Requirement for distinct vesicle-associated membrane proteins in insulin- and AMP-activated protein kinase (AMPK)-induced translocation of GLUT4 and CD36 in cultured cardiomyocytes.

AIMS/HYPOTHESIS: Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation. METHODS: Five VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured. RESULTS: Three VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation). CONCLUSIONS/INTERPRETATION: The involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs.

Divergent effects of rosiglitazone on protein-mediated fatty acid uptake in adipose and in muscle tissues of Zucker rats.

Thiazolidinediones (TZDs) increase tissue insulin sensitivity in diabetes. Here, we hypothesize that, in adipose tissue, skeletal muscle, and heart, alterations in protein-mediated FA uptake are involved in the effect of TZDs. As a model, we used obese Zucker rats, orally treated for 16 days with 5 mg rosiglitazone (Rgz)/kg body mass/day. In adipose tissue from Rgz-treated rats, FA uptake capacity increased by 2.0-fold, coinciding with increased total contents of fatty acid translocase (FAT/CD36; 2.3-fold) and fatty acid transport protein 1 (1.7-fold) but not of plasmalemmal fatty acid binding protein, whereas only the plasmalemmal content of FAT/CD36 was changed (increase of 1.7-fold). The increase in FA uptake capacity of adipose tissue was associated with a decline in plasma FA and triacylglycerols (TAGs), suggesting that Rgz treatment enhanced plasma FA extraction by adipocytes. In obese hearts, Rgz treatment had no effect on the FA transport system, yet the total TAG content decreased, suggesting enhanced insulin sensitivity. Also, in skeletal muscle, the FA transport system was not changed. However, the TAG content remained unaltered in skeletal muscle, which coincided with increased cytoplasmic adipose-type FABP content, suggesting that increased extramyocellular TAGs mask the decline of intracellular TAG in muscle. In conclusion, our study implicates FAT/CD36 in the mechanism by which Rgz increases tissue insulin sensitivity.

Long-chain fatty acid uptake by skeletal muscle is impaired in homozygous, but not heterozygous, heart-type-FABP null mice.

Previous studies with cardiac myocytes from homozygous heart-type fatty acid (FA)-binding protein (H-FABP) -/- mice have indicated that this intracellular receptor protein for long-chain FA is involved in the cellular uptake of these substrates. Based on the knowledge that muscle FA uptake is a process highly sensitive to regulation by hormonal and mechanical stimuli, we studied whether H-FABP would play a role in this regulation. A suitable model system to answer this question is provided by H-FABP +/- mice, because in hindlimb muscles the content of H-FABP was measured to be 34% compared to wild-type mice. In these H-FABP +/- skeletal muscles, just as in H-FABP -/- muscles, contents of FA transporters, i.e., 43-kDa FABPpm and 88-kDa FAT/CD36, were similar compared to wild-type muscles, excluding possible compensatory mechanisms at the sarcolemmal level. Palmitate uptake rates were measured in giant vesicles prepared from hindlimb muscles of H-FABP -/-, H-FABP +/-, and H-FABP +/+ mice. For comparison, giant vesicles were isolated from liver, the tissue of which expresses a distinct type of FABP (i.e., L-FABP). Whereas in H-FABP -/- skeletal muscle FA uptake was reduced by 42-45%, FA uptake by H-FABP +/- skeletal muscle was not different from that in wild-type mice. In contrast, in liver from H-FABP -/- and from H-FABP +/- mice, FA uptake was not altered compared to wild-type animals, indicating that changes in FA uptake are restricted to H-FABP expressing tissues. It is concluded that H-FABP plays an important, yet merely permissive, role in FA uptake into muscle tissues.

Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism.

