Differing cardioprotective efficacy of the Na+/Ca2+ exchanger inhibitors SEA0400 and KB-R7943

KB-R7943 and SEA0400 are Na(+)/Ca(2+) exchanger (NCX) inhibitors with differing potency and selectivity. The cardioprotective efficacy of these NCX inhibitors was examined in isolated rabbit hearts (Langendorff perfused) subjected to regional ischemia (coronary artery ligation) and reperfusion. KB-R7943 and SEA0400 elicited concentration-dependent reductions in infarct size (SEA0400 EC(50): 5.7 nM). SEA0400 was more efficacious than KB-R7943 (reduction in infarct size at 1 microM: SEA0400, 75%; KB-R7943, 40%). Treatment with either inhibitor yielded similar reductions in infarct size whether administered before or after regional ischemia. SEA0400 (1 microM) improved postischemic recovery of function (+/-dP/dt), whereas KB-R7943 impaired cardiac function at >/=1 microM. At 5-20 microM, KBR-7943 elicited rapid and profound depressions of heart rate, left ventricular developed pressure, and +/-dP/dt. Thus the ability of KB-R7943 to provide cardioprotection is modest and limited by negative effects on cardiac function, whereas the more selective NCX inhibitor SEA0400 elicits marked reductions in myocardial ischemic injury and improved +/-dP/dt. NCX inhibition represents an attractive approach for achieving clinical cardioprotection.     

Nitroxide Radicals Prevent Metal-aggravated Reperfusion Injury in Isolated Rat Heart

The effects of Cu(II) and the stable nitroxide radical 4-OH-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TPL) on reperfusion injury following global myocardial ischemia have been studied using the isolated rat heart model in the Langendorff configuration. Hearts were equilibrated with Krebs-Henseleit buffer (KH-buffer) for 10 min and subjected to 18 min of normothermic global ischemia. After 20 min reperfusion, hemodynamic parameters recovered as follows: ventricular developed pressure (77%), dP/dt (71%) and -dP/dt (80%), heart rate (91%), and work index (70%). End-diastolic pressure was 16 mm Hg. When 10μM Cu-nitrilotriacetate or Cu-(histidine)₂ was included in the perfusate before, during, and following ischemia, the heart injury was more extensive and the work index only recovered to 17% of the preischemic value. The inclusion of 100μM TPL during reperfusion abolished the copper-induced sensi-tization. In the absence of copper, TPL did not provide any protection against ischemia-reperfusion damage to the heart. The inclusion of 100μM 1, 4-dihydroxy-2, 2, 6, 6-tetramethylpiperidine (TPL-H) during reperfusion, partially abolished the copper-induced sensitization. Since conversion between TPL and TPL-H takes place, the fact that both forms provide protection can increase their protective efficacy.     

Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

These experiments examined the independent effects of short-term exercise and heat stress on myocardial responses during in vivo ischemia-reperfusion (I/R). Female Sprague-Dawley rats (4 mo old) were randomly assigned to one of four experimental groups: 1) control, 2) 3 consecutive days of treadmill exercise [60 min/day at 60-70% maximal O2 uptake (VO2 max)], 3) 5 consecutive days of treadmill exercise (60 min/day at 60-70% VO2 max), and 4) whole body heat stress (15 min at 42 degrees C). Twenty-four hours after heat stress or exercise, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was maintained for 30-min followed by a 30-min period of reperfusion. Compared with control, both heat-stressed animals and exercised animals (3 and 5 days) maintained higher (P < 0.05) left ventricular developed pressure (LVDP), maximum rate of left ventricular pressure development (+dP/dt), and maximum rate of left ventricular pressure decline (-dP/dt) at all measurement periods during both ischemia and reperfusion. No differences existed between heat-stressed and exercise groups in LVDP, +dP/dt, and -dP/dt at any time during ischemia or reperfusion. Both heat stress and exercise resulted in an increase (P < 0.05) in the relative levels of left ventricular heat shock protein 72 (HSP72). Furthermore, exercise (3 and 5 days) increased (P < 0.05) myocardial glutathione levels and manganese superoxide dismutase activity. These data indicate that 3-5 consecutive days of exercise improves myocardial contractile performance during in vivo I/R and that this exercise-induced myocardial protection is associated with an increase in both myocardial HSP72 and cardiac antioxidant defenses.     

