Daniel S. Lee

Ischemic Preconditioning Decreases Mitochondrial Proton Leak and Reactive Oxygen Species Production in the Postischemic Heart

BACKGROUND Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. MATERIALS AND METHODS Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red. RESULTS IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates. CONCLUSIONS Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.

Ischemic preconditioning preserves mitochondrial membrane potential and limits reactive oxygen species production

BACKGROUND Mitochondrial superoxide radical (O(2)(•¯)) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O(2)(•¯) production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O(2)(•¯) production; mitochondrial depolarization decreases O(2)(•¯) formation. We examined the relationship between O(2)(•¯) production and mΔΨ during IR and IPC. MATERIALS/METHODS Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O(2)(•¯) production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high-pressure liquid chromatography. RESULTS IPC preserved mΔΨ at End I (-156 ± 5 versus -131 ± 6 mV, P < 0.001) and End RP (-168 ± 2 versus -155 ± 2 mV, P < 0.05). At End RP, IPC attenuated O(2)(•¯) production (2527 ± 221 versus 3523 ± 250 AU/mg protein, P < 0.05). IPC preserved cytochrome c levels (351 ± 14 versus 269 ± 16 picomoles/mg protein, P < 0.05) at End RP, and decreased mitochondrial cristae disruption (10% ± 4% versus 33% ± 7%, P < 0.05) and amorphous density formation (18% ± 4% versus 28% ± 1%, P < 0.05). CONCLUSION We conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O(2)(•¯) production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O(2)(•¯) production, our results indicate that preservation of mΔΨ is associated with decreased O(2)(•¯) and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential.

Ischemic Postconditioning Does Not Provide Cardioprotection from Long-Term Ischemic Injury in Isolated Male or Female Rat Hearts1

BACKGROUND Ischemic postconditioning (PoC) is a cardio-protective strategy in which initial reperfusion is interrupted by episodes of ischemia. It is unclear whether PoC can be achieved in the Langendorff perfused rat heart model. We investigated (1) whether postconditioning occurs in Langendorff perfused rat heart and (2) whether there is a gender-specific response to PoC. MATERIALS AND METHODS Male/female rat hearts (n = 8/group) were subjected to 30 min of equilibration, 30 min of ischemia, and 120 min of reperfusion (Control). PoC was induced by 6 cycles (PoC 6c10s), 3 cycles (PoC 3c10s), or 2 cycles (PoC 2c10s) of 10 s reperfusion/10 s ischemia. Rate pressure product (RPP) and infarct size were measured. Male rats (n = 7/group) were subjected in vivo to 30 min left coronary ligation followed by 24 h of reperfusion (Control) or PoC 6c10s and 24 h of reperfusion. RESULTS Recovery of RPP was 18% ± 4% in male Control versus 17% ± 2% for 6c10s, 16% ± 1% for 3c10s, and 15% ± 3% for 2c10s. Female Control hearts recovered 25% ± 3% of their RPP versus 21% ± 2% for 6c10s. Infarct size was 25% ± 3% for male Control versus 26% ± 3% for 6c10s, 30% ± 2% for 3c10s, 28% ± 1% for 2c10s, and 30% ± 2% for female Control versus 29% ± 2% in 6c10s. In vivo infarct size for Control and PoC 6c10s was 44% ± 3% and 28% ± 5%, respectively (P < 0.05). CONCLUSIONS In the Langendorff perfused rat hearts, none of the PoC protocols improved myocardial tolerance to ischemia reperfusion injury nor decreased infarct size; however, in vivo postconditioning did confer protection. The lack of protection in the isolated hearts was not gender specific.