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Notes: Summary The purpose of this report is to describe the contribution of propionate as an adjunct source of oxidative metabolism in aerobic myocardium. In the first series of studies, six groups of isolated working rat hearts (n=6–8 per group) were perfused for 40 minutes with Krebs-Henseleit media containing 11 mM glucose. Propionate treatment was provided to the media at a constant dose per heart group and extended over a range of dosages, including: 0 (placebo control), 0.1, 0.5, 1.0, 5.0, and 10.0 mM, buffered to pH 7.4. Average aerobic coronary blood flow for all groups was 21.5\+-0.6 ml/min; average left ventricular peak systolic pressure was 123.7\+-1.4 mmHg. There were no significant differences among groups compared with placebo hearts for aortic flow, heart rate x aortic pressure product, or myocardial oxygen consumption, although performance tended to decline in the 10 mM group. A clear dose-response relationship was observed in 14CO2 production from labeled propionate, with a 12-fold increase between the 0.1 and 10 mM groups. Most of the increase occurred at the lower dosages, with a relative leveling off at the 1.0, 5.0, and 10.0 mM doses. In part 2, prpionate was examined as a sole substrate. At 1.0 mM without glucose, proionate per se was unable to support mechanical function over the course of the perfusions, but still maintained high rates of oxidation, comparable to that of the 1.0 mM group with glucose in part 1. Thus propionate proved to be a useful intermediate for substrate oxidation in mammalian hearts, and its resulting contribution to energy metabolism may provide one mechanism to understand the benefits of the propionyl-L-carnitine compound.

Notes: Summary A method for estimating regional contractility is described using the end-systolic relationships between left ventricular pressure and myocardial segment-lengths in rapidly volume-loaded beats. The approach was based on the success of previously developed end-systolic relationships between left ventricular used to describe glopal contractility in beating hearts. The regional end-systolic relationship was more complicated than its global counterpart, which was load independent, and appeared curvilinear to rapid volume loading. As an approximation of this relationship, a linear slope was constructed between maximum and minimum (pre-ejection) loaded beats of equal cycle length. Because of its load dependency and in order to compare slope relationships between interventions, slope functions were derived only from similarly loaded beats either within or between interventions. Slopes generated by this technique had a reasonable constancy at control conditions and coronary flows with an average SEM of 9.1% of the slope means. End-systolic slopes also appeared sensitive to changes in contractile state, increasing appropriately following treatments with dobutamine and decreasing after propranolol. Following shifts in the end-systolic slopes were unreliable, however, in describing the regional changes in contractility with ischemia. At milder levels of flow restriction, the slopes declined as expected. At moderate levels of flow restriction, the pressure-segment loops shifted markedly rightward and the slope increased. At advanced levels of ischemia, the loops were so distorted, that end-systole could not be identified accurately and the loops essentially described the diastolic compliance characteristics of the left ventricle. Thus the slope estimates of regional contractility as described in this report provided a reliable assessment of inotropic background during modifications with positive and negative inotropic drugs but became invalid as systolic shortening was replaced by aneurysmal bulging high-grade ischemia.

Notes: Summary Fatty acid metabolites (long-chain esters of CoA and carnitine) which collect in ischemic myocardium can form amphiphiles capable of disrupting subcellular performance. It is important to document the role of these amphiphiles in intact tissue. D-Octanoylcarnitine was chosen because of its previously described effects on inhibiting palmitoylcarnitine transferasc (PCT-II) inin vitro andin vivo liver preparations. This inhibition will shift tissue levels of CoA and carnitine intermediates and thus alter amphiphile levels. The compound's actions in cardiac muscle are unknown. Dose response curves were developed in intact hearts to test the influence of D-octanoylcarnitine at pharmacological concentrations. Measurements were obtained in working, extracorporeally perfused, swine hearts. Drug was administered either systemically (IV) or via dircct intracoronary (IC) infusions into the left anterior descending coronary circulation. Excess fatty acids were provided to ensure adequate fatty acid substrate for oxidation. Coronary flow was controlled at aerobic levels. Systemic administration of D-octanoylcarnitine (0.8–6.8 mM) resulted in transient peripheral hypotension which caused correlative decreascs in14CO2 production from labeled palmitate. Infusion of D-octanoylcarnitine (0.5–3.9 mM) IC did not cause appreciable hypotension and was not associated with suppression of fatty acid oxidation. No build-up of carnitine esters was noted in treated hearts but acyl CoA levels were reduced (p〈-0.002). This latter finding was modestly related to increasing dose schedule of the compound in the IC group. The lack of suppression in fatty acid oxidation argues against significant inhibition of PCT II and lessens the attractiveness of using D-octanoylcarnitine in intact myocardium to selectively bloock fatty acid utilization at this locus.