Protection Against Free Radical-Induced and Transition Metal-Mediated Damage: The Use of “Pull” and “Push” Mechanisms

Free radicals have been incriminated in a variety of injurious processes including the toxicity of the herbicide paraquat and the damage following ischemia and reperfusion of different organs.

Based on the assumption that iron and copper could serve as mediators for the transformation of relatively low reactive species (such as superoxide radicals, hydrogen peroxide, axorbate, and others) to the highly reactive species, in the site-specific metal-mediated mechanism, two new modes for intervention have been tried out. The first is the introduction of specific chelators that “pull” out redox-active and available metals, and by this reduce the apparent damage. Desferrioxamine was shown to protect bacterial cells and mammals against the poisonous effects of paraquat. Using the retrogradly perfused isolated rat heart, we have demonstrated that the chelator neocuproine, which effectively binds both iron and copper provides a major protection against hydrogen peroxide-induced cardiac damage and against ischemia/ reperfusion-induced arrhythmias. Likewise, TPEN a heavy metal chelator. provides almost total (> 90%) protection against ischemia/reperfusion-induced arrhythmias.

The other mode of intervention is the use of redox-inactive metal ions that could compete for the binding sites of iron and copper, and by this “push” these metal ions out, lead to their displacement, and divert the site of free radical attack. Applying Zn(II) complexes provided a marked protection against metal mediated free radical-induced damage in the copper-mediated paraquat toxicity to E. coli, and in the arrhythmias induced by ischemia and reperfusion.

It is proposed that the complex zinc-desferrioxamine would be the ultimate protector being effective by both the “pull” and “push” mechanisms.     

Organ blood flow and cardiac contractility in anaesthetized cats at 5 bar (500 kPa) ambient pressure

Previous in vivo and in vitro experiments have demonstrated increased cardiac contractility and increased total myocardial blood flow (Qmyocardial) when rats were exposed to normoxic 5-bar (500 kPa) ambient pressure. In the present study, regional blood flow was measured using the microsphere method on nine anaesthetized cats at surface and normoxic 5-bar (500 kPa) ambient pressure. Left ventricular pressure (LVP) and cardiac contractility, measured as peak left ventricular +dP/dt and -dP/dt were measured in six of the cats. Arterial pressure, heart rate and cardiac output remained unchanged after compression, but total Qmyocardial increased by 29% (P less than 0.01) and cerebral blood flow increased by 66% (P less than 0.05). At the same time +dP/dt and -dP/dt was increased by 83% and 102%, respectively (P less than 0.01), while LVP was enhanced by 14% (P less than 0.05). Except for a moderate decrease in partial pressure of oxygen, acid base status in arterial blood remained unchanged. The results indicate that the effects of increased ambient pressure on the heart are general physiological phenomena, which are not only limited to the laboratory rat.     

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-epsilon, may slow the rates of cardiac contraction and relaxation (+dP/dt and -dP/dt). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser(43) and Ser(45) and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater -dP/dt than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/dt increased by 33% and -dP/dt increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.     

Cardioprotective effects of stretch are mediated by activation of sarcolemmal, not mitochondrial, ATP-sensitive potassium channels

To determine whether sarcolemmal and/or mitochondrial ATP-sensitive potassium (K(ATP)) channels (sarcK(ATP), mitoK(ATP)) are involved in stretch-induced protection, isolated isovolumic rat hearts were assigned to the following protocols: nonstretched hearts were subjected to 20 min of global ischemia (Is) and 30 min of reperfusion, and before Is stretched hearts received 5 min of stretch + 10 min of no intervention. Stretch was induced by a transient increase in left ventricular end-diastolic pressure (LVEDP) from 10 to 40 mmHg. Other hearts received 5-hydroxydecanoate (5-HD; 100 microM), a selective inhibitor of mitoK(ATP), or HMR-1098 (20 microM), a selective inhibitor of sarcK(ATP), before the stretch protocol. Systolic function was assessed through left ventricular developed pressure (LVDP) and maximal rise in velocity of left ventricular pressure (+dP/dt(max)) and diastolic function through maximal decrease in velocity of left ventricular pressure (-dP/dt(max)) and LVEDP. Lactate dehydrogenase (LDH) release and ATP content were also measured. Stretch resulted in a significant increase of postischemic recovery and attenuation of diastolic stiffness. At 30 min of reperfusion LVDP and +dP/dt(max) were 87 +/- 4% and 92 +/- 6% and -dP/dt(max) and LVEDP were 95 +/- 9% and 10 +/- 4 mmHg vs. 57 +/- 6%, 53 +/- 6%, 57 +/- 10%, and 28 +/- 5 mmHg, respectively, in nonstretched hearts. Stretch increased ATP content and did not produce LDH release. 5-HD did not modify and HMR-1098 prevented the protection achieved by stretch. Our results show that the beneficial effects of stretch on postischemic myocardial dysfunction, cellular damage, and energetic state involve the participation of sarcK(ATP) but not mitoK(ATP).     

