Theo H.M. Roemen

Uptake and tissue content of fatty acids in dog myocardium under normoxic and ischemic conditions.

The effect of ischemia on the myocardial content of nonesterified fatty acids (NEFA), triacylglycerol, cholesteryl esters, and phospholipids assayed with gas-liquid chromatography was studied in an open-chest dog preparation. Ischemia was induced by partial occlusion of the left interventricular coronary artery during 120 minutes (n=20). Tissue content of the lipid classes was assessed in biopsies taken from ischemic and normoxic areas of the left ventricular free wall. Local venous blood from the concomitant vein of the left interventricular coronary artery was collected to determine myocardial extraction of lipids. In eight other dogs, no ischemia was induced (control group). Under normoxic conditions, NEFA appeared to be present in trace amounts: about 25 nmol/ g wet weight of tissue, representing less than 0.1% of total myocardial fatty acids. During ischemia, NEFA increased in the affected area. This accumulation was most pronounced in the least perfused layer: the subendocardium (up to 172 nmol/g). Blood flow, estimated with radioactively labeled microspheres fell from 0.55 to 0.06 ml/min per g in this particular layer. The uptake of NEFA by the ischemic myocardium was decreased, indicating that enhanced lipolysis of endogenous lipids or reduced combustion may be held responsible for the accumulation of NEFA in ischemic tissue. Since arachidonic and linoleic acids showed the highest relative increase, lipolysis of endogenous phospholipids, rich in these fatty acids, seems to be reasonable. Ischemia had no significant effect on the content of triacylglycerol and cholesteryl esters. Phospholipids tended to decrease in the affected subendocardial layers.

Lipid alterations in isolated, working rat hearts during ischemia and reperfusion: its relation to myocardial damage.

Disturbances in lipid metabolism may play an important role in the onset of irreversible myocardial damage. To investigate the effect of ischemia and reperfusion on lipid homeostasis and to delineate its possible consequences for myocardial damage, Krebs-Henseleit-perfused, working rat hearts were subjected to various periods of no-flow ischemia (10 to 90 minutes) with or without 30 minutes of reperfusion. During ischemia, the rise in nonesterified fatty acids (NEFAs) was preceded by the accumulation of substantial amounts of glycerol, indicating the presence of an active triacylglycerol-NEFA cycle. The subsequent rise in NEFAs (from 0.25 to 1.64 μmol/g dry residue wt after 90 minutes [means]) coincided with the reduction of ATP to values lower than 10 μmol/g dry wt and the rise of AMP, a potent inhibitor of acyl-coenzyme A synthetase, to values exceeding 2 μmol/g dry wt, making the latter compound a good candidate to hamper the turnover of endogenous lipids during prolonged ischemia. Reperfusion resulted in an additional rise in NEFAs (up to 4.1 μmol/g dry residue wt after 60 minutes of ischemia). Neither ischemia nor reperfusion resulted in significant decreases in the tissue content of triacylglycerols and the various phospholipids. During reperfusion recovery of stroke volume was still adequate at tissue NEFA levels thought to be incompatible with normal mitochondrial function.37 A positive correlation (r=0.81) was found between NEFA content of reperfused hearts and cumulative release of lactate dehydrogenase during reperfusion. Accordingly it is concluded that 1) reperfusion results in additional changes in myocardial lipid homeostasis, 2) the accumulating NEFAs are compartmentalized, possibly at the cellular level, and 3) the accumulation of NEFAs is a sensitive marker for myocardial cell damage.

Assessment of fatty acids in dog left ventricular myocardium

The concentration and composition of fatty acids in four lipid classes in biopsies of dog left ventricular myocardium were determined, using gas-liquid chromatography. When precautions were taken to minimize lipolysis during storage of the tissue and the homogenization process, the following results were obtained: 29 +/- 10 nmol non-esterified fatty acids, 2.98 +/- 2.41 mumol triacylglycerol, 149 +/- 51 nmol cholesteryl esters and 23.76 +/- 3.38 mumol phospholipid (expressed as fatty acid moiety per gram of wet tissue). The concentration of non-esterified fatty acids was 15 to 300 times lower than reported in literature. The main constituents of the non-esterified fatty acids were palmitic, stearic and oleic acid. Triacylglycerol consisted of approximately 40% esterified oleic acid. Linoleic acid accounted for 40% of the fatty acids in the cholesteryl-esters class. More than half of the fatty acid moiety of total phospholipids was linoleic acid and arachidonic acid.