Notes: Summary Toxic peroxides result when oxygen radicals with various peroxidic precursors in tissue or blood. A colorimetric method based on reactions of malondialdehyde (MDA), a key intermediate in the formation of peroxides, has been described in fresh tissues. The present report adapts this assay to measure MDA levels in frozen heart muscle from rats and swine. Mean tissue values of MDA ranged from 154–353 nmol/g (n=13) in fresh rat hearts perfused by the Langendorff technique. Values in frozen samples were 228±14 nmol MDA/g (n=22). When mechanical function was increased in isolated working rat hearts, tissue MDA levels decreased by-25

Notes: Abstract The purpose of this precis is to state and support a personal viewpoint concerning intermediary metabolism and substrate utilization in stunned and reperfused myocardium following an interval of nonnecrotizing myocardial ischemia. The data in support of this viewpoint were derived from an intact, working pig heart preparation which includes extracorporeal control of regional coronary perfusion, and which has been used in our laboratory for almost two decades. Metabolism was characterized using steady-state labeling of myocardium with tracer isotopes including [U-14C]palmitate, [9,10-3H]palmitate, [6-14C] glucose, [5-3H]glucose, [2-14C]pyruvate, [14C]lactate, and [1-14C]acetate either singly or in combination. Myocardium was rendered mild to moderately ischemic for 30–40 min in separate protocols, and then aerobically reperfused for 40–60 min. Rates of substrate utilization and/or oxidation were routinely measured during the preischemic period, the ischemic interval, and as emphasized in this overview the subsequent period of metabolic adjustments during aerobic reperfusion. The composite data compiled from these experiments indicate that in stunned myocardium metabolism is in rapid transition with greater restoration of aerobic oxidation than mechanical function, a hierarchical shift in preferred substrate relationships back toward aerobic utilization with beneficial washout of noxious amphiphiles, and an overshoot in fatty acid oxidation, presumably due to persistent alterations in regulatory mechanisms affected by the preceding ischemic stress. In select reperfusion studies and depending somewhat on the levels of exogenous fatty acid substrate in perfusate, fatty acid oxidation approximately doubled over preischemic values (6). This augmentation occurred in part from fatty acids made available from release of intra cellular stores, principally triacyclglycerols, as we showed (8) and in part from increased uptake of exogenous suostrate as demonstrated by Saddik and Lopaschuk (12). This rapid ascendancy in fatty acid preference, however, does not imply a substrate immunity from the normal regulatory inhibitions imposed by competing substrates. In separate studies we provided to recovering myocardium excess propionate to influence the kinetcs of anaplerotic entrance to the citric acid cycle (4). This competing substrate effected a 38% decrease in the oxidation of fatty acids which was not explained by the scavenging of coenzyme A units otherwise destined for fatty acid activation It has been argued from data collected in isolated preparations that the resurgency in fatty acid oxidation is of no benefit but rather a negative inotropic intluence on mechanical recovery (1, 7). However, in our intact model using whole blood perfusate this was not the case (6). A second objective in our experiments was to place in perspective the role of carbohydrate substrate utilizations using glucose, pyruvate, and lactate as representative analogues and to define their relationships to those of fatty acids as regulated substrates. Glucose flux and glucose oxidation were highly suppressed during preischemia, presumably due to allosteric and product inhibitions imposed by the preferred use of fatty acids (2, 3, 9). In moderately ischemic pig hearts with a 60% reduction in regional coronary flow, glycolysis rose nearly tenfold, and even glucose oxidation was increased slightly (2, 9). When multiple exposures of brief ischemic pretreatments were applied in protocols designed to simulate preconditioning (2), flux rates for glycolysis were significantly reduced. During reperfusion, glucose utilization and oxidation fell toward normal preischemic values and lactate release into perfusate, which had been observed during ischemia, reverted to lactate extraction, consistent with aerobic myocardium. Increased levels of glucose in perfusate tended to maintain somewhat higher rates of glycolysis in reperfusion (3). The shift in glucose utilization toward normal preischemic levels were compatible with the allosteric and product inhibitions imposed by the rebound in fatty acid metabolism. To further test this hypothesis, oxfenicine (S-4-hydroxyphenylglycine), an inhibitor of fatty acid utilization and specifically of carnitine palmitoyltransferase I, was administered in separate studies (9). With relief of the regulatory influence of fatty acids, glucose oxidation appropriately increased twofold in stunned myocardium. It was suggested also that pyruvate and lactate oxidation responded predictably to the regulatory influences of fatty acids (10, 11). Utilization rates of both substrates were decreased from preischemic levels during withdrawal of oxygen supply in moderate ischemia but remained depressed during aerobic reperfusion. Treatment with oxfenicine in separate studies caused an appropriate restoration of pyruvate oxidation during reflow to near preischemic values (10). It is noteworthy that if one compares the metabolic adjustments in stunned, acutely reperfused myocardium with those of an acutely reperfused, chronic pig model simulating hibemating myocardium (5), important distinctions are evident. In the latter group heart rate was increased, but oxidative capacity was maintained and myocardial oxygen consumption was higher than in acutely developed sham hearts paced at similar rates. Fatty acid oxidation was in the aerobic range compatible with values obtained in acute experiments during early reperfusion following moderate ischemia (6). In chronic hearts, however, in contrast to stunned hearts, glycolytic flux during reflow remained, elevated and was sixfold greater than rates in acute sham hearts. This latter finding suggests a disruption in the normal regulatory controls of fatty acid utilization upon constituent steps within the glycolytic pathway. In summary, in stunned and reperfused myocardium after a reversible period of moderate ischemia rapid adjustments in substrate utilization occur dominated by the return of the preferred use of long-chain fatty acids for energy metabolism. This recovery of fatty acid oxidation may include an overshoot above preischemic aerobic values. Mechanisms of regulation are maintained among substrates such that glucose, pyruvate, and lactate utilization are normally suppressed during reperfusion but may be up-regulated by the inhibition of fatty acid oxidation. Patterns of glucose utilization appear different between stunned and hibernating myocardium.

