Gregory K. Asimakis

Effects of atractyloside and palmitoyl coenzyme a on calcium transport in cardiac mitochondria

Abstract Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.

Myocardial Ischemia: Correlation of Mitochondrial Adenine Nucleotide and Respiratory Function

State 3 respiration of rat heart mitochondria decreased approximately 60% after 20 min normothermic in vitro ischemia. After 20 min ischemia, the levels of intramitochondrial adenine nucleotides (ATP + ADP + AMP) decreased to approximately 20% of control values, with a rapid loss between 10 and 20 min. Also, the exchangeable adenine pool of the mitochondria decreased 60% after 20 min ischemia. State 4 respiration was not affected by the ischemic insult. The adenine nucleotide translocase activities of mitochondria from control and ischemic hearts were too high to measure accurately. Therefore, the effects of ischemia on adenine nucleotide translocase activity could not be established. However, 1 microM carboxyatractyloside did not impair state 3 respiration of control mitochondria, but did inhibit the adenine translocase activity by at least 80%. Moreover, titration of state 3 respiration with carboxyatractyloside produced sigmoidal curves for mitochondria from control and ischemic tissue. State 3 respiration correlated well with the total mitochondrial adenine nucleotides and the exchangeable adenine pool (r = 0.63 and 0.78, respectively). Data collected from isolated perfused rat hearts also showed a good correlation between state 3 respiration and the exchangeable adenine nucleotides (r = 0.92). In this study, mitochondria were isolated from hearts that were either perfused, made ischemic for 30 min by aortic cross-clamp, or reperfused for 10 min after the aortic cross-clamp. The slopes and y-intercepts of the regression lines were similar for the in vitro ischemic and the perfusion studies. There was no significant difference between the effects of ischemia on the state 3 and uncoupled respiratory rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioprotection by Local Heating: Improved Myocardial Salvage After Ischemia and Reperfusion

BACKGROUND Previous studies have shown that expression of the inducible 70-kD heat-shock protein (HSP72) by whole-body hyperthermia is associated with protection against ischemia-reperfusion injury. To develop techniques for regional elevation of heat-shock proteins that prevent extracardiac sequelae during whole-body hyperthermia, we sought to determine if local heating of the heart in vivo provides protection against ischemia-reperfusion injury in the rat. METHODS A thermal probe was used to locally heat rat hearts at two adjacent sites on the epicardial surface of the left ventricle. Rats were subjected to either 30 minutes of sham surgery (control; n = 10) or two local applications of the probe at 42.5 degrees to 43.5 degrees C for 15 minutes each (n = 9). After 4 hours, rats were subjected to 30 minutes of regional ischemia followed by 120 minutes of reperfusion. Hearts were removed and area at risk and infarct area were determined. RESULTS Localized heat stress resulted in a significant limitation of infarct size in heat-treated animals versus controls (mean +/- standard error of the mean infarct area/area at risk = 4.3% +/- 0.85 versus 19.2% +/- 3.4%; p < 0.005). Western blot experiments confirmed elevated HSP72 expression in left (heated) and right (nonheated) ventricular samples from treated animals (n = 6; left ventricular = 5.5-fold; right ventricular = 3.7-fold) compared with sham-operated controls. Controls treated with the probe at 37 degrees C (n = 4) showed no increases in HSP72. CONCLUSIONS Local heating of the heart is associated with elevated levels of HSP72 and improved myocardial salvage. The increase in expression of HSP72 is not limited to the heated region, but extends into nonheated regions of the heart as well. This may lead to the development of new techniques that improve methods of myocardial revascularization and heart transplantation procedures.

Hemodynamic action of calcitonin gene-related peptide in the isolated rat heart

The effects of calcitonin gene-related peptide (CGRP) on heart rate, coronary flow, pressure development, and time to ischemic contracture were studied in the isolated, perfused rat heart. A bolus of CGRP (2640 pmols) caused significant increases in heart rate and coronary flow; these effects were sustained for at least five minutes after injection. The increase in coronary flow was independent of heart rate, since CGRP caused an increase in coronary flow in non-beating (potassium-arrested) hearts. The dose-response of CGRP was studied using five doses (65, 218, 658, 1320 and 2640 pmols) given as bolus injections. Although the increase in heart rate was apparently dose-dependent, significant increases above baseline were observed only with the two highest doses. In contrast, coronary flow increased significantly above baseline with the injection of all but the lowest dose of CGRP. Ten minutes after injection of CGRP, all hearts were made ischemic. The time to onset of ischemic contracture was approximately 11 minutes for those hearts that received 65 pmols of CGRP; however, for those hearts receiving all other doses of CGRP, the time to onset of contracture was approximately 8 minutes. We conclude that CGRP significantly decreases the resistance of the coronary vascular bed, and that it may be an important regulator of regional blood flow in the heart.

