Hypothermic perfusion at 17 degrees C caused moderate and reversible changes in mitochondrial function. However, hypothermia protects during ischemia, as shown by preservation of mitochondrial NADH energy balance and prevention of deleterious increases in m[Ca2+] and ROS formation. The close temporal relations of these factors during cooling and during ischemia suggest a causal link between mCa2+, mitochondrial energy balance, and ROS production.

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We measured the effects of global ischemia and reperfusion on intracellular Na(+), NADH, cytosolic and mitochondrial (subscript mito) Ca(2+), relaxation, metabolism, contractility, and Ca(2+) sensitivity in the intact heart. Langendorff-prepared guinea pig hearts were crystalloid perfused, and the left ventricular (LV) pressure (LVP), first derivative of LVP (LV dP/dt), coronary flow, and O(2) extraction and consumption were measured before, during, and after 30-min global ischemia and 60-min reperfusion. Ca(2+), Na(+), and NADH were measured by luminescence spectrophotometry at the LV free wall using indo 1 and sodium benzofuran isophthalate, respectively, after subtracting changes in tissue autofluorescence (NADH). Mitochondrial Ca(2+) was assessed by quenching cytosolic indo 1 with MnCl(2). Mechanical responses to changes in cytosolic-systolic (subscript sys), diastolic (subscript dia), and mitochondrial Ca(2+) were tested over a range of extracellular [Ca(2+)] before and after ischemia-reperfusion. Both [Ca(2+)](sys) and [Ca(2+)](dia) doubled at 1-min reperfusion but returned to preischemia values within 10 min, whereas [Ca(2+)](mito) was elevated over 60-min reperfusion. Reperfusion dissociated [Ca(2+)](dia) and [Ca(2+)](sys) from contractile function as LVP(sys-dia) and the rise in LV dP/dt (LV dP/dt(max)) were depressed by one-third and the fall in LV dP/dt (LV dP/dt(min)) was depressed by one-half at 30-min reperfusion, whereas LVP(dia) remained markedly elevated. [Ca(2+)](sys-dia) sensitivity at 100% LV dP/dt(max) was not altered after reperfusion, but [Ca(2+)](dia) at 100% LV dP/dt(min) and [Ca(2+)](mito) at 100% LV dP/dt(max) were markedly shifted right on reperfusion (ED(50) +36 and +125 nM [Ca(2+)], respectively) with no change in slope. NADH doubled during ischemia but returned to normal on initial reperfusion. The intracellular [Na(+)] ([Na(+)](i)) increased minimally during ischemia but doubled on reperfusion and remained elevated at 60-min reperfusion. Thus Na(+) and Ca(2+) temporally accumulate during initial reperfusion, and cytosolic Ca(2+) returns toward normal, whereas [Na(+)](i) and [Ca(2+)](mito) remain elevated on later reperfusion. Na(+) loading likely contributes to Ca(2+) overload and contractile dysfunction during reperfusion.

In this study, we determined 1) whether ROS scavenging only during cold perfusion before global ischemia improves mitochondrial and myocardial function, and 2) which ROS leads to compromised cardiac function during ischemia and reperfusion (I/R) injury. Using fluorescence spectrophotometry, we monitored redox balance (NADH and FAD), O 2•Ϫ levels and mitochondrial Ca 2ϩ (m[Ca 2ϩ ]) at the left ventricular wall in 120 guinea pig isolated hearts divided into control (Con), MnTBAP (a superoxide dismutase 2 mimetic), MnTBAP (M) ϩ catalase (C) ϩ glutathione (G) (MCG), CϩG (CG), and N G -nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor) groups. After an initial period of warm perfusion, hearts were treated with drugs before and after at 27°C. Drugs were washed out before 2 h at 27°C ischemia and 2 h at 37°C reperfusion. We found that on reperfusion the MnTBAP group had the worst functional recovery and largest infarction with the highest m [Ca 2ϩ ], most oxidized redox state and increased ROS levels. The MCG group had the best recovery, the smallest infarction, the lowest ROS level, the lowest m [Ca 2ϩ ], and the most reduced redox state. CG and L-NAME groups gave results intermediate to those of the MnTBAP and MCG groups. Our results indicate that the scavenging of cold-induced O 2•Ϫ species to less toxic downstream products additionally protects during and after cold I/R by preserving mitochondrial function. Because MnTBAP treatment showed the worst functional return along with poor preservation of mitochondrial bioenergetics, accumulation of H 2 O 2 and/or hydroxyl radicals during cold perfusion may be involved in compromised function during subsequent cold I/R injury. hypothermic ischemia; mitochondrial Ca 2ϩ ; reactive oxygen species HYPOTHERMIA of the arrested, ischemic heart improves its function on reperfusion compared with the normothermic arrested ischemic heart. The strategy behind hypothermic protection against ischemia, i.e., better tissue perfusion, and improved metabolic and mechanical function on reperfusion, is the reduced mitochondrial respiration and oxidative phosphorylation during ischemia that results in better mitochondrial respiration and regeneration of ATP on reperfusion. For example, we demonstrated that NADH, m [Ca 2ϩ ], and reactive O 2 species (ROS) levels were less altered during and after 30 min of ischemia at 17°C vs. 37°C (39). We reported that the more severe the hypothermia, the later is the onset of deleterious changes in mitochondrial function (1,2,16,22,31).Although hypothermia is very protective against ischemia, hypothermic perfusion, e.g., before subsequent cardiac ischemia, may cause injury due to altered cellular ion homeostasis resulting from impaired membrane ion pumps and exchangers and/or to reduced activity of enzymes responsible for mitochondrial respiration, scavenging of ROS, and contractile activity. A well-known effect of hypothermia is hypercontracture with elevated cytosolic [Ca 2ϩ ] (42). Another is a decreasing temperature-depende...

We determined in intact hearts whether inhibition of Na(+)/H(+) exchange (NHE) decreases intracellular Na(+) and Ca(2+) during ischemia and reperfusion, improves function during reperfusion, and reduces infarct size. Guinea pig isolated hearts were perfused with Krebs-Ringer solution at 37 degrees C. Left ventricular (LV) free wall intracellular Na(+) concentration ([Na(+)](i)) and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured using fluorescence dyes. Hearts were exposed to 30 min of ischemia with or without 10 microM of benzamide (BIIB-513), a selective NHE-1 inhibitor, infused for 10 min just before ischemia or for 10 min immediately on reperfusion. At 2 min of reperfusion, BIIB-513 given before ischemia decreased peak increases in [Na(+)](i) and [Ca(2+)](i), respectively, from 2.5 and 2.3 times (controls) to 1.6 and 1.3 times pre-ischemia values. At 30 min of reperfusion, BIIB-513 increased systolic-diastolic LV pressure (LVP) from 49 +/- 2% (controls) to 80 +/- 2% of pre-ischemia values. BIIB-513 reduced ventricular fibrillation by 54% and reduced infarct size from 64 +/- 1% to 20 +/- 3%. First derivative of the LVP, O(2) consumption, and cardiac efficiency were also improved by BIIB-513. Similar results were obtained with BIIB-513 given on reperfusion. These data show that Na(+) loading is a marker of reperfusion injury in intact hearts in that inhibiting NHE reduces Na(+) and Ca(2+) loading during reperfusion while improving function. These results clearly implicate the ionic basis by which inhibiting NHE protects the guinea pig intact heart from ischemia-reperfusion injury.