In order to use giant vesicles for substrate uptake studies in metabolically important tissues, we characterized giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue. We investigated which cell types and which plasma membrane regions are involved in giant vesicle formation and we examined the presence of transporters for metabolic substrates. Analysis of giant vesicles with markers specific for distinct cell types and distinct domains of the plasma membrane reveals that the plasma membrane of parenchymal cells, but not endothelial cells, are the source of the vesicle membranes. In addition, plasma membrane regions enriched in caveolae and involved in docking of recycling vesicles from the endosomal compartment are retained in giant vesicles, indicating that KCl-induced alterations in recycling processes are involved in giant vesicle formation. Giant vesicles contain vesicular lumen consisting of the soluble constituents of the cytoplasm including, fatty-acid binding proteins. Furthermore, giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue are similar in size (10-15 microm) and shape and do not contain subcellular organelles, providing the advantage that substrate fluxes in the different organs can be studied independently of the surface/volume ratio but most importantly in the absence of intracellular metabolism.

Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes.

Recently, we established that cellular contractions increase long-chain fatty-acid (FA) uptake by cardiac myocytes. This increase is dependent on the transport function of an 88 kDa membrane FA transporter, FA translocase (FAT/CD36), and, in analogy to skeletal muscle, is likely to involve its translocation from an intracellular pool to the sarcolemma. In the present study, we investigated whether cAMP-dependent signalling is involved in this translocation process. Isoproterenol, dibutyryl-cAMP and the phosphodiesterase (PDE) inhibitor, amrinone, which markedly raised the intracellular cAMP level, did not affect cellular FA uptake, but influenced the fate of intracellular FAs by directing these to mitochondrial oxidation in electrostimulated cardiac myocytes. The PDE inhibitors 3-isobutyl-1-methylxanthine, milrinone and dipyridamole each significantly stimulated FA uptake as well as intracellular cAMP levels, but these effects were quantitatively unrelated. The stimulatory effects of these PDE inhibitors were antagonized by sulpho- N -succinimidylpalmitate, indicating the involvement of FAT/CD36, albeit that the different PDE inhibitors use different molecular mechanisms to stimulate FAT/CD36-mediated FA uptake. Notably, 3-isobutyl-1-methylxanthine and milrinone increased the intrinsic activity of FAT/CD36, possibly through its covalent modification, and dipyridamole induces translocation of FAT/CD36 to the sarcolemma. Elevation of intracellular cGMP, but not of cAMP, by the PDE inhibitor zaprinast did not have any effect on FA uptake and metabolism by cardiac myocytes. The stimulatory effects of PDE inhibitors on cardiac FA uptake should be considered when applying these agents in clinical medicine.

Impaired muscular contractile performance and adenine nucleotide handling in creatine kinase-deficient mice.

Creatine kinase (CK) forms a small family of isoenzymes playing an important role in maintaining the concentration of ATP and ADP in muscle cells. To delineate the impact of a lack of CK activity, we studied contractile performance during a single maximal tetanic contraction and during 12 repeated tetanic contractions of intact dorsal flexors of CK knockout (CK(-/-)) mice. To investigate the effect on ATP regeneration, muscular high-energy phosphate content was determined at rest, immediately after the contraction series, and after a 60-s recovery period. Maximal torque of the dorsal flexors was significantly lower in CK(-/-) mice than in wild-type animals, i.e., 23.7 +/- 5.1 and 33.3 +/- 6.8 mN. m. g(-1) wet wt, respectively. Lower muscle ATP (20.1 +/- 1.4 in CK(-/-) vs. 28.0 +/- 2.1 micromol/g dry wt in controls) and higher IMP (1.2 +/- 0.5 in CK(-/-) vs. 0.3 +/- 0.1 micromol/g dry wt in controls) levels at the onset of contraction may contribute to the declined contractility in CK(-/-) mice. In contrast to wild-type muscles, ATP levels could not be maintained during the series of 12 tetanic contractions of dorsal flexors of CK(-/-) mice and dropped to 15.5 +/- 2.4 micromol/g dry wt. The significant increase in tissue IMP (2.4 +/- 1.1 micromol/g dry wt) content after the contraction series indicates that ATP regeneration through adenylate kinase was not capable of fully compensating for the lack of CK. ATP regeneration via the adenylate kinase pathway is a likely cause of reduced basal adenine nucleotide levels in CK(-/-) mice.

Ischemic-reperfused isolated working mouse hearts: membrane damage and type IIA phospholipase A2.

