Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium

The role of nuclear membrane phospholipids as targets of phospholipases resulting in the generation of nuclear signaling messengers has received attention. In the present study, we have exploited the utility of electrospray ionization mass spectrometry to determine the phospholipid content of nuclei isolated from perfused hearts. Rat heart nuclei contained choline glycerophospholipids composed of palmitoyl and stearoyl residues at the sn-1 position with oleoyl, linoleoyl, and arachidonoyl residues at the sn-2 position. Diacyl molecular species were the predominant molecular subclass in the choline glycerophospholipids, with the balance of the molecular species being plasmalogens. In the ethanolamine glycerophospholipid pool from rat heart nuclei approximately 50% of the molecular species were plasmalogens, which were enriched with arachidonic acid at the sn-2 position. A 50% loss of myocytic nuclear choline and ethanolamine glycerophospholipids was observed in hearts rendered globally ischemic for 15 min followed by 90 min of reperfusion in comparisons with the content of these phospholipids in control perfused hearts. The loss of nuclear choline and ethanolamine glycerophospholipids during reperfusion of ischemic myocardium was partially reversed by the calcium-independent phospholipase A(2) (iPLA(2)) inhibitor bromoenol lactone (BEL), suggesting that the loss of nuclear phospholipids during ischemia/reperfusion is mediated, in part, by iPLA(2). Western blot analyses of isolated nuclei from ischemic hearts demonstrated that iPLA(2) is translocated to the nucleus after myocardial ischemia. Taken toghether, these studies have demonstrated that nuclear phospholipid mass decreases after myocardial ischemia by a mechanism that involves, at least in part, phospholipolysis mediated by iPLA2.     

Increased severity of reperfusion arrhythmias in mouse hearts lacking histamine H3-receptors

We had previously reported that activation of histamine H(3)-receptors (H(3)R) on cardiac adrenergic nerve terminals decreases norepinephrine (NE) overflow from ischemic hearts and alleviates reperfusion arrhythmias. Thus, we used transgenic mice lacking H(3)R (H(3)R(-/-)) to investigate whether ischemic arrhythmias might be more severe in H(3)R(-/-) hearts than in hearts with intact H(3)R (H(3)R(+/+)). We report a greater incidence and longer duration of ventricular fibrillation (VF) in H(3)R(-/-) hearts subjected to ischemia. VF duration was linearly correlated with NE overflow, suggesting a possible cause-effect relationship between magnitude of NE release and severity of reperfusion arrhythmias. Thus, our findings strengthen a protective antiarrhythmic role of H(3)R in myocardial ischemia. Since malignant tachyarrhythmias cause sudden death in ischemic heart disease, attenuation of NE release by selective H(3)R agonists may represent a new approach in the prevention and treatment of ischemic arrhythmias.     

Mechanical function and fatty acid oxidation in the neonatal pig heart with ischemia and reperfusion

We investigated mechanical function and exogenous fatty acid oxidation in neonatal pig hearts subjected to ischemia, followed by reperfusion. Isolated, isovolumically-beating hearts, from pigs 12 h to 2 days of age, were perfused with an erythrocyte-enriched (hematocrit approximately 15%) solution (37 degrees C). All hearts were studied for 30 min. with a perfusion pressure of 60 mmHg (pre-ischemia). One group of hearts (low-flow ischemia, N = 12) was then perfused for 30 min. with a perfusion pressure of approximately 12 mmHg. In the other group (no-flow ischemic arrest, N = 9), the perfusion pressure was zero for 30 min. Following ischemia in both groups, the perfusion pressure was restored to 60 mmHg for 40 min. (reperfusion). Pre-ischemia parameters for all hearts averaged: left ventricular peak systolic pressure, 99.0 +/- 2.0 mmHg; end diastolic pressure, 1.9 +/- 0.2 mmHg; coronary flow, 3.4 +/- 0.1 ml/min per g; myocardial oxygen consumption, 56.6 +/- 1.6 microliter/min per g and fatty acid oxidation, 33.4 +/- 1.4 nmol/min per g. During low-flow ischemia, hearts released lactate, and the corresponding parameters decreased to: 30.7 +/- 0.9 mmHg; 1.2 +/- 0.3 mmHg; 0.8 +/- 0.1 ml/min per g; 26.6 +/- 2.3 microliters/min per g and 12.9 +/- 1.1 nmol/min per g, respectively. Early in reperfusion in both groups, all parameters, except for fatty acid oxidation, exceeded pre-ischemia values, before recovering to near pre-ischemia values. Late in reperfusion, however, rates of fatty acid oxidation exceeded pre-ischemia rates by approximately 60%. Thus, the neonatal pig heart demonstrated similar recovery following 30 min of low-flow ischemia or no-flow ischemic arrest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dendroaspis natriuretic peptide protects the post-ischemic myocardial injury

