Introduction:
In a pig model of myocardial infarction (MI), intracoronary delivered Poloxamer (P) 188 significantly reduces ischemia/reperfusion (IR) injury when given immediately upon reperfusion, with improved mitochondrial function as a predominant effect. As mitochondria are heavily damaged during IR, a direct effect of P188 on mitochondria may lead to better therapy options during reperfusion. To show not only a similar reduction of IR injury by P188 in the brain, but also a direct P188 effect on mitochondria, we established an in-vitro model of IR that consists of damaging isolated rat brain mitochondria with hydrogen peroxide (H
2
O
2
), one component of ischemia, then applying P188, and analyzing mitochondrial function.
Methods:
Male Sprague-Dawley rat brains were removed, and the mitochondria isolated by differential centrifugation and Percoll gradients, then kept on ice to slow their bioenergetics prior to any experimental treatments. Mitochondria were exposed to 200 μM H
2
O
2
for 10 min at room temperature with slight agitation; controls received no H
2
O
2
. Samples were then diluted ½ with buffer ± P188 (250 μM after dilution) to simulate reperfusion and treatment, and kept at room temperature for 10 further minutes. ATP synthesis was measured in a luminometer using a luciferase enzymatic assay. Oxygen consumption was measured by closed cell respirometry with an oxygen meter. In both assays, Complex I and Complex II were examined; Complex I substrates glutamate and malate, Complex II substrate succinate plus the Complex I inhibitor rotenone. Statistics: Data are expressed as mean ± SEM. One-Way ANOVA, SNK-Test; Kruskal-Wallis-Test; α=0.05, * vs control.
Results:
In both Complex I and II, mitochondrial function was significantly impaired by H
2
O
2
, with ATP synthesis affected more at Complex I and oxygen consumption affected more at Complex II. Addition of P188 did not provide any significant improvement in mitochondrial function.
Conclusions:
Although P188 significantly reduced IR injury when given during reperfusion in a pig model of MI, it does not appear to provide direct protection to mitochondria in this in-vitro model. Whether the exposure to H
2
O
2
causes the appropriate injury for P188 to become effective remains to be elucidated.
ATP synthesis kinetics and mitochondrial function in the postischemic myocardium as studied by 31P NMR.