For the murine heart the relationships between ischemia-reperfusion-induced loss of cardiac function, enzyme release, high-energy phosphate (HEP), and membrane phospholipid metabolism are ill-defined. Accordingly, isolated ejecting murine hearts were subjected to varying periods of ischemia, whether or not followed by reperfusion. On reperfusion, hemodynamic function was almost completely restored after 10 min of ischemia [83 +/- 14% recovery of cardiac output (CO)], but was severely depressed after 15 and 20 min of ischemia (40 +/- 24 and 31 +/- 24% recovery of CO, respectively). Reperfusion was associated with partial recovery of HEP stores and enhanced degradation of phospholipids as indicated by the accumulation of fatty acids (FA). Myocardial FA content and enzyme release during reperfusion were correlated (r = 0.70), suggesting that membrane phospholipid degradation and cellular damage are closely related phenomena. To investigate the role of type IIA secretory phospholipase A2 (sPLA2) in this process, hearts from wild-type and sPLA2-deficient mice were subjected to ischemia-reperfusion. Postischemic functional recovery, ATP depletion, enzyme release, and FA accumulation were not significantly different between wild-type and sPLA2- deficient hearts. These findings argue against a prominent role of type IIA sPLA2 in the development of irreversible cell damage in the ischemic-reperfused murine myocardium.

Atrial high energy phosphate content and mitochondrial enzyme activity during chronic atrial fibrillation.

OBJECTIVE: Prolonged atrial fibrillation (AF) results in (ultra)structural remodelling of atrial cardiomyocytes resembling alterations seen in ischemia-induced ventricular hibernation. The mechanisms underlying these changes are incompletely understood. In the present study we explored the hypothesis that a profound imbalance in energy status during chronic AF acts as a stimulus for structural remodelling. METHODS AND RESULTS: The content of high energy-phosphates and related compounds together with a selected number of mitochondrial enzymes, known to be altered under ischemic conditions, were determined in tissue samples taken from atria of goats in sinus rhythm (SR) and after 1, 2, 4, 8 and 16 weeks of AF maintained by burst pacing. Atrial remodelling was quantified by counting the percentage of cells with >10% myolysis. During AF structural remodelling developed progressively, after 8 weeks about 40% of the atrial myocytes were affected. The concentration of adenine nucleotides and their degradation products did not change significantly during AF. Also the activity of mitochondrial cytochrome c oxidase activity was similar during AF and SR. Mitochondrial NADH-oxidase and proton-translocating ATPase activities were not induced by AF. The tissue content of phosphocreatine decreased during the first week by 60%, but completely recovered between 8 and 16 weeks of AF. CONCLUSIONS: The analysis of adenine nucleotides during AF provided no indication for the development of severe atrial ischemia. This notion is supported by enzyme cytochemical findings. However, AF-induced atrial remodelling was associated with a transient lowering of phosphocreatine content, suggesting an increase in energy demand during the early phase of AF. The subsequent recovery of the phosphocreatine pool indicates restoration of the balance between energy demand and supply in chronically fibrillating atria.

Depletion of endogenous dopamine stores and shift in beta-adrenoceptor subtypes in cardiac tissue following five weeks of chronic denervation.

Surgical ablation of extrinsic cardiac nerve fibers results in a chronically denervated state of the left ventricle of the heart. The present study was performed to elucidate the effect of a period of 5 weeks of chronic denervation on cardiac catecholamine levels in general and dopamine in particular. Moreover, the possible effect on cardiac beta-adrenoceptor subtypes was investigated. Experiments were performed on adult dogs. In addition to adrenaline and noradrenaline the tissue levels of dopamine were found to be severely depressed. A significant shift from beta1- to beta2-adrenoceptor subtype was observed, while the total beta-adrenoceptor density remained unaffected. The present findings indicate that catecholamine synthesis in chronically denervated hearts is impaired upstream of dopamine and that a shift in beta-adrenoceptor subtype occurs already within a relatively short period of five weeks of denervation, and suggest that the lack of endogenous catecholamines influence the relative expression levels of the two subtypes of beta-adrenoceptors present in cardiac tissue.