Inhibition of PLC improves postischemic recovery in isolated rat heart

The Ca2+-dependent PLC converts phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Because these products modulate Ca2+ movements in the myocardium, PLC may also contribute to a self-perpetuating cycle that exacerbates cardiomyocyte Ca2+-overload and subsequent cardiac dysfunction in ischemia-reperfusion (I/R). Although we have reported that I/R-induced changes in PLC isozymes might contribute to cardiac dysfunction, the present study was undertaken to examine the beneficial effects of the PLC inhibitor, U-73122, as well as determining the role of Ca2+ on the I/R-induced changes in PLC isozymes. Isolated rat hearts were subjected to global ischemia 30 min, followed by 5 or 30 min of reperfusion. Pretreatment of hearts with U-73122 (0.5 microM) significantly inhibited DAG and Ins(1,4,5)P3 production in I/R and was associated with enhanced recovery of cardiac function as indicated by measurement of left ventricular (LV) end-diastolic pressure (EDP), LV diastolic pressure (LVDP), maximum rate of pressure development (+dP/dtmax), and maximum rate of LV pressure decay (-dP/dtmax). Verapamil (0.1 microM) partially prevented the increase in sarcolemmal (SL) PLC-beta1 activity in ischemia and the decrease in its activity during the reperfusion phase as well as elicited a partial protection of the depression in SL PLC-delta1 and PLC-gamma1 activities during the ischemic phase and attenuated the increase during the reperfusion period. Although these changes were associated with an improved myocardial recovery after I/R, verapamil was less effective than U-73122. Perfusion with high Ca2+ resulted in the activation of the PLC isozymes studied and was associated with a markedly increased LVEDP and reduced LVDP, +dP/dtmax, and -dP/dtmax. These results suggest that inhibition of PLC improves myocardial recovery after I/R.     

Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.     

Maintenance of left ventricular function (90%) after twenty-four-hour heart preservation with deferoxamine.

During 24-h in vitro heart preservation and reperfusion, irreversible tissue damage occurs caused by reactive oxygen intermediates, such as superoxide radicals, singlet oxygen, hydrogen peroxide, hydroperoxyl, hydroxyl radicals, as well as the peroxynitrite radical. Reduction of the related oxidative damage of reperfused ischemic tissue by free radical scavengers and metal chelators is of primary importance in maintaining heart function. We assessed whether deferoxamine (DFR) added to a cardioplegia solution decreased free radical formation during 24-h cold (5 degrees C) heart preservation and normothermic reperfusion (37 degrees C) in the Langendorff isolated perfused rat heart. The deferoxamine treated hearts were significantly (p less than .001) better preserved than the control hearts after 24 h of preservation with regard to recovery of left ventricular diastolic pressure, contractility (+dP/dt), relaxation (-dP/dt), creatine kinase release, and lipid peroxidation. DFR preserved cell membrane integrity and maintained 93% of left ventricular contractility. The evidence suggests that DFR reduces lipid peroxidation damage by reducing free radical formation and thereby maintaining normal coronary perfusion flow and myocardial function.     

Gender differences in the cardiac response to dietary conjugated linoleic acid isomers

The present study was undertaken to assess the heart function, by the in vivo catheterization technique, of healthy male and female Sprague-Dawley rats fed different conjugated linoleic acid (CLA) isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. Whereas the triglyceride form of the CLA isomer mix lowered the heart rate, the rate of contraction (+dP/dt) and rate of relaxation (-dP/dt), systolic and diastolic pressures, mean arterial pressure, and the left ventricular systolic pressure were higher in male rats as compared with all the other dietary groups. In contrast, there were no significant effects in the cardiac function of the female rats in response to the CLA isomer mix in triglyceride form. Whereas the heart rate, +dP/dt, and left ventricular systolic pressure were lower in male rats fed the t10,c12 CLA isomer alone, the heart rate of the female rats was higher, but the systolic pressure, +dP/dt, and mean arterial pressure were lower compared with the control group. Also, the left ventricular end-diastolic pressure was specifically higher in the female rats in response to free fatty acids-containing CLA mix. Furthermore, an additive effect of the free fatty acids-containing CLA mix was seen in the +dP/dt and -dP/dt of female rats compared with the control group. These results indicate that CLA isomers exert differential effects on heart function and suggest the need for a complete evaluation of the benefits, interactions, and potential side effects of each isomer.     