The present study used isolated rat hearts to investigate whether (1) Dendroaspis natriuretic peptide (DNP) is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effects of DNP is related to alteration of Bcl-2 family protein levels. The excised hearts of Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg) and coronary flow (CF, ml/min) were continuously monitored. In the presence of 50 nM DNP, all hearts were perfused for a total of 100 min consisting of a 20 min pre-ischemic period followed by a 30 min global ischemia and 50 min reperfusion. Lactate dehydrogenase (LDH) activity in the effluent was measured during reperfusion. Treatment with DNP alone improved the pre-ischemic LVEDP and post-ischemic LVEDP significantly comparing with the untreated control hearts during reperfusion. However, DNP did not affect the LVDP, heart rate (HR, beats/min), and CF. Bcl-2, an anti-apoptotic protein expressed in ischemic myocardium of DNP+ischemia/reperfusion (I/R) group, was higher than that in I/R alone group. Bax, a pro-apoptotic protein expressed in ischemic myocardium of DNP+I/R group, has no significant difference compared with I/R alone group. These results suggest that the protective effects of DNP against I/R injury would be mediated, at least in part, through the increased ratio of Bcl-2 to Bax protein after ischemia-reperfusion.     

Accumulation of long-chain acylcarnitine and 3-hydroxy acylcarnitine molecular species in diabetic myocardium: identification of alterations in mitochondrial fatty acid processing in diabetic myocardium by shotgun lipidomics

Diabetic cardiomyopathy is the result of maladaptive changes in energy homeostasis. However, the biochemical mechanisms underlying dysfunctional lipid metabolism in diabetic myocardium are incompletely understood. Herein, we exploit shotgun lipidomics to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which was reversible upon insulin treatment. Analysis of acylcarnitine molecular species in myocardium unexpectedly identified acylcarnitine molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species. Synthesis of 3-hydroxy acylcarnitine identified the natural products as the 3-hydroxylated acylcarnitines through comparisons of diagnostic fragmentation patterns of synthetic and naturally occurring constituents using tandem mass spectrometry. Diabetes induced an increase of both calcium-independent phospholipase A(2) (iPLA(2)) mRNA and iPLA(2) activity in rat myocardium. Cardiac ischemia in myocardium genetically engineered to overexpress iPLA(2) dramatically increased the amount of acylcarnitine present in myocardium. Moreover, mechanism-based inactivation of iPLA(2) in either wild-type or transgenic myocardium ablated a substantial portion of the acylcarnitine increase. Collectively, these results identify discrete insulin remediable abnormalities in mitochondrial fatty acid processing in diabetic myocardium and identify iPLA(2) as an important enzymatic contributor to the pool of fatty acids that can be used for acylcarnitine synthesis and energy production in myocardium.     