Metabolic alterations in the chronically denervated dog heart.

OBJECTIVE: Previous studies have shown that chronic cardiac denervation impairs myocardial glucose oxidation. To investigate this further we tested whether the tissue content of glucose transporters, activity of glycolytic enzymes or metabolic capacity of pyruvate dehydrogenase were altered. Moreover, we investigated whether the decline in glucose utilization was associated with an upregulation of proteins and enzymes involved in fatty acid handling. Chronic cardiac denervation results also in decreased left ventricular efficiency. We explored whether alterations in mitochondrial properties could be held responsible for this phenomenon. METHODS: Twelve adult dogs were included in the study. In 6 of them chronic cardiac denervation was accomplished by surgical ablation of the extrinsic nerve fibers. The other 6 dogs were sham-operated. Biopsies were obtained from the left ventricle after 4-5 weeks of denervation. The content or enzymatic activity of proteins involved in fatty acid and glucose handling was assessed. Features of glutamate oxidation were measured in freshly isolated mitochondria. RESULTS: The content or activity of a set of fatty acid handling proteins did not change during chronic cardiac denervation. In contrast GLUT1 content significantly increased in the chronically denervated left ventricle, while the active form of pyruvate dehydrogenase declined (p < 0.05). Glutamate oxidation characteristics in freshly isolated mitochondria were not affected by chronic denervation. CONCLUSION: The impairment of glucose oxidation in the chronically denervated myocardium is most likely caused by a decline of pyruvate dehydrogenase in its active form. It is unlikely that the decrease in work efficiency is caused by alterations in mitochondrial properties.

The influence of lactate, pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion.

Previous studies have shown that exogenous lactate impairs mechanical function of reperfused ischaemic hearts, while pyruvate improves post-ischaemic recovery. The aim of this study was to investigate whether the diverging influence of exogenous lactate and pyruvate on functional recovery can be explained by an effect of the exogenous substrates on endogenous protecting mechanisms against oxygen-derived free radicals. Isolated working rat hearts were perfused by a Krebs-Henseleit bicarbonate buffer containing glucose (5 mM) as basal substrate and either lactate (5 mM) or pyruvate (5 mM) as cosubstrate. In hearts perfused with glucose as sole substrate the activity of glutathione reductase was decreased by 32% during 30 min of ischaemia (p < 0.10 versus control value), while the activity of superoxide dismutase and catalase was reduced by 27 and 35%, respectively, during 5 min of reperfusion (p < 0.10 versus control value). The GSH level in the glucose group was reduced by 29% following 30 min of ischaemia and 35 min of reperfusion (p < 0.10). In lactate- and pyruvateperfused hearts there were no significant decreases of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activity during 30 min of ischaemia, 5 min of reperfusion or 35 min of reperfusion. In pyruvate-perfused hearts the glutathione peroxidase activity was even increased by 43% during 30 min of ischaemia (p < 0.05). Glutathione levels (reduced and oxidized) did not markedly change in the lactate and pyruvate groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The hydrolysis of glycerol-3-phosphate into glycerol in cardiac tissue: possible consequences for the validity of glycerol release as a measure of lipolysis.

Glycerol release has been generally accepted as an index of lipolysis in the intact heart. The glycerol moiety of glycerol-3-phosphate (glycerol-3-P) is incorporated into triacylglycerols, which are then hydrolysed with release of glycerol. This study investigates the possibility that glycerol may be derived directly from glycerol-3-P instead of passing through the triacylglycerol pool. The cardiac capacity for hydrolysis of glycerol-3-P into glycerol was determined in homogenates of rat hearts. Glycerol-3-P hydrolysis activity in homogenates increased with decreasing pH. The activity was approximately four times higher at pH 5.0 than at pH 7.2 (0.94 +/- 0.11 and 0.25 +/- 0.03 mumol.g wet weight-1.min-1 respectively). The substrate concentration at which half-maximal glycerol-3-P hydrolysis activity was reached did not significantly differ at pH 5.0 and pH 7.2 (4.2 +/- 1.1 mM and 2.9 +/- 1.0 mM respectively). In the intact heart, the pH and substrate conditions found under ischaemia are favourable for direct conversion of glycerol-3-P into glycerol. The glycerol-3-P hydrolysis activity measured in vitro was sufficiently high to account for glycerol production in the ischaemia heart. However, the lack of a stoichiometric relation between cardiac glycerol-3-P and glycerol levels in ischaemia indicates that production of glycerol cannot be explained solely by hydrolysis.

