Abstract
Ischemia-reperfusion injury (IRI) is associated with mitochondrial permeability transition pore (mPTP) opening and impaired mitochondrial respiration. Hypothermia attenuates IRI. We examined mitochondrial function in mitochondria obtained from isolated hearts subjected to warm or cold ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs-Ringer's solution at 37°C and subjected to 30 min global ischemia at 37°C or 17°C. After 5 min of reperfusion mitochondria were isolated. Mitochondrial [Ca2+]m, membrane potential (ΔΨm), and NADH were measured by spectrophotometry at appropriate wavelengths with indo-1, BCECF, rhodamine 123 fluorescent dyes, and autofluorescence, respectively. After energizing with pyruvic acid, 0-100 μM CaCl2 (0.03-60 μM free [Ca2+]e) was added followed by 250 μM ADP. Ca2+ -induced mPTP opening was assessed by collapse of ΔΨm. 10 μM [Ca2+]e resulted in mPTP opening after 37°C IRI, but only at 35 μM [Ca2+]e after 17°C IRI. ADP decreased ΔΨm and NADH and increased [Ca2+]m in all mitochondria, but the fall in ΔΨm was greater and the responses to ADP with Ca2+ overloading were worse after 37°C IRI vs. 17°C IRI. The incidence of no state 4 respiration was 25% with no added CaCl2 after 37°C IRI and 0% after 17°C IRI. This study shows that hypothermia prevents IRI damage through pathways restricting mitochondrial Ca2+ loading and preserves mitochondrial redox state and respiration. Moreover, mitochondria protected during ischemia with hypothermia were more resistant to Ca2+-induced mPTP opening and oxidative phosphorylation was better preserved. Hypothermia might prevent conformational changes in the F1F0-ATPsynthase and the ADP/ATP carrier, leading to better mitochondrial function and a resistance to mPTP opening as the ADP/ATP carrier is associated with mPTP opening.
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