The effect of zinc on reperfusion arrhythmias in the isolated perfused rat heart

Considerable evidence suggests that free radicals engendered by redox-active metals, particularly iron and copper, are causative agents in reperfusion injury following ischemia. This study demonstrates that perfusion of the isolated rat heart with a buffer containing zinc, a non-redox active metal similar to copper in its coordination chemistry, inhibits the development of ventricular arrhythmias during reperfusion. Zinc was employed as the bishistidine complex, Zn--His2, to maintain solubility and permeability. Zn--His2 exerted an antiarrhythmic activity as hearts spent a longer time in normal sinus rhythm and a shorter time in ventricular fibrillation during reperfusion following 10 min of regional ischemia. However, Zn--His2 also produced a negative inotropic and chronotropic effect, evident during equilibration and ischemia. In the course of experiments which began in Israel and continued in the U.S. it was necessary to use two different sources of rats. Hearts from the two sources manifested different sensitivities to the concentrations of Zn--His2, although their physiological effects were similar. Differential activity responses were noted for antiarrhythmic activity, negative inotropic and chronotropic properties, and toxicity. In both groups of untreated hearts the incidence of ventricular fibrillation after ischemia was 100%. Ventricular fibrillation was reduced to 17% at 37.5 microM Zn--His2 in the U.S.-bred rat hearts and to 9% at 200 microM Zn--His2 in those from Israel. These changes in Zn--His2 treated animals were accompanied by a decrease in lactate dehydrogenase release from the myocardium during reperfusion. None of the protective effects was due to histidine alone. These results indicate that zinc prevents ventricular arrhythmias during reperfusion following regional ischemia and may prevent membrane damage, possibly, by reduction of free radical formation.     

Increased incidence of isoproterenol-induced ventricular fibrillation in aging rats

Prolonged beta-adrenergic stimulation obtained by subcutaneous injection of isoproterenol in unanesthetized, unrestrained rats elicited ventricular fibrillation in approximately 80% of animals at 10-12 months of age. Ventricular fibrillation failed to occur in 1-month-old rats and involved only 12% of rats at 2 months. Senescence appeared not to increase the frequency of ventricular fibrillation since a similar incidence was seen in rats at 10-12 and 19-21 months. In all instances, ventricular fibrillation was preceded by ECG changes consistent with acute subendocardial ischemia. To evaluate whether acute beta-adrenergic stimulation elicits comparable cardiovascular effects in animals of different age, a dose-response curve to intravenous injection of isoproterenol was performed in anesthetized rats. Changes in heart rate, systemic arterial pressure, left ventricular pressure, and dP/dt were not different among animal groups. It was concluded that the arrhythmogenic potential of isoproterenol may not be related to differences in cardiac beta-receptor sensitivity with age as suggested by the comparable changes in the inotropic and chronotropic actions of isoproterenol in the animal groups studied.     

Hydrogen peroxide enhanced inron-induced injury in isolated heart and ventricular cardiomyocyte in rats

To explore the cardiac effects of iron with or without hydrogen peroxide, the isolated perfused rat heart and enzymatically isolated ventricular cardiomyocyte were used. It was shown that treatment with cell-permeable iron (Fe-HQ) for 10 min reduced the contractile amplitude and velocity and end diastolic cell length in the cardiomyocyte and increased the contents of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary effluent and malondialdehyde (MDA) in the myocardium. The left ventricular developed pressure (LVDP), ± dP/dtmax, and heart rate and coronary flow are showed a biphasic phase, an increase at first followed by a decline. Treatment with hydrogen peroxide for 10 min following Fe-HQ augmented the effect of iron with an increase in coronary LDH and CK release and myocardial MDA content, and decrease in LVDP, ± dP/dtmax and heart rate. Perfusion of reduced glutathione with hydrogen peroxide counteracted these effects of Fe-HQ and hydrogen peroxide while dimethyl sulfoxide had no effect on the injury induced by Fe-HQ and hydrogen peroxide in the isolated rat heart. This suggests that augmentation of myocardial injury as a result of an increase in intracellular iron by hydrogen peroxide might involve the dysfunction of sulfydryl group containing proteins but not the hydroxyl radicals.     