Differences in ischaemia tolerance between hypertrophied hearts of adult and aged spontaneously hypertensive rats.

OBJECTIVE: The aim was to examine differences between the postischaemic functional and biochemical recovery of adult and aged hypertrophied hearts. METHODS: Isolated hypertrophied hearts of adult and aged spontaneously hypertensive rats (SHRadult; SHRaged) and normal hearts of age matched Wistar-Kyoto rats (WKYadult; WKYaged) were perfused in an ejecting heart preparation. Haemodynamic function was monitored before and after 45 min of ischaemia. Coronary effluent samples and tissue biopsies were taken for biochemical analysis. RESULTS: After ischaemia, in SHRadult and WKYadult the maximum positive first derivative of the left ventricular pressure (dP/dtmax) was restored to 105% and 97% respectively of the preischaemic values. Left ventricular developed pressure recovered to 80% (SHRadult) and 97% (WKYadult), while cardiac output reached 71% (SHRadult) and 99% (WKYadult) of preischaemic levels. In SHRaged and WKYaged the dP/dtmax recovered to 26% and 60% respectively (both p < 0.05 compared to the preischaemic values). The left ventricular developed pressure recovered to 36% in SHRaged and to 73% in WKYaged (both p < 0.05), while cardiac output was restored to 6% in SHRaged and 38% in WKYaged (both p < 0.05). Throughout reperfusion, left ventricular end diastolic pressure remained significantly elevated in SHRaged, and was associated with a prominent subendocardial underperfusion, suggesting an impaired diastolic functional recovery. Overall haemodynamic recovery was significantly better in the WKYaged than in the SHRaged. The preischaemic total adenine nucleotides content was comparable in all groups, but creatine phosphate levels were significantly lower in both aged groups than in adult groups. In all but the WKYadult, the total adenine nucleotides were depressed upon reperfusion, while creatine phosphate normalised, except in SHRaged. SHRaged lost more lactate dehydrogenase and tended to lose more xanthine and uric acid than other groups. CONCLUSIONS: The aged hypertrophied heart shows a higher vulnerability to ischaemic damage than the adult hypertrophied heart. This phenomenon is associated with subendocardial underperfusion, increased membrane damage and inadequate recovery of creatine phosphate levels.

Substrate-induced changes in the lipid content of ischemic and reperfused myocardium. Its relation to hemodynamic recovery.

To investigate the effect of lactate, pyruvate, and glucose on the endogenous levels of lipids in the normoxic, ischemic, and reperfused myocardium, isolated working rat hearts were exposed to various grades of ischemic insult (15, 30, or 45 minutes). Glucose was present as the basal substrate in the perfusion medium, and lactate (5 mM) or pyruvate (5 mM) was added as the cosubstrate. Lipid metabolism was evaluated by fatty acid accumulation, triacylglycerol turnover, and phospholipid homeostasis. Exogenous lactate significantly increased fatty acid content above preischemic levels after 45 minutes of ischemia. In glucose-perfused hearts, fatty acid levels were even slightly higher than in lactate-perfused hearts, whereas pyruvate-perfused hearts demonstrated less accumulation of fatty acids. By reperfusion, fatty acid levels in glucose-perfused hearts returned to control values. In lactate- and pyruvate-perfused hearts, fatty acid accumulation was further enhanced by reperfusion. When the fatty acid content exceeded 400 nmol/g dry wt during reperfusion, hemodynamic function was impaired, whereas fatty acid levels below 400 nmol/g dry wt did not correlate with hemodynamic recovery. The total triacylglycerol content did not change during ischemia and reperfusion. However, accumulation of glycerol was remarkable during the first 15 minutes of ischemia in all hearts, and release of glycerol by reperfusion was considerable in lactate-perfused hearts after 30 minutes of ischemia and in all groups of hearts after 45 minutes of ischemia. Release of glycerol in association with maintained levels of triacylglycerols suggests turnover of the triacylglycerol pool. The rate of triacylglycerol cycling correlated poorly with hemodynamic recovery. Accumulation of arachidonic acid revealed disturbances in phospholipid turnover. Arachidonic acid accumulation during reperfusion demonstrated a strong relation with impairment of cardiac function. Hence, derangements in phospholipid homeostasis during reperfusion might be involved in myocardial damage, which is influenced by the substrates available.