Notes: Summary The focus of this review centered on describing the effects of excess fatty acids on myocardial recovery during reperfusion following ischemic stress. Effects on mechanical function were modest in our studies and are likely to remain difficult/impossible to measure due to the independent phenomenon of stunning which obfuscates and no doubt dominates the influences of other mechanical determinants. Mitochondria appear capable of again using long-chain fatty acids as a preferred substrate and in the presence of restored oxygen delivery can produce normal levels of CO2. These changes in oxidative metabolism are not mirrored by equal recoveries in mitochondrial energetics. Because of inefficiencies in electron transport and oxidative phosphorylation together with moderate uncoupling of electron transport from oxidative phosphorylation, ATP resynthesis is blunted. This explains in part the absolute decrease in contents of exchangeable nucleotides noted both in cytosol and mitochondria. Further impairments in recovery reside in the inability of the mitochondria to exchange adenine nucleotides into cytosol through the adenine nucleotide translocase antiport. These findings contribute to our understanding of mechanical stunning and may be of value in designing future strategies to optimize the handling of substrates during myocardial reperfusion.

Notes: Abstract The objective of this study was to augment myocardial tissue levels of amphiphiles using a treatment protocol of pantothenic acid, cysteine and dithiothreitol (DTT) in 24hr fasted pigs and to test their influence on mechanical recovery in reperfusion. Eighteen pig hearts were extracorporeally perfused aerobically, subjected to regionally reversible ischemia in the left anterior descending perfusion system and reperfused. Nine hearts served as a placebo group; nine hearts were treated. All hearts received trace-labeled palmitate to measure fatty acid oxidation and were perfused with an infusion of 20% Intralipid to augment perfusate levels of fatty acids. Fasting alone in the presence of carbon substrates in the coronary perfusate was not sufficient to de-inhibit pantothenic acid kinase such that CoA synthesis was not enhanced. Tissue contents of triacylglycerols and phospholipids in reperfused myocardium were no different than in aerobic heart muscle but free CoA and free and total carnitine were reduced, suggesting a leakage of cytosolic contents across injured sarcolemma. Treatment significantly impaired mechanical recovery during reflow, presumable due to the noxious properties of DTT whose reported effects in heart muscle are wide ranging, difficult to predict in intact hearts and may be harmful.