Transgenic MMP-2 expression induces latent cardiac mitochondrial dysfunction

Matrix metalloproteinases (MMPs) are central to the development and progression of dysfunctional ventricular remodeling after tissue injury. We studied 6 month old heterozygous mice with cardiac-specific transgenic expression of active MMP-2 (MMP-2 Tg). MMP-2 Tg hearts showed no substantial gross alteration of cardiac phenotype compared to age-matched wild-type littermates. However, buffer perfused MMP-2 Tg hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion had a larger infarct size and greater depression in contractile performance compared to wild-type hearts. Importantly, cardioprotection mediated by ischemic preconditioning (IPC) was completely abolished in MMP-2 Tg hearts, as shown by abnormalities in mitochondrial ultrastructure and impaired respiration, increased lipid peroxidation, cell necrosis and persistently reduced recovery of contractile performance during post-ischemic reperfusion. We conclude that MMP-2 functions not only as a proteolytic enzyme but also as a previously unrecognized active negative regulator of mitochondrial function during superimposed oxidative stress.     

Myocardial ischemic postconditioning: a brief ischemia causes conversion of resistent reperfusion-induced ventricular fibrillation into the normal rhythm

Brief episodes of myocardial ischemia-reperfusion were shown to be protective against reperfusion injury when used during early reperfusion after a prolonged ischemic episode. This phenomenon has been termed myocardial ischemic postconditioning. In this study, an effect of ischemic postconditioning on persistent reperfusion-induced ventricular fibrillation was studied in the rat isolated heart. 2 minutes of global ischemia on the 15th minute of reperfusion after 30 minutes of regional ischemia effectively abolished the persistent ventricular fibrillation. In non-postconditioned hearts, the ventricular fibrillation continued to the end of reperfusion. The ischemic postconditioning seems to exert a strong antiarrhythmic effect protecting the heart against persistent reperfusion-induced ventricular tachyarrhythmias.     

Protective role of protein kinase C epsilon activation in ischemia-reperfusion arrhythmia

PURPOSE: Ischemic heart disease carries an increased risk of malignant ventricular tachycardia (VT), fibrillation (VF), and sudden cardiac death. Protein kinase C (PKC) epsilon activation has been shown to improve the hemodynamics in hearts subjected to ischemia/reperfusion. However, very little is known about the role of epsilon PKC in reperfusion arrhythmias. Here we show that epsilon PKC activation is anti-arrhythmic and its inhibition is pro-arrhythmic. METHOD: Langendorff-perfused isolated hearts from epsilonPKC agonist (epsilonPKC activation), antagonist (epsilonPKC inhibition) transgenic (TG), and wild-type control mice were subjected to 30 min stabilization period, 10 min global ischemia, and 30 min reperfusion. Action potentials (APs) and calcium transients (CaiT) were recorded simultaneously at 37 degrees C using optical mapping techniques. The incidence of VT and VF was assessed during reperfusion. RESULTS: No VT/VF was seen in any group during the stabilization period in which hearts were perfused with Tyrode's solution. Upon reperfusion, 3 out of the 16 (19%) wild-type mice developed VT but no VF. In epsilonPKC antagonist group, in which epsilonPKC activity was downregulated, 10 out of 13 (76.9%) TG mice developed VT, of which six (46.2%) degenerated into sustained VF upon reperfusion. Interestingly, in epsilonPKC agonist mice, in which the activity of epsilonPKC was upregulated, no VF was observed and only 1 out of 12 mice showed only transient VT during reperfusion. During ischemia and reperfusion, CaiT decay was exceedingly slower in the antagonist mice compared to the other two groups. CONCLUSION: Moderate in vivo activation of epsilonPKC exerts beneficial antiarrhythmic effect vis-a-vis the lethal reperfusion arrhythmias. Abnormal CaiT decay may, in part, contribute to the high incidence of reperfusion arrhythmias in the antagonist mice. These findings have important implications for the development of PKC isozyme targeted therapeutics and subsequently for the treatment of ischemic heart diseases.     

