Novel difference in IF1 reactivity to Zn2+ in rabbit versus rat cardiomyocytes, mitochondria, and submitochondrial particles.

Zn2+ has a paradoxical effect on IF1-ATPase interaction in cardiac muscle mitochondria in so-called slow heart-rate mammalian species like rabbit. In such slow heart-rate mammalian species, it completely prevents IF1-mediated ATPase inhibition regardless of pH while concomitantly causing full IF1 binding to the ATPase, again, regardless of pH (Rouslin et al. (1993) J. Bioenerget. Biomembr. 25, 297-306). While our earlier study suggested that there are two kinds of IF1-ATPase interaction, a docking interaction and an ATPase inhibitory interaction with Zn2+ promoting docking and interfering with inhibition, it did not yield information on whether Zn2+ interacted primarily with IF1, with the ATPase, or with both. In the present study we show that, in contrast to its effects in rabbit cardiomyocytes, mitochondria, and SMP in which Zn2+ fully blocked IF1-mediated ATPase inhibition, Zn2+ actually enhanced ATPase inhibition in rat cardiomyocytes, although the extent of this effect was limited by the low level of IF1 in rat cardiomyocytes. Moreover, Zn2+ had no effect on IF1-mediated ATPase inhibition in rat heart mitochondria and, as suggested by inter and intra-species IF1 binding to SMP, the different effects of Zn2+ in rabbit versus those in rat appear to be mediated primarily through the different reactivities of rabbit and rat IF1 to Zn2+.

IF1 function in situ in uncoupler-challenged ischemic rabbit, rat, and pigeon hearts.

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.

Isoform-independent heart rate-related variation in cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase activity.

In the present study, we compared the activities of the cardiac myofibrillar Ca(2+)-activated Mg(2+)-ATPase and the content of cardiac muscle mitochondrial ATPase inhibitor protein (IF1) of several mammalian species covering broad ranges of body mass and heart rate, i.e., from beef cattle to mouse. The cardiac myofibrillar ATPase from each species was assayed over a range of pCa values at pH 7.4. While the cardiac myofibrillar ATPase from all species examined showed essentially identical Ca2+ concentration dependencies with the ATPase in each species activating steeply between pCa 6.5 and 5.5, the maximal ATPase specific activity reached varied considerably from species to species, and this variation was largely independent of the predominant cardiac myosin ATPase isoform present. Thus, while adult beef cattle, pig, dog, and rabbit all contain predominantly the slow cardiac myosin ATPase isoform the cardiac myofibrillar ATPase specific activities of these four species varied over approximately a fourfold range. Moreover, there was a fairly smooth curvilinear relationship between maximum Ca(2+)-activated myofibrillar ATPase activity and median conscious heart rate for the slow cardiac myosin ATPase-possessing species examined. This smooth continuum also extended to include two species possessing the fast cardiac myosin ATPase isoform, rat and mouse. This relationship between myofibrillar ATPase activity and heart rate that appears to be applicable to a broad range of species suggests that the myofibrillar ATPase is specifically modeled or fine-tuned to the kinetic (heart rate) demand of each species and, within slow and fast heart rate ranges, is essentially independent of myosin ATPase isoform per se. Only hearts containing predominantly the slow myosin ATPase isoform contained functional levels of IF1. Finally, while it has been reported that the ratio of myosin Ca(2+)-ATPase to actomyosin Mg(2+)-ATPase activity is a good index of the percent of the fast myosin ATPase in rabbit myofibrillar preparations, we found that this relationship may be applicable to only some species.

ATPase activity, IF1 content, and proton conductivity of ESMP from control and ischemic slow and fast heart-rate hearts.

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.

Content and binding characteristics of the mitochondrial ATPase inhibitor, IF1, in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms.

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)

Analysis of factors affecting functional assays for estimating IF1, the mitochondrial ATPase inhibitor.

Functional assays for IF1 have been in use for more than 30 years, i.e., since the initial report of Pullman and Monroy in 1963 on the inhibition of soluble F1-ATPase by the purified bovine heart inhibitor. However, beginning with the report of Horstman and Racker in 1970 and for approximately 17 years thereafter, workers in many laboratories routinely used IF1-depleted bovine heart submitochondrial particles (SMP) prepared from slaughterhouse material for the assay of IF1-containing extracts and preparations. Then, in 1987 we introduced the use of submitochondrial particles prepared from a species naturally poor in IF1 for this purpose. Thus, rat heart SMP which are largely depleted of IF1 in their native state were found to allow the performance of particularly linear and reproducible IF1 titration assays regardless of the species source of the IF1 titrated on them. The present study presents the first systematic comparison of the effects of a variety of factors upon functional assays for IF1. These include variations in IF1 functional assays due to seasonal effects on bovine A particles as well as to the species source of the IF1-depleted particles used in the assays. Interestingly, bovine heart A particles prepared during cold weather were considerably more active than those prepared during warmer weather. Moreover, the more active cold-weather particles allowed the performance of better IF1 assays. Also, the larger the species of origin of the IF1-depleted heart muscle SMP, the less IF1 was required to produce a given amount of ATPase inhibition regardless of both seasonal effects and the species source of the IF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Zn2+ on the activity and binding of the mitochondrial ATPase inhibitor protein, IF1.