Muscular long-chain fatty acid content during graded exercise in humans

We measured the content of long-chain fatty acids (LCFA) in biopsies obtained from the vastus lateralis muscle in humans at rest and after different exercise intensities. Nine volunteers exercised at 65% of maximal oxygen uptake (VO2 max) for 40 min and at 90% of VO2 max for another 15 min on a Krogh bicycle ergometer. LCFA measured in muscle tissue averaged 76 +/- 5 nmol/g wet wt at rest, decreased significantly after exercise at 65% VO2 max to 48 +/- 4 nmol/g wet wt, and increased to 68 +/- 5 nmol/g wet wt (P < 0.05) after high-intensity exercise. The calculated myocyte LCFA content at rest amounted to 69 +/- 5 nmol/g wet wt, decreased by 43% (P < 0.05) after exercise at 65% of VO2 max, and subsequently increased by 54% after exercise at 90% of VO2 max (P < 0.05) compared with the values obtained at the lower workload. The blood plasma LCFA concentration during the low-intensity exercise (366 +/- 23 nmol/ml) was similar to the values obtained at rest (372 +/- 31 nmol/ml) but decreased significantly during the high-intensity workload (249 +/- 49 nmol/ml). From these data it is proposed that 1) in human skeletal muscle, metabolism rather than cellular availability of LCFA governs the rate of LCFA utilization at rest and during exercise, and 2) consequently reduction in muscle LCFA oxidation during high-intensity exercise (e.g., 90% VO2 max) is due primarily to a decrease in mitochondrial LCFA oxidation rate rather than an insufficient cellular availability of LCFA.We measured the content of long-chain fatty acids (LCFA) in biopsies obtained from the vastus lateralis muscle in humans at rest and after different exercise intensities. Nine volunteers exercised at 65% of maximal oxygen uptake (V˙o 2 max) for 40 min and at 90% of V˙o 2 max for another 15 min on a Krogh bicycle ergometer. LCFA measured in muscle tissue averaged 76 ± 5 nmol/g wet wt at rest, decreased significantly after exercise at 65% V˙o 2 max to 48 ± 4 nmol/g wet wt, and increased to 68 ± 5 nmol/g wet wt ( P < 0.05) after high-intensity exercise. The calculated myocyte LCFA content at rest amounted to 69 ± 5 nmol/g wet wt, decreased by 43% ( P < 0.05) after exercise at 65% ofV˙o 2 max, and subsequently increased by 54% after exercise at 90% ofV˙o 2 max( P < 0.05) compared with the values obtained at the lower workload. The blood plasma LCFA concentration during the low-intensity exercise (366 ± 23 nmol/ml) was similar to the values obtained at rest (372 ± 31 nmol/ml) but decreased significantly during the high-intensity workload (249 ± 49 nmol/ml). From these data it is proposed that 1) in human skeletal muscle, metabolism rather than cellular availability of LCFA governs the rate of LCFA utilization at rest and during exercise, and 2) consequently reduction in muscle LCFA oxidation during high-intensity exercise (e.g., 90%V˙o 2 max) is due primarily to a decrease in mitochondrial LCFA oxidation rate rather than an insufficient cellular availability of LCFA.

Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.

Aim:  To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice.

Ketone bodies disturb fatty acid handling in isolated cardiomyocytes derived from control and diabetic rats.

According to the current paradigm, fatty acid (FA) utilization is increased in the diabetic heart. Since plasma levels of competing substrates such as ketone bodies are increased during diabetes, the effect of those substrates on cardiac FA handling was explored. Cardiomyocytes were isolated from control and streptozotocin-treated diabetic rats and incubated with normal (80 microM) and elevated (160 microM) palmitate concentrations in the absence or presence of ketone bodies, including acetoacetate (AcAc). Comparing control cardiomyocytes under normal conditions (80 microM, no AcAc) with diabetic cardiomyocytes (160 microM, 3 mM AcAc) showed that palmitate uptake was increased from 35.2 +/- 4.8 to 60.2 +/- 14.0 nmol x 3 min(-1) x g wet weight(-1) respectively. Under these conditions, palmitate oxidation rates were comparable (58.9 +/- 23.6 versus 53.2 +/- 18.5 nmol x 30 min(-1) x g wet weight(-1)). However, in the absence of AcAc, palmitate oxidation was significantly enhanced in diabetic cardiomyocytes, indicating that ketone bodies are able to suppress cardiac FA oxidation in diabetes. The concomitantly increased FA uptake in diabetic cells, mainly due to the elevated extracellular FA levels, may be responsible for the accumulation of FA and triacylglycerol, as observed in the diabetic heart in situ.