Urotensin II protects ischemic reperfusion injury of hearts through ROS and antioxidant pathway

Urotensin II (UII) is a vasoactive peptide which is bound to a G protein-coupled receptor. UII and its receptor are upregulated in ischemic and chronic hypoxic myocardium, but the effect of UII on ischemic reperfusion (I/R) injury is still controversial. The aim of the present study was to investigate whether UII protects heart function against I/R injury. Global ischemia was performed using isolated perfused Langendorff hearts of Sprague-Dawley rats. Hearts were perfused with Krebs-Henseleit buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Pretreatment with UII (10nM) for 10min increased recovery percentage of the post-ischemic left ventricular developed pressure and ±dp/dt, and decreased post-ischemic left ventricular end-diastolic pressure as compared with I/R group. UII decreased infarct size and an increased lactate dehydrogenase level during reperfusion. Cardioprotective effects of UII were attenuated by pretreatment with UII receptor antagonist. The hydrogen peroxide activity was increased in UII-treated heart before ischemia. The Mn-SOD, catalase, heme oxygenase-1 and Bcl-2 levels were increased, and the Bax and caspase-9 levels were decreased in UII-treated hearts. These results suggest that UII has cardioprotective effects against I/R injury partly through activating antioxidant enzymes and reactive oxygen species.     

Effect of phosphocreatine on the lysophosphoglyceride levels in total ischemia of the rat myocardium

The phospholipid composition of the crude plasma membrane fraction of Langendorff perfused rat hearts has been studied. The effect of phosphocreatine (PCr) and 3-phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI) on lysophosphoglycerides (LPG) level in this fraction isolated from hearts that were totally ischemic for 8 minutes, has been examined. The absolute and relative contents of LPG were significantly increased in ischemic hearts: the lysophosphatidylcholine content was elevated by 94% and that of lysophosphatidylethanolamine--by 77%. Accumulation of these LPG in ischemic myocardium was completely inhibited in the presence of 10 mM PCr or PIMOI in the perfusate. LPG may play a key role in the destruction of sarcolemma. Therefore, these data allow to assume that the protective effect of PCr and PIMOI on the sarcolemma of ischemic myocardium may be the result of their influence on the phospholipid metabolism in the ischemic region of the heart.     

Cholera toxin enhances ischemia-induced arrhythmias in the isolated rat heart--involvement of a guanine nucleotide binding protein (Gs)

Cholera toxin (CTX) at a dose, which disturbed the intestinal functions, was administered into the rat via the tail vein. At 3 hr after injection, the heart was removed and perfused or subject to global ischemia in the Langendorff isolated heart preparation. Electrocardiogram (ECG) was recorded throughout the experiment. The myocardial cAMP content was measured in the intact non-ischemic heart, and in the isolated ischemic heart at 2.5, 5 and 10 min after ischemia. It was found that the incidence and severity of malignant ventricular arrhythmias including ventricular tachycardia (VT) and ventricular fibrillation (VF) was significantly increased during ischemia in the CTX treated group. The cAMP content was also significantly increased in the CTX treated group in both intact non-ischemic and ischemic hearts, indicating an activation of the guanine nucleotide regulatory protein (Gs). The results of the present study provide evidence that activation of Gs during ischemia may also contribute to the genesis of arrhythmia.     

Low over-expression of TNFalpha in the mouse heart increases contractile performance via TNFR1

TNFalpha is a cytokine wit pleiotropic functions in many organs. In the heart increased TNFalpha levels are not only associated with heart failure, but also, paradoxically, with protection from ischemic damage. To test whether the protective role of TNFalpha in the heart is concentration-dependent, we studied two mouse heart models with low (two- to threefold) over-expression of endogenous TNFalpha: mice deficient in a translational repressor of TNFalpha mRNA, TIA-1(-/-), and mice over-expressing human TNFalpha. Hearts lacking TIA-1 were characterized for their endogenous TNFalpha over-expression during normal Langendorff perfusion. To define which TNFalpha receptor mediates cardiac protection, we also used mice lacking the TNFR1 receptor. Contractile function was assessed in isolated hearts perfused in the isovolumic Langendorff mode during and following global no-flow ischemic stress and in response to varying extracellular [Ca(2+)] to determine their contractile response and Ca(2+) sensitivity. All hearts with low over-expression of TNFalpha, independent of human or murine origin, have improved contractile performance and increased Ca(2+) sensitivity (by 0.2-0.26 pCa). Hearts lacking TNFR1 have contractile performance equal to wild type hearts. Recovery from ischemia was greater in TIA-1(-/-) and was diminished in TNFR1(-/-). Better contractile function in TNFalpha over-expressing hearts is not due to improved cardiac energetics assessed as [ATP] and glucose uptake or to differences in expression of SERCA2a or calmodulin. We suggest that low levels of TNFalpha increase the Ca(2+) sensitivity of the heart via a TNFR1-mediated mechanism.     

