Glutathione preconditioning ameliorates mitochondria dysfunction during warm pulmonary ischemia-reperfusion injury

Abstract

Objectives: Reduced glutathione (GSH) has been shown to improve pulmonary graft preservation. Mitochondrial dysfunction is regarded to be the motor of ischemia-reperfusion injury (IR) in solid organs. We have shown previously that IR induces pulmonary mitochondrial damage. This study elucidates the impact of GSH preconditioning on the integrity and function of pulmonary mitochondria in the setting of warm pulmonary IR.

Methods: Wistar rats were subjected to control, sham, and to two-study-group conditions (IR30/60 and GSH-IR30/60) receiving IR with or without GSH preconditioning. Rats were anesthetized and received mechanical ventilation. Pulmonary in situ clamping followed by reperfusion generated IR. Mitochondria were isolated from pulmonary tissue. Respiratory chain complexes activities (I-IV) were analyzed by polarography. Mitochondrial viability (Ca2+-induced swelling) and membrane integrity (citrate synthase assay) were determined. Subcellular-fractional cytochrome C-content (Cyt C) was quantified by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was analyzed by fluorescence-activated cell sorting (FACS) after energizing and uncoupling. Inflammatory activation was determined by myeloperoxidase activity (MPO), matrix-metalloproteinase 9 (MMP-9) activity by gel zymography.

Results: Pulmonary IR significantly reduced mitochondrial viability in combination with ΔΨm hyper-polarization. GSH preconditioning improved mitochondrial viability and normalized ΔΨm. Cyt C was reduced after IR; GSH protected from Cyt C liberation. Respiratory chain complex activities (I, II, III) declined during IR; GSH protected complex II function. GSH also protected from MMP-9 and neutrophil sequestration (P>.05).

Conclusions: GSH preconditioning is effective to prevent mitochondrial death and improves complex II function during IR, but not mitochondrial membrane stability. GSH-mediated amelioration of ΔΨm hyper-polarization appears to be the key factor of mitochondrial protection.

Figure 1:

Mitochondrial viability by Ca²⁺-induced mitochondrial swelling: differences between study groups were highly significant (P < .001). Compared to controls mitochondrial viability was significantly impaired in IR30/60 (P < .01). Compared to IR30/60 GSH preconditioning (GSH-IR30/60) significantly protected mitochondrial viability during IR (P < .01). Data are expressed as means.

Figure 2:

FACS-analysis of ΔΨ_m in energized mitochondria and after uncoupling with CCCP: Values reflect the ratio of J-aggregate⁺-mitochondria and JC1⁺-mitochondria. Compared to controls membrane potential (ΔΨ_m) of succinate-energized mitochondria demonstrated hyper-polarization during IR (IR30/60). After uncoupling with CCCP ΔΨ_m declined faster in IR30/60-mitochondria when compared to controls. GSH preconditioning (GSH-IR30/60) prevented hyper-polarization and accelerated decline after CCCP comparing to IR30/60. Differences between groups reached statistical significance 3 min after CCCP addition (P < .05). Compared to controls values differed significantly in IR30/60 (P < .05). Differences between IR30/60 and GSH-IR30/60 did not differ significantly (P > .05). Data are expressed means ± SEM.

Figure 3:

Mitochondrial cytochrome C (Cyt C) content determined from sub-cellular fractions: regarding Cyt C, inter-group differences differed significantly (P = .003). Between controls and sham no statistical significant difference on Cyt C was detectable (P > .05). Cyt C content was high in controls and significantly declined during IR (P < .01). GSH preconditioning preserved tissue from Cyt C loss without reaching statistical significance when compared to controls or IR30/60 (P > .05). Data are expressed means ± SEM.

Figure 4:

Mitochondrial integrity reflected by the citrate synthase ratio (CSR): differences of CSR demonstrated statistical significance (P = .002). There was no statistical significant difference between control and sham (P > .05). The CSR declined during ischemia–reperfusion injury when compared to sham (P < .01). Compared to IR30/60 decay of CSR remained detectable in GSH-IR30/60 (P > .05). Data are expressed as means ± SEM.

Figure 5:

Activity of matrix metalloproteinase-9 determined by gelatin in vitro zymography. Values represent MMP-9 semi-quantitatively. Activities between groups differed significantly (P = .0003). Control and sham did not differ statistically significant (P > .05). Differences between control and IR30/60 reached a statistically significant level (P < .001). Differences between sham and IR30/60 showed a trend toward significance (P > .05). GSH preconditioning (GSH-IR30/60) effectively reduced MMP-9-activity during IR and showed to trend toward statistical significance when compared to IR30/60 (P > .05). Values of IR-IR30/60 almost equaled values of sham (P > .05). Data are expressed means ± SEM.

Figure 6:

Myeloperoxidase assay (MPO) to assess sequestration of neutrophil granulocytes from lung tissue: MPO-activities of groups differed significantly (P < .0003). MPO activity remained low in controls and peaked in IR30/60 (P < .001). GSH-preconditioning (GSH-IR30/60) effectively suppressed MPO-activity during IR without reaching statistical significance when compared to controls or IR30/60 (P > .05). Data are expressed means ± SEM.