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions.

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.

Differences in metabolic response of dog and goat latissimus dorsi muscle to chronic stimulation.

The latissimus dorsi (LD) muscle is considered suitable to assist ventricular mechanical function in either cardiomyoplasty or extra-aortic-assist devices. Such application requires that this mixed-type skeletal muscle be transformed into a fatigue-resistant muscle, the adaptation of which can be elicited by chronic stimulation. In this study the LD muscles of dog and goat were subjected in situ to 12 wk of continuous electrical stimulation through intramuscular electrodes, and their myofibrillar and metabolic adaptations were compared. A gradual increase in the contraction rate of the muscle (in 10 wk from 30 to 80 contractions/min) caused the proportion of immunohistochemically identified type I fibers to increase in dog muscle from 30 to 74% and in goat muscle from 21 to 99%. Correspondingly, the anaerobic-glycolytic activity (fructose-6-phosphate kinase and lactate dehydrogenase activities) decreased by approximately 75% in both dog and goat muscles, whereas the oxidative capacity (fatty acid oxidation and citrate synthase activity) increased two- to threefold in goat LD muscle but remained unaltered in dog LD muscle. Muscular contents of high-energy phosphates and endogenous substrates were maintained, but the L-carnitine content decreased by 43% in both dog and goat. Our data further indicate that, for the monitoring of the metabolic adaptation of skeletal muscle, the ratio of activities of the oxidative and anaerobic-glycolytic pathways (e.g., citrate synthase to fructose-6-phosphate kinase activities) is a useful parameter in both dog and goat.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acid accumulation during ischemia and reperfusion: effects of pyruvate and POCA, a carnitine palmitoyltransferase I inhibitor.

The present study was designed to evaluate the effects of POCA, a carnitine palmitoyltransferase I (CPT I) inhibitor, and pyruvate, a substrate inhibiting fatty acid (FA) oxidation, on post-ischemic cardiac FA accumulation on the one hand, and hemodynamic recovery and loss of cellular integrity on the other. To this end isolated, working rat hearts, receiving glucose (11 mM) as substrate, were subjected to 45 min of no-flow ischemia and 30 min of reperfusion. Hearts were perfused with or without POCA (10 microM) and/or pyruvate (5 mM). In the control group the FA content increased significantly during ischemia and remained elevated during reperfusion. Administration of POCA did not affect functional recovery and LDH release significantly, but resulted in about two-fold increased FA levels upon reperfusion as compared to glucose-perfused hearts. Pyruvate markedly improved functional recovery. Addition of this substrate did not affect lactate dehydrogenase (LDH) release, but enhanced FA accumulation during reperfusion. The combined administration of pyruvate and POCA nullified the positive effect of pyruvate on hemodynamic recovery, aggravated LDH release, and further enhanced the accumulation of FAs. The adenine nucleotide content of reperfused hearts was comparable for all groups investigated. In conclusion, during transient ischemia POCA and pyruvate markedly increased cardiac FA accumulation through inhibition of the oxidation of FAs released from endogenous lipid pools. No clear relation was found between the FA content of reperfused hearts and post-ischemic functional recovery.

The effects of global ischemia and reperfusion on compensated hypertrophied rat hearts.

