In the present study, isolated dog and rat hearts were perfused in the Langendorff mode with Krebs bicarbonate buffer in the absence and presence of 10(-5) M oligomycin. The perfusion protocols employed allowed tissue pH to drop during subsequent ischemic incubations essentially as it would in blood-perfused hearts. Tissue pH, ATP, lactate, and mitochondrial respiratory function were measured during the course of subsequent zero-flow ischemic incubations. The adenosinetriphosphatase (ATPase) activities attributable to both mitochondrial and nonmitochondrial ATPases in sonicated heart homogenates and the actomyosin ATPase in isolated cardiac myofibrils were measured in both species. Consistent with earlier results with a different model in which tissue pH was buffered during the ischemic incubations [W. Rouslin, J. L. Erickson, and R. J. Solaro. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H503-H508, 1986], the inhibition of the mitochondrial ATPase in situ by oligomycin markedly slowed both tissue ATP depletion and the loss of mitochondrial function during ischemia in the dog. However, oligomycin had only a very small and transient effect on ATP depletion and mitochondrial function in the rat. This was apparently so because of the fivefold higher rate of glycolytic ATP production as well as the nearly threefold higher total nonmitochondrial ATPase activity of ischemic rat compared with ischemic dog heart. These results suggest that although the inhibition of the mitochondrial ATPase makes a major contribution to ATP conservation in ischemic dog heart, it makes only a very small contribution in rat.

Rabbit, rat, and pigeon are species representative of three cardiac muscle mitochondrial ATPase regulatory classes, a, b and c, respectively. Class a species contain a full complement of higher affinity ATPase inhibitor subunit, IF1, in their cardiac muscle mitochondria and show marked IF1-mediated mitochondrial ATPase inhibition during myocardial ischemia. Class b species contain low levels of higher affinity IF1 and show very little IF1-mediated ATPase inhibition during ischemia. Class c species contain a full complement of a lower affinity form of IF1 and show a low-to-moderate level of IF1- mediated ATPase inhibition during ischemia. In the present study we perfused hearts of a member of each regulatory class through the coronary arteries with the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), before making them ischemic. We then compared net rates of cell ATP depletion during ischemia in the FCCP-treated hearts to identically treated FCCP-free hearts. Thus, we tested the relative capacities of cardiac muscle mitochondria of the three species to avert a potentially greatly increased net rate of cell ATP depletion due to ATP hydrolysis by the fully uncoupled mitochondrial ATPase. We found that FCCP-uncoupling in situ had a relatively small effect on ATP depletion during ischemia in rabbit hearts, that it dramatically accelerated ATP depletion in ischemic rat hearts, and that it had an intermediate effect on ATP depletion in ischemic pigeon hearts. These results demonstrate for the first time the relative extents to which IF1-mediated mitochondrial ATPase inhibition can slow cell ATP depletion due to the fully uncoupled mitochondrial ATPase in these three classes of hearts. They show that, in contrast to the situation in rabbit hearts, the low level of higher affinity IF1 present in the cardiac muscle mitochondria of the rat is, under these conditions, essentially nonfunctional, while the full complement of the lower affinity form of IF1 present in the cardiac muscle mitochondria of the pigeon is partially functional in that it appeared to provide an intermediate level of protection against rapid cell ATP depletion.

We determined the IF1 contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF1-mediated inhibition of the ATPase activity of IF1-depleted rabbit heart mitochondrial particles (RHMP) by IF1-containing extracts from these same eight species. The IF1 binding experiments were run at both species-endogenous IF1 levels and at an IF1 level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF1 or either species-endogenous or normalized levels of pig heart IF1 were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF1-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF1 were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF1-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF1 inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF1 inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF1, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF1, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF1.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study we compared the quantitatively most important, Pi-activated mechanisms for conserving ATP during ischemia in dog and rat cardiac muscle. Earlier studies by ourselves showed that dog heart, like all slow heart rate mammalian hearts examined, possesses the ability to inhibit its mitochondrial ATPase by binding IF1, the ATPase inhibitor protein, during ischemia. Rat heart, like other fast heart rate mammalian hearts studied, does not. The present study demonstrated that this IF1-mediated ATPase inhibition in ischemic dog heart, as in other slow heart rate hearts, appears to depend on matrix space acidification mediated largely by Pi-H+ symport via the mitochondrial Pi carrier. The present study further confirmed that maximal glycolytic flux rates are five- to sixfold greater in ischemic rat than in ischemic dog heart. Both of these systems are activated by increasing Pi concentration ([Pi]) during ischemia, and both appear to be regulated somewhat differently in dog than in rat heart. Thus intact dog heart mitochondria exhibited a [Pi]-dependent ATPase inhibition at low external pH, whereas rat heart mitochondria did not. The [Pi] required for maximal ATPase inhibition in dog heart mitochondria was approximately 6 mM. Although both dog and rat heart phosphofructokinase were stimulated by Pi, the enzyme in dog heart was maximally activated by approximately 6 mM Pi, whereas the rat heart enzyme required only approximately 3 mM Pi for its maximal stimulation under otherwise identical conditions. The most active nonmitochondrial ATPase in ischemic dog and rat cardiac muscle, the Ca(2+)-activated actomyosin ATPase, accounted for approximately one-half of the total nonmitochondrial ATPase activity in each species.(ABSTRACT TRUNCATED AT 250 WORDS)

Earlier studies by Rouslin and coworkers showed that, during myocardial ischemia in slow heart-rate species which include rabbits and all larger mammals examined including humans, there is an IF1-mediated inhibition of the mitochondrial ATPase due to an increase in the amount of IF1 bound to the ATPase (Rouslin, W., and Pullman, M.E., J. Mol. Cell. Cardiol. 19,661-668, 1987). Earlier work by Guerrieri and colleagues demonstrated that IF1 binding to bovine heart ESMP was accompanied by parallel decreases in ATPase activity and in passive proton conduction (Guerrieri, F., et al., FEBS Lett. 213, 67-72, 1987). In the present study rabbit was used as the slow heart-rate species and rat as the fast heart-rate species. Rat is a fast heart-rate species that contains too little IF1 to down regulate the ATPase activity present. Mitochondria were prepared from control and ischemic hearts and ESMP were made from aliquots by sonication at pH 8.0 with 2 mM EDTA. Oligomycin-sensitive ATPase activity and IF1 content were measured in SMP prepared from the control and ischemic mitochondrial samples. After identical incubation procedures, oligomycin-sensitive ATPase activity, oligomycin-sensitive proton conductivity, and IF1 content were also measured in ESMP samples. The study was undertaken to corroborate further what appear to be fundamental differences in ATPase regulation between slow and fast heart-rate mammalian hearts evident during total myocardial ischemia. Thus, passive proton conductivity was used as an independent measure of these regulatory differences. The results show that, consistent with the low IF1 content of rat heart cardiac muscle mitochondria, control rat heart ESMP exhibit approximately twice as much passive proton conductivity as control rabbit heart ESMP regardless of the pH of the incubation and assay. Moreover, while total ischemia caused an increase in IF1 binding and a commensurate decrease in passive proton conductivity in rabbit heart ESMP regardless of pH, neither IF1 content nor proton conductivity changed significantly in rat heart ESMP as a result of ischemia.