Zn2+ caused a noninhibitory binding of IF1 to mitochondrial membranes in both rabbit heart SMP and intact rabbit heart mitochondria. This Zn(2+)-induced IF1 binding required the presence of at least trace amounts of MgATP and was essentially independent of pH between 6.2 and 8.2. Addition of Zn2+ after the formation of fully inhibited IF1-ATPase complexes very slowly reversed IF1-mediated ATPase inhibition without causing significant IF1 release from the membranes. When Zn2+ was added during the state 4 energization of ischemic mitochondria in which IF1 was already functionally bound, it slowed somewhat energy-driven ATPase activation. This slowing was probably due to the fairly large depressing effect Zn2+ had upon membrane potential development, but Zn2+ did not decrease the degree of ATPase activation eventually reached at 20 min of state 4 incubation. Zn2+ also preempted normal IF1 release from the membranes, causing what little inhibitor that was released to rebind to the enzyme in noninhibitory IF1-ATPase complexes. The data suggest that IF1 can interact with the ATPase in two ways of through two kinds of sites: (a) a noninhibitory interaction involving a non-inhibitory IF1 conformation and/or an IF1 docking site on the enzyme and (b) an inhibitory interaction involving an inhibitory IF1 conformation and/or a distinct ATPase activity regulatory site. Zn2+ appears to have the dual effect of stabilizing the noninhibitory IF1-ATPase interaction and possibly a noninhibitory IF1 conformation while concomitantly preventing the formation of an inhibitory IF1-ATPase interaction and possibly an inhibitory IF1 conformation, regardless of pH. While the data do not rule out direct effects of Zn2+ on either free IF1 or the free enzyme, they suggest that Zn2+ cannot interact readily with either the inhibitor or the enzyme once functional IF1-ATPase complexes are formed.

Factors affecting the species-homologous and species-heterologous binding of mitochondrial ATPase inhibitor, IF1, to the mitochondrial ATPase of slow and fast heart-rate hearts.

We examined the effects of a variety of conditions upon the IF1-mediated inhibition of the ATPase in both intact and sonicated mitochondria and in IF1-depleted submitochondrial particles (SMP) in species-homologous and species-heterologous combinations of IF1 and ATPase. IF1-mediated ATPase inhibition occurred in intact rabbit heart mitochondria at low matrix pH and low membrane potential, but not in intact pigeon and rat heart mitochondria under the same conditions. IF1-mediated ATPase inhibition was, however, demonstrable in both the rabbit and pigeon heart systems in sonicated mitochondria incubated at low ionic strength. The rat heart system failed to exhibit significant IF1-mediated ATPase inhibition in either intact or sonicated mitochondria due to the low amount of IF1 present. When rabbit heart IF1-containing extracts were incubated with IF1-depleted rabbit heart SMP over a range of KCl concentrations, increasing the [KCl] to 100 mM had little effect on IF1-mediated ATPase inhibition. When pigeon heart IF1-containing extracts were incubated with IF1-depleted pigeon heart SMP under the same conditions, increasing [KCl] to 100 mM nearly completely blocked IF1-mediated ATPase inhibition. While the species-endogenous level of rat heart IF1 (i.e., 1x IF1) inhibited IF1-depleted rat heart SMP virtually not at all at any [KCl] examined, the 8x rat heart IF1 was nearly as inhibitory as the 1x rabbit heart IF1 at varying ionic strengths. When rabbit, pigeon, or rat heart IF1 was bound to rabbit versus pigeon IF1-depleted SMP, the effect of varying ionic strength on IF1-mediated ATPase inhibition was related to the species source of the IF1, not to the species source of the enzyme; 1x bovine heart IF1 purified to homogeneity behaved much like 1x crude rabbit heart IF1 when binding to either the rabbit or the pigeon heart enzyme. This suggests that an IF1-ATPase complex stabilizing factor such as has been isolated from baker's yeast cells in neither lacking in the pigeon heart system nor required for the more ionic-strength-resistant binding of IF1 observed in slow heart-rate mammalian heart mitochondria.

Mechanisms of ATP conservation during ischemia in slow and fast heart rate hearts.

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)