Effects of nicotinic acid and mepacrine on fatty acid accumulation and myocardial damage during ischemia and reperfusion

To assess the nature of ischemia- and reperfusion-induced lipid changes and their consequences for myocardial function and integrity, Krebs-Henseleit perfused, isolated, working rat hearts were treated with nicotinic acid or mepacrine, putative inhibitors of triacylglycerol and phospholipid hydrolysis, respectively. In non-treated hearts 60 min ischemia resulted in a marked rise in myocardial fatty acid (FA) content. The FA content sharply increased further during 30 min reperfusion. Seven out of 16 (44%) hearts fibrillated continuously during reperfusion. Post-ischemic recovery of cardiac output (CO) of the non-fibrillating hearts amounted to 68 +/- 15% of the preischemic value. Nicotinic acid (10 microM) significantly reduced FA accumulation during ischemia (P less than 0.05), but not during reperfusion (0.05 less than P less than 0.10). Post-ischemic recovery of CO was improved (87 +/- 12%). This was neither associated with preservation of myocardial adenine nucleotide content, nor significant reduction of enzyme release. Mepacrine (1 microM) completely abolished reperfusion arrhythmias and improved recovery of CO (88 +/- 7% of pre-ischemic value). The reduction of FA content in ischemic and reperfused hearts did not reach the level of significance. Enzyme release was not attenuated. At 10 microM, mepacrine completely prevented accumulation of FAs during ischemia and reperfusion, abolished reperfusion-arrhythmias, and reduced enzyme release. No concomitant preservation of adenine nucleotides was observed. In conclusion, nicotinic acid and mepacrine are able to reduce ischemia- and reperfusion-induced changes in myocardial lipid metabolism. In addition, both drugs improve post-ischemic functional recovery. It remains to be established whether these effects are causally related.

HEAT STRESS PRETREATMENT MITIGATES POSTISCHEMIC ARACHIDONIC ACID ACCUMULATION IN RAT HEART

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage.The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min.Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p < 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.

The content of non-esterified fatty acids in rat myocardial tissue. A comparison between the Dole and Folch extraction procedures

Oliver and coworkers hypothesized that under certain circumstances NEFA (non-esterified fatty acids = FFA = free fatty acids) might be toxic for myocardial function. Unambiguous conclusions on the putative detrimental effect of intracellularly localized NEFA are hampered by contradictory values published for the NEFA content in normoxic myocardial tissue. From studies in which the assay procedures were carefully evaluated, one might conclude that the NEFA content in dog and rat myocardial tissue will not exceed 60 and 150 nmol/g wet weight, respectively. However, recently Victor and coworkers found considerably higher NEFA values in rat myocardial tissue and suggested that the low NEFA values as measured, for example, in our laboratory, resulted from incomplete extraction when the Folch medium was applied instead of the Dole mixture. Since Victor and coworkers used a modified Dole procedure as described by Hagenfeldt, we evaluated the Folch procedure as well as the original Dole technique and the modified version. Our findings indicate that the lower values found by the Folch technique are more likely to be correct. Incomplete extraction of NEFA did not occur, whereas hydrolysis and transmethylation of phospholipid fatty acids were observed in case of the (modified) Dole procedures.

Chronic catecholamine depletion switches myocardium from carbohydrate to lipid utilisation.

AbstractPurpose: Chronic cardiac transplantation denervation (i.e., global sympathetic denervation with myocardial catecholamine depletion, plus parasympathetic denervation) is known to inhibit myocardial oxidation of glucose. It is not known whether this is due to increased utilization of lactate, lipid or ketone bodies. The purpose of the present study was to test the hypothesis that the extraction and contribution of blood-borne fatty acids (FA) to overall oxidative energy conversion is increased. Methods: In anaesthetised dogs (control n = 6, cardiac denervated n = 6), we investigated fatty acid (FA) utilization. The studies were made at least four weeks after surgical cardiac denervation. Measurements were made of total FAs and with a radio-labelled tracer (U-14C palmitate). Results: The contribution of FA utilisation to overall substrate oxidation rose from 31% (control) to 48% (cardiac denervated). The increase in the ratio (%) of CO2 production from palmitate oxidation to total CO2 production increased from 4.0 ± 1.8 (control) to 10.6 ± 5.8 (denervated, p = 0.04). The time from uptake of FA to release of CO2 product was unaltered. Conclusion: We conclude that the contribution of FA oxidation to overall energy conversion is increased in chronically denervated hearts, which is postulated to result from a decline in the active form of pyruvate dehydrogenase. This would appear to be a result of chronic catecholamine depletion.