Abdominal aorta constriction was performed in 10-week-old Lewis rats (Aoband). Ten weeks later the hearts were isolated and attached to a non-recirculating perfusion apparatus. The hearts could eject against a diastolic aortic pressure of either 60 or 100 mmHg. The functional recovery was compared with that of hearts of sham-operated (Sham) rats. After 45 min of global ischemia, Sham hearts regained cardiac output up to 75% and 70% of the pre-ischemic levels at 60 and 100 mmHg, respectively. At 60 mmHg Aoband hearts showed a minor recovery of ejection function. However, at 100 mmHg the recovery of Aoband hearts was completely comparable with that of Sham hearts. At 60 mmHg but not at 100 mmHg, the pre-ischemic and post-ischemic coronary flow was lower in Aoband than in Sham hearts (P less than or equal to 0.05). During the initial reperfusion phase Sham hearts, perfused at 60 mmHg, released more degradation products of adenine nucleotides and lactate dehydrogenase (LDH) than Aoband hearts (P less than or equal to 0.05), while the Aoband hearts lost more degradation products and LDH than the Sham hearts later during the reperfusion phase (P less than or equal to 0.05). In the groups perfused at 60 mmHg, higher tissue levels of ATP were found in Sham than in Aoband hearts at the end of the reperfusion period (P less than or equal to 0.05). However, at 100 mmHg comparable levels were found in the Sham and Aoband hearts. It is concluded that the height of the coronary perfusion pressure is of critical importance for the post-ischemic functional recovery of the compensated hypertrophied heart. At sufficiently high perfusion pressure levels, the functional and biochemical recovery of the hypertrophied heart is at least as good as in the non-hypertrophied heart. However, in the hypertrophied heart a coronary perfusion pressure which is too low leads to relative underperfusion during the initial reperfusion period which is associated with severely depressed cardiac performance and delayed wash-out of metabolites and intracellular enzymes.

Changes in myocardial high-energy phosphate stores and carbohydrate metabolism during intermittent aortic crossclamping in dogs on cardiopulmonary bypass at 34 degrees and 25 degrees C.

The effect of cooling to 25 degrees C on myocardial metabolism was studied during four periods of global ischemia (10 minutes each) followed by 15 minutes of reperfusion in dogs on cardiopulmonary bypass. Systemic and heart temperature at normothermia (group N, 34 degrees C; n = 15) was compared with general hypothermia (group H, 25 degrees C; n = 16). Before and at the end of each aortic crossclamp period in small myocardial biopsy specimens the adenosine triphosphate, creatine phosphate, inorganic phosphate, glycogen, and lactate content was analyzed. Also, lactate and inorganic phosphate were measured in the coronary effluents during the repetitive periods of reperfusion. Hemodynamic function was not different at 60 minutes after cardiopulmonary bypass compared with pre-cardiopulmonary bypass values, and was not different between the groups N and H. The tissue content of adenosine triphosphate and glycogen decreased progressively during the experimental period, resulting in slightly depressed values in both groups at the end of cardiopulmonary bypass. Pronounced effects of ischemia and reperfusion on tissue content of creatine phosphate, inorganic phosphate, and lactate were observed after each period of ischemia. The net decrease in tissue creatine phosphate content was not different between groups N and H (41 +/- 4 versus 38 +/- 4 mumol.gm-1 dry weight; mean +/- standard error of the mean) after 10 minutes of ischemia. However, during ischemia the net inorganic phosphate increase in myocardial tissue was significantly higher in group H (70 +/- 7 mumol.gm-1) than in group N (44 +/- 3 mumol.gm-1). These findings do not support the notion that myocardial protection is improved during hypothermia. Moreover, quantitatively the release of inorganic phosphate and lactate did not correlate with the amount accumulated in the myocardial tissue during the preceding periods of ischemia. The release appeared to be temperature dependent, that is, significantly reduced at 25 degrees C. The present data demonstrate why clinical outcome is satisfactory in both surgical procedures, when in general the periods of aortic crossclamping do not exceed 10 minutes each and the reperfusion periods in between the ischemic episodes last about 15 minutes. Besides, the findings indicate that hypothermia is not strictly necessary under these circumstances.

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.