,5-triphenyltetrazolium chloride and phthalol blue dye to delineate area at risk (red plus light yellow areas) and infarct size (light yellow areas). *P < 0.05 versus control (n = 7 mice/group).

Figure 4

Isoflurane postconditioning produced concentration-dependent increases in cardiac function in Langendorff-perfused wild-type hearts subjected to 30 min of global ischemia followed by 2 h of reperfusion. A: LVDP (left ventricular developed pressure); B: +dP/dt (maximum rate of increase of LVDP); C: -dP/dt (maximum rate of decrease of LVDP); D: LVEDP (LV end-diastolic pressure); E: coronary flow; F: heart rate. ISO_0.5 = 0.5 minimum alveolar concentration of isoflurane; ISO_1.0 = 1.0 minimum alveolar concentration of isoflurane; ISO_1.5 = 1.5 minimum alveolar concentration of isoflurane. *P < 0.05 versus control (n = 7-8 hearts/group).

Figure 5

Nitric oxide is both a trigger and a mediator of isoflurane postconditioning-induced protection in wild-type hearts subjected to 30 min of ischemia followed by 60 min of reperfusion. A: LVDP (left ventricular developed pressure); B: +dP/dt (maximum rate of increase of LVDP); C: -dP/dt (maximum rate of decrease of LVDP). REP₃₀ = 30 min after reperfusion; REP₆₀ = 60 min after reperfusion. Buffer containing 1.0 minimum alveolar concentration of isoflurane (ISO_1.0) was administered in the ISO_1.0, trigger, and mediator groups. To inhibit nitric oxide synthesis before and after myocardial exposure to isoflurane, the hearts were perfused with 30 μM N^G-nitro-L-arginine methyl ester (a non-selective endothelial nitric oxide synthase inhibitor) for 20 min either prior to ischemia (trigger group) or at the onset of reperfusion (mediator group). *P < 0.05 versus control; ^# P < 0.05 versus ISO_1.0 (n = 7 hearts/group).

Figure 6

Inhibition of the mitochondrial permeability transition pore opening by isoflurane postconditioning in wild-type (WT) hearts but not in endothelial nitric oxide synthase knockout (eNOS^-/-) hearts subjected to 30 min of ischemia followed by 30 min of reperfusion. A: the amount of in vitro Ca²⁺ overload necessary to open the mitochondrial permeability transition pore in WT hearts and in eNOS^-/- hearts; B: representative tracings showing the changes in membrane potential (ΔΨ_m) of mitochondria isolated from WT hearts after in vitro Ca²⁺ loading; C: tracings showing the changes in ΔΨ_m of mitochondria isolated from the eNOS^-/- hearts after in vitro Ca²⁺ loading. Opening of the mitochondrial permeability transition pore was assessed after in vitro Ca²⁺ overload by following changes in ΔΨ_m using the fluorescent dye rhodamine 123. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was added at the arrows to depolarize mitochondria. *P < 0.05 versus sham; ^# P < 0.05 versus control (n = 10/group).

Figure 7

Isoflurane postconditioning improved cardiac function in wild-type (WT) hearts but not in endothelial nitric oxide synthase knockout (eNOS^-/-) hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion. A: LVDP (left ventricular developed pressure) in WT hearts; B: LVDP in eNOS^-/- hearts; C: +dP/dt (maximum rate of increase of LVDP) in WT hearts; D: +dP/dt in eNOS^-/- hearts; E: -dP/dt (maximum rate of decrease of LVDP) in WT hearts; F: -dP/dt in eNOS^-/- hearts. ISO_1.0 = 1.0 minimum alveolar concentration of isoflurane; REP₃₀ = 30 min after reperfusion. *P < 0.05 versus sham; ^# P < 0.05 versus control (n = 10/group).