Postischemic recovery of mitochondrial adenine nucleotides in the heart.

BackgroundAdenine nucleotides (AdNs) are lost from the mitochondrial fraction of the heart cell during ischemia. It is unknown whether this pool of AdNs can be replenished after reperfusion. The purpose of this study was to evaluate the postischemic recovery of the mitochondrial AdN pool. Methods and ResultsThe left anterior descending coronary artery (LAD) of the canine heart was occluded for 30 minutes followed by either no reflow, 30-minute reflow, 1-day reflow, or 7-day reflow. Systolic shortening in the TAD-supplied region was absent during occlusion but recovered to approximately 30% of preocclusion values during early reperfusion. Mitochondrial and tissue AdNs (ATP, ADP, and AMP) were determined in the LAD-supplied and left circumflex-supplied (control) regions of the heart. The AdN content (expressed as percent of control values) of mitochondria from the LAD region was 55±101% (p<0.002), 64±7% (p<0.001), 81±6% (p<0.03), and 94±8% for the no-reflow, 30-minutereflow, 1-day-reflow, and 7-day-reflow groups, respectively. The AdN content (expressed as percent of control values) of tissue samples from the LAD region was 52±9% (p<0.002), 48±12% (p<0.02), 68±5% (p<0.002), and 70±9% for the no-reflow, 30-minute-reflow, 1-day-reflow, and 7-day-reflow groups, respectively. There was a good correlation between mitochondrial and tissue AdN (r=0.95). Using initial exchange rates, adenine nucleotide translocase activities of mitochondria from the LAD and control regions were not significantly different. State 3 respiration of LAD mitochondria was depressed (approximately 25%, p<0.05) only in the no-reflow group. Acceptor control ratios of the LAD mitochondria were not significantly different from control values in any group. Conclsions. After 30 minutes of regional ischemia, postischemic restoration of the mitochondrial AdN pool occurs between 1 and 7 days; this restoration is preceded by recovery of respiratory and adenine nucleotide translocase functions. Although the abnormally low levels ofAdN persist in the mitochondrial compartment during the early reperfusion period, postischemic contractile dysfunction cannot be explained by depressed mitochondrial respiratory activity.

Heat shock improves recovery and provides protection against global ischemia after hypothermic storage

BACKGROUND Improved methods of donor heart preparation before preservation could allow for prolonged storage and permit remote procurement of these organs. Previous studies have shown that overexpression of heat-shock protein 72 provides protection against ischemic cardiac damage. We sought to determine whether rats subjected to heat stress with only 6-hour recovery could acquire protection to a subsequent heart storage for 12 hours at 4 degrees C. METHODS Three groups of animals (n = 10 each) were studied: control, sham-treated, and heat-shocked rats (whole-body hyperthermia 42 degrees C for 15 minutes). After 12-hour cold ischemia hearts were reperfused on a Langendorff column. To confirm any differences in functional recovery, hearts were then subjected to an additional 15-minute period of warm global ischemia after which function and lactate dehydrogenase enzyme leakage were measured. RESULTS Heat-shocked animals showed marked improvements compared with controls in left ventricular developed pressure (63+/-4 mm Hg versus 44+/-4 mm Hg, p<0.05) heart rate x developed pressure (13,883+/-1,174 beats per minute x mm Hg versus 8,492+/-1,564 beats per minute x mm Hg, p<0.05), rate of ventricular pressure increase (1,912+/-112 mm Hg/second versus 1,215+/-162 mm Hg/second, p<0.005), rate of ventricular pressure decrease (1,258+/-89 mm Hg/second versus 774+/-106 mm Hg/second, p<0.005). Diastolic compliance and lactate dehydrogenase release were improved in heatshocked animals compared with controls and sham-treated animals. Differences between heat-shocked animals and control or sham-treated animals were further increased after the additional 15-minute period of warm ischemia. Western blot experiments confirmed increased heat-shock protein 72 levels in heat-shocked animals (>threefold) compared with sham-treated animals and controls. CONCLUSIONS Heat shock 6 hours before heart removal resulted in marked expression of heat-shock protein 72 and protected isolated rat hearts by increased functional recovery and decreased cellular necrosis after 12-hour cold ischemia in a protocol mimicking that of heart preservation for transplantation. Protection was further confirmed after an additional 15-minute period of warm ischemia.