Energy metabolism and contractile function of the heart in diabetic cardiomyopathy: effect of ischemia and reperfusion]

Physiological parameters, rates of mitochondrial respiration, high energy phosphate levels and creatine phosphokinase (CPK) activity were investigated in the hearts from control and alloxan-induced diabetic rabbits before and after 40-min total ischemia and reperfusion. Diabetic hearts demonstrated significant decreases in the rates of contraction (+dP/dt) and relaxation (-dP/dt), heart rates and cardiac work compared to control hearts. Determination of mitochondrial respiration rates in saponin-skinned fibers showed a low mitochondrial respiratory function in diabetic hearts. It was found that the ATP and ADP levels and the total and mitochondrial isoenzyme activities of CPK in diabetic hearts were lowered in comparison with control. A post-ischemic recovery of cardiac performance for diabetic hearts was better than in controls. After reperfusion diabetic hearts had increased ATP levels. The data obtained demonstrate some abnormalities of both cardiac performance and energy metabolism in the hearts of diabetic animals and a decreased sensitivity of the latter to ischemic injury.     

The role of NO in ischemia/reperfusion injury in isolated rat heart

Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dt(max) (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 +/- 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 +/- 5.7 %, p < 0.05). Recovery of both CF and dP/dt(max) after TI was also significantly higher in the adapted hearts (101.5 +/- 5.8 % and 83.64 +/- 3.92 % ) as compared with the controls (71.9 +/- 6.3 % and 35.7 +/- 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 +/- 3.1 %; dP/dt(max) 67.5 +/- 5.92 %) and increased CF to 82.4 +/- 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 +/- 3.1 %; CF 70.3 +/- 3.4 % and dP/dt(max) 43.25 +/- 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) - 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). In conclusion: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.     

N-oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia-reperfusion injury via activation of TRPV1

N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloid that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild-type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. Hearts of WT or TRPV1(-/-) mice were Langendorffly perfused with OLDA (2 x 10(-9) M) in the presence or absence of CGRP8-37 (1 x 10(-6) M), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; RP-67580 (1 x 10(-6) M), a selective neurokinin-1 receptor antagonist; chelerythrine (5 x 10(-6) M), a selective protein kinase C (PKC) antagonist; or tetrabutylammonium (TBA, 5 x 10(-4) M), a nonselective K(+) channel antagonist, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF), and left ventricular peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1(-/-) hearts by increasing LVDP, CF, and +dP/dt and by decreasing LVEDP. CGRP8-37, RP-67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of substance P (SP) and CGRP after OLDA treatment was higher in WT than in TRPV1(-/-) hearts, which was blocked by chelerythrine or TBA. Thus OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC or K(+) channel antagonists. The protective effect of OLDA is void in TRPV1(-/-) hearts, supporting the notion that TRPV1 mediates OLDA-induced protection against cardiac I/R injury.     

Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury

During postischemic reperfusion, free radicals are produced and have deleterious effects in isolated rat hearts. We investigated whether melatonin (MEL) reduces the production of hydroxyl radical (*OH) in the effluent and aids in recovery of left ventricular (LV) function. Hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. Salicylic acid (SAL) was used as the probe for *OH, and its derivatives 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were quantified using HPLC. In addition, thiobarbituric acid reactive substances (TBARS) in the myocardium was measured. Plateaus in the measurement of 2,5- and 2,3-DHBA were seen from 3 to 8 min after reperfusion in each group. The group that received 100 microM MEL+ SAL had significantly reduced amounts of 2,5- and 2,3-DHBA by multiple folds, compared to the SAL group. TBARS was significantly decreased in the 100 microM MEL group (1.20+/-0.36 vs 1.85+/-0.10 micromol/g of drug-free group, p<0.001). More importantly, the 100 microM MEL group significantly recovered in LV function (LV developed pressure, +dp/dt, and -dp/dt; 63.0%, 60.3%, and 59.4% in the 100 microM MEL group; 30.2%, 29.7%, and 31.5% in the drug-free group, respectively; p<0.05). Duration of ventricular tachycardia or ventricular fibrillation significantly decreased in the 100 microM MEL group (100 microM MEL, 159+/-67 sec; drug-free, 1244+/-233 sec; p<0.05). As a result of scavenging *OH and reducing the extent of lipid peroxidation, MEL is an effective agent for protection against postischemic reperfusion injury.     