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.     

Basic FGF reduces stunning via a NOS2-dependent pathway in coronary-perfused mouse hearts

Basic fibroblast growth factor (FGF-2) may protect the heart from ischemia-reperfusion injury (stunning) by stimulating nitric oxide (NO) production. To test this hypothesis, we pretreated coronary-perfused mouse hearts with 1 microg/ml FGF-2 or vehicle control before the onset of ischemia. Intracellular calcium (Ca(i)(2+)) was estimated by aequorin, and NO release was measured with an NO-selective electrode. Hearts perfused with FGF-2 maintained significantly better left ventricular (LV) function during ischemia than hearts perfused with vehicle. FGF-2 significantly delayed the onset of ischemic contracture and improved LV recovery during reperfusion. Ca(i)(2+) was similar in both groups at baseline during ischemia and reperfusion. L-N(6)-(1-iminoethyl)lysine, a selective inhibitor of inducible NO synthase (NOS2), obliterated the protective effects of FGF-2. In transgenic hearts deficient in the expression of NOS2 (NOS2-/-), FGF-2 did not attenuate ischemia-induced LV dysfunction. Measurements of NO release demonstrated that FGF-2 perfusion significantly increased NO in wild-type but not in NOS2-/- hearts. We conclude that basic FGF attenuates myocardial stunning independent of alterations in Ca(i)(2+) by stimulating NO production via an NOS2-dependent pathway.     

Myocardial function following cold ischemic storage is improved by cardiac-specific overexpression of A1-adenosine receptors

Cold ischemic storage of hearts for transplantation is limited to 4-6 h, and therefore the development of strategies to extend preservation time may increase the donor pool of hearts. Overexpression of A1-adenosine receptors (A1AR) can protect hearts from acute ischemic injury, and the purpose of this study was to test the hypothesis that overexpression of A1AR will improve tolerance to longer periods of cold ischemic preservation. Hearts from 18 wild type and 16 transgenic mice with overexpression of A1AR (A1AR Trans) were isolated and perfused, and then subjected to 18 h of preservation in 5 degrees C University of Wisconsin solution followed by 2 h of reperfusion. Left ventricular end diastolic pressure and left ventricular developed pressure were measured as indices of ventricular function. Cell viability was assessed by determination of infarct size and myocardial cell apoptosis. A1AR Trans hearts showed improved function following 18 h of ischemia, as shown by lower end diastolic pressure (p < 0.05) and higher recovery of left ventricular developed pressure (p < 0.05) during reperfusion. A1AR Trans hearts had markedly reduced infarct size (p < 0.05) and decreased apoptosis (p < 0.05). Overexpression of cardiac A1AR imparts cardioprotection during long-term cold ischemic preservation.     

Free oxygen radicals contribute to high incidence of reperfusion-induced arrhythmias in isolated rat heart

Early period of reperfusion of ischemic myocardium is associated with a high incidence of severe tachyarrhythmias including ventricular tachycardia and fibrillation (VT and VF). Free oxygen radicals (FOR) have been identified as one of the principal factors responsible for reperfusion-induced events. However, their role in arrhythmogenesis is not clear. In the present study, in isolated Langendorff-perfused rat hearts subjected to 30 min global ischemia, the onset of reperfusion induced 100% incidence of both VT and VF with their gradual cessation over 5 min of reperfusion. Generation of H2O2 in the myocardium in the first minutes of reperfusion was visualized by means of cerium cytochemistry and confirmed by X-ray microanalysis. The mechanism of the arrhythmogenic effect of FOR may involve inhibition of the sarcolemmal Na+/K+-ATPase, as demonstrated in the rat heart sarcolemmal fraction subjected to FOR-generating system (H2O2 + FeSO4).     