Intermittent ischemia produces a cumulative depletion of mitochondrial adenine nucleotides in the isolated perfused rat heart.

The purpose of the present study was to determine if repetitive myocardial ischemia would result in the cumulative loss of mitochondrial adenine nucleotides. Isolated perfused rat hearts were subjected to continuous or intermittent ischemia. A single 5-minute period of continuous ischemia did not result in a significant decrease in the mitochondrial adenine nucleotide pool; a single 10-minute period of ischemia resulted in a decrease of approximately 17%. Next, the adenine nucleotide content of mitochondria from preischemic and 30-minute continuous ischemic hearts was compared with two groups of hearts undergoing intermittent ischemia (both groups receiving a total of 30 minutes of ischemia). One group received three 10-minute episodes of ischemia interrupted by 5-minute periods of reperfusion (3 x 10-minute intermittent ischemia); the other intermittent ischemic group received six 5-minute episodes of ischemia interrupted by 5-minute periods of perfusion (6 x 5-minute intermittent ischemia). The mitochondrial adenine nucleotide content (expressed as nanomoles per nanomole cytochrome a) for the preischemic and 30-minute continuous ischemic hearts was 14.7 +/- 0.6 and 8.0 +/- 0.4, respectively. The mitochondrial adenine nucleotide content of the 3 x 10-minute intermittent ischemia group (8.5 +/- 0.5) was not significantly different from the 30-minute continuous ischemic group. The mitochondrial adenine nucleotide content of the 6 x 5-minute intermittent ischemia group (11.0 +/- 0.6) was significantly larger than that of the 30-minute continuous and the 3 x 10-minute intermittent ischemia groups (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphate-induced efflux of adenine nucleotides from rat-heart mitochondria: evaluation of the roles of the phosphate/hydroxyl exchanger and the dicarboxylate carrier

Upon the addition of inorganic phosphate, isolated rat-heart mitochondria released endogenous adenine nucleotides. To elucidate the mechanism of this phosphate-induced efflux, we evaluated the relative roles of three inner mitochondrial membrane carriers: the adenine nucleotide translocase, the phosphate/hydroxyl exchanger, and the dicarboxylate carrier. Atractyloside (a specific inhibitor of the adenine nucleotide translocase) prevented this efflux, but did not inhibit mitochondrial swelling. Inhibitors of the phosphate/hydroxyl exchanger (200 microM n-ethylmaleimide and 10 microM mersalyl) did not inhibit phosphate-induced efflux. 200 microM mersalyl (which inhibited both the phosphate/hydroxyl exchanger and the dicarboxylate carrier) inhibited the rate of efflux approx. 65% Phenylsuccinate and 2-n-butylmalonate (inhibitors of the dicarboxylate carrier) partially inhibited phosphate-induced efflux and adenine nucleotide translocase activity. Mersalyl (200 microM) had no effect on adenine nucleotide translocase activity. Partial inhibition of the adenine nucleotide translocase by phenylsuccinate and butylmalonate could not explain the extent of inhibition of phosphate-efflux by these agents. Moreover, the rates of adenine nucleotide efflux in the presence of phenylsuccinate, butylmalonate, or mersalyl correlated well with the ability of these agents to inhibit succinate-supported respiration. We conclude that phosphate-induced efflux of adenine nucleotides from rat heart mitochondria occurs over the adenine nucleotide translocase, and that the site of action of the phosphate is not the phosphate/hydroxyl exchanger, but is likely the dicarboxylate carrier.

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Medical Education 2010: 44: 1232–1240

Release of AMP and adenosine from rat heart mitochondria.

Release of AMP and adenosine from rat heart mitochondria was studied. The rate of appearance of extramitochondrial adenosine was independent of the extramitochondrial phosphate concentration between 5 and 20 mM. In the absence of exogenous, respiratory substrates or in the presence of glutamate/malate plus rotenone, the rate of appearance of adenosine was relatively low when phosphate was not added. The appearance of extramitochondrial AMP + adenosine was found to be directly proportional to the extra-mitochondrial phosphate concentration. Zn2+ (10 mM) decreased the rate of adenosine appearance by 90% and increased the rate of AMP appearance 6-fold. The mitochondrial preparations dephosphorylated exogenous AMP; this activity was inhibited by 10 mM Zn2+. We conclude that the adenosine appearing in the extramitochondrial space was not due to a direct release from the matrix, but instead was due to adenine nucleotide release with subsequent conversion to adenosine in the extramitochondrial space.