Time window of nitroxide effect on myocardial ischemic-reperfusion injury potentiated by iron

Transition metals such as iron and copper potentiate the postischemic reperfusion (I/R) injury induced by oxygen-derived radical and nonradical toxic species (ROS). Various natural and synthetic antioxidants have been previously tested to ameliorate such injury, yet the limitations of the common antioxidants are well known. An alternative strategy for combating oxidative damage is presented wherein cell-permeable, nitroxide stable radicals, which act as SOD-mimics and oxidize reduced metals thus prompting the Fenton-like chemistry, are investigated for utility in ameliorating I/R injury. Our study concentrates on the early effect of nitroxide on the myocardial I/R injury. Isolated rat hearts in the Langendorff configuration were equilibrated with Krebs-Henseleit buffer and then subjected to 18 min of normothermic global ischemia followed by 20 min reperfusion. Iron administered as Fe(III)-citrate (10 microM) did not affect the cardiac function under normoxia but did potentiate I/R injury and decreased the recovery during reperfusion. The iron-induced damage was manifested by further deterioration of the cardiac hemodynamic function and the energy status as reflected by decreased tissue level of phosphorylated nucleotides. Nitroxide at 200 microM protected against the iron-potentiated I/R injury by improving the recovery of the hemodynamic function and the cardiac energy status. Exogenously added iron requires bioreduction to form deleterious Fe(II) bound to critical cellular sites. The nitroxide, which enters the cell and oxidizes the reduced metal instantaneously, provided protection even when administered 2 or 3.5, but not 5 min, after the onset of reperfusion. Thus, its narrow therapeutic time window provides insight into the schedule of the I/R injurious process.     

Antioxidant and lysosomotropic properties of acute D-propranolol underlies its cardioprotection of postischemic hearts from moderate iron-overloaded rats

The benefits of acute D-propranolol (D-Pro, non-beta-adrenergic receptor blocker) pretreatment against enhanced ischemia/reperfusion (I/R) injury of hearts from moderate iron-overloaded rats were examined. Perfused hearts from iron-dextran-treated rats (450 mg/kg/week for 3 weeks, intraperitoneal administration) exhibited normal control function, despite iron treatment that elevated plasma iron and conjugated diene levels by 8.1-and 2.5-fold, respectively. However, these hearts were more susceptible to 25 mins of global I/R stress compared with non-loaded hearts; the coronary flow rate, aortic output, cardiac work, left ventricular systolic pressure, positive differential left ventricular pressure (dP/dt), and left ventricular developed pressure displayed 38%, 60%, 55%, 13%, 41%, and 15% lower recoveries, respectively, and a 6.5-fold increase in left ventricular end-diastolic pressure. Postischemic hearts from iron-loaded rats also exhibited 5.6-, 3.48-, 2.43-, and 3.45-fold increases in total effluent iron content, conjugated diene levels, lactate dehydrogenase (LDH) activity, and lysosomal N-acetyl-beta-glucosaminidase (NAGA) activity, respectively, compared with similarly stressed non-loaded hearts. A comparison of detection time profiles during reperfusion suggests that most of the oxidative injury (conjugated diene) in hearts from iron-loaded rats occurred at later times of reperfusion (8.5-15 mins), and this corresponded with heightened tissue iron and NAGA release. D-Pro (2 microM infused for 30 mins) pretreatment before ischemia protected all parameters compared with the untreated iron-loaded group; pressure indices improved 1.2- to 1.6-fold, flow parameters improved 1.70- to 2.96-fold, cardiac work improved 2.87-fold, and end-diastolic pressure was reduced 56%. D-Pro lowered total release of tissue iron, conjugated diene content, LDH activity, and NAGA activity 4.59-, 2.55-, 3.04-, and 4.14-fold, respectively, in the effluent of I/R hearts from the iron-loaded group. These findings suggest that the enhanced postischemic dysfunction and tissue injury of hearts from iron-loaded rats was caused by excessive iron-catalyzed free radical stress, and that the membrane antioxidant properties of D-Pro and its stabilization of sequestered lysosomal iron by D-Pro may contribute to the cardioprotective actions of D-Pro.