Protaglandins and the antiarrhythmic effect of preconditioning in the isolated rat heart

Our study evaluated the relationship between the endogenous production of prostacyclin and the antiarrhythmic effect of ischemic preconditioning against ischemic and reperfusion-induced tachyarrhythmia. Langendorff perfused rat hearts underwent 30 min regional ischemia with reperfusion. Preconditioning was induced by a single episode of 5 min ischemia and 15 min reperfusion. Prostaglandin 6-keto F₁ (a stable metabolite of prostacyclin) was determined in the coronary effluent.In the control group the incidence of tachyarrhythmia was 31 % during ischemia and 67% during reperfusion. Preconditioning did not affect ischemic arrhythmias but attenuated arrhythmias a reperfusion (8%, p < 0.01) and was associated with increased release of prostacyclin prior to reperfusion. Aspirin abolished the antiarrhythmic effect of preconditioning against reperfusion tachyarrhythmias. However, no relationship was found between suppression of prostacyclin production and the occurrence of arrhythmia in individual hearts.Thus, our findings suggest that metabolites of arachidonic acid via the cyclooxygenase pathway are involved in the protective effect of ischemic preconditioning against reperfusion-induced tachyarrhythmias. (Mol Cell Biochem 160/161: 249–255, 1996)     

The ischemic rat heart releases S100B

S100B is an astrocytic protein assessed in cerebrospinal fluid and serum as a biochemical marker of cerebral injuries. However, increasing evidences suggest the influence of extra cerebral sources on its serum levels. Since it was reported that the injured myocardium expresses S100B, we investigated whether the isolated heart releases this protein. The rat hearts were excised and perfused by the Langendorff technique of isolated heart perfusion. After stabilization, 10 hearts (ischemic group) were submitted to 20 minutes of ischemia and 30 minutes of reperfusion, and 5 hearts (control group) were submitted to 50 minutes of perfusion. The perfusion fluid was collected at pre-ischemia, and 0, 5, 10, 15 and 30 min after ischemia (or equivalent in controls) for S100B and cardiac troponin T (a heart injury marker) assays. In the ischemic group, S100B and troponin T levels increased significantly at time 0 min: S100B values [mug/L, median (IQ25/IQ75)] increased from < or = 0.02 (< or = 0.02/0.03) to 0.38 (0.22/0.84), while troponin T values [mug/L, median (IQ25/IQ75)] increased from 0.31 (0.15/0.45) to 2.84 (2.00/3.63). Our results point to the ischemic heart as an extra cerebral source of S100B.     

Effects of L-carnitine on mechanical recovery of isolated rat hearts in relation to the perfusion with glucose and palmitate

The purpose of this study was to investigate the effects of L-carnitine on the hemodynamic parameters of Langendorff hearts. Isolated rat hearts were perfused with various solutions containing high or low concentrations of fatty acids, additional glucose or no glucose, and L-carnitine or no L-carnitine. The most interesting part of the experiments was the behaviour of the hearts in the reperfusion period after no-flow ischemia of 20 min. The results were: (1) With glucose and high fatty acid concentrations the hearts showed an improved recovery of the left ventricular functions in the reperfusion period compared with low fatty acid concentrations. Without glucose the left ventricular pressure is much lower in the reperfusion period. (2) Addition of L-carnitine improved the recovery of the ischemically damaged hearts. This improvement is especially impressive at low fatty acid concentrations. L-carnitine addition at high fatty acid concentrations but without glucose strongly improved reperfusion behaviour. (3) The coronary flow is increased by 2 experimental conditions: (i) perfusion at low levels of fatty acids, carnitine and with glucose and (ii) high levels of fatty acids and carnitine but without glucose. These findings suggest that supplementation of L-carnitine has a beneficial effect on the isolated heart under various conditions, and possibly on specific human heart diseases.