Nonischemic Hearts Research Articles

Nitric oxide regulates the apoptotic pathway in explanted failing human hearts

Cardiomyocyte apoptosis causes ventricular remodeling in heart failure. Caspases are intracellular enzymes that mediate apoptosis. We investigated nitric oxide (NO) mediated regulation of caspase activity in failing human myocardium to determine if loss of NO production in heart failure contributes to apoptosis. Methods: Ventricular myocardium was obtained from 10 explanted failing human hearts at heart transplantation. 5 patients (pts) were on chronic angiotensin-converting enzyme inhibitors (ACEI) prior to transplantation. Caspase-3 activity was induced using recombinant caspases 8 and 9 in cytosolic extracts of myocardium and measured colorimetrically. Effect of following on caspase activity was measured-exogenous NO donor, S-nitroso-N-acetyl penicillamine (SNAP, 10-5M, 10-4M), endogenous NO inhibitor, nitro-L-arginine methyl ester (L-NAME, 10-4M), and ACEI (enalapril, 10-4M). Results: (i) Caspase 8 and 9 induced caspase-3 activity in myocardial cell lysates (5.44+/-0.6 pmol/10 mcg/min). Exogenous NO donor, SNAP, caused a direct, dose-dependent decrease in induced caspase 3 activity downstream of caspases 8 and 9 (0.52+/-0.5 pmol/10mcg/min at highest dose, p<0.01) (ii) Caspase-3 activity was lower in the 5 pts on chronic ACEI therapy and increased significantly with addition of L-NAME, indicating that ACEIs may decrease caspase activity through increased NO production. This was confirmed by the ability of exogenously added enalapril to reduce myocardial caspase-3 activity (2.48+/-0.4 pmol/10mcg/min). (iii) Caspase activity was significantly higher in nonischemic (9.86+/-1) versus ischemic hearts (5.14+/-0.6) (p<0.05) including caspase-9 induced caspase-3 activity suggesting upregulation of the mitochondrial pathway of apoptosis. Conclusion: NO regulates apoptosis by inhibiting downstream caspase-3 activity in explanted failing human hearts and can potentially rescue a cell from apoptosis even after activation of the caspase cascade. This may have implications for use of NO agonists to inhibit apoptotis in heart failure.

Regulation of ventricular fibrillation by heme oxygenase in ischemic/reperfused hearts.

We have assessed the relationship between reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO) mRNA expression using northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme activity in isolated working ischemic/reperfused rat hearts. Isolated hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. Upon reperfusion with VF, cardiac function was registered (n = 6 in each group), and HO mRNAs and enzyme activities were measured at the end of reperfusion in hearts that showed VF or did not develop VF. The expression of HO-1 mRNA (about fourfold) was observed in ischemic/reperfused nonfibrillated myocardium in comparison with the nonischemic control hearts. In those hearts when VF was developed, the expression of HO-1 mRNA was not observed in comparison with the nonischemic control myocardium. The results measured by RT-PCR and enzyme analysis support the data obtained by northern blotting. In additional studies, we decided to approach the question from a different angle. Thus, the purpose of our work was also to study the role of HO expression and enzyme activity in electrically fibrillated hearts without the ischemic/reperfused protocol. To simulate the period of 10 min of reperfusion-induced VF, hearts were electrically fibrillated, then defibrillated, and perfused for an additional 110 min, and HO-1 mRNA expression and enzyme activities were determined. Thus, electrically induced VF resulted in about 60%, 60%, and 70% reduction in HO-1 mRNA expression, RT-PCR signal intensity, and enzyme activity, respectively, compared with the nonfibrillated ischemic/reperfused group. In conclusion, our data provide evidence that the development of reperfusion-induced VF inhibits HO-1 mRNA expression and enzyme activity in both electrically fibrillated myocardium and ischemic/reperfused fibrillated hearts. The results clearly show that HO-1 mRNA expression and enzyme activity were increased in ischemic/reperfused nonfibrillated myocardium, suggesting that interventions that are able to increase HO-1 mRNA expression and enzyme activity may prevent the development of VF.

Adenosine induced direct negative inotropic effect is abolished during global ischemia: role of protein kinase C and prostacyclin

Adenosine acts as a cardioprotective agent by producing coronary vasodilation, decreasing heart rate and by antagonizing the cardiostimulatory effect of catecholamines; adenosine also exerts a direct negative inotropic effect. Myocardial ischemia is known to be associated with enhanced levels of adenosine, increased protein kinase C (PKC) activity and prostacyclin (PGI2) release. The present study was conducted to determine if myocardial ischemia alters the cardioprotective effect of adenosine by increasing PKC activity and PGI2release in the isolated rat heart perfused at 10 ml/min with Krebs–Henseleit buffer (KHB; 95% O2+5% CO2). Adenosine (10 mmol/min) reduced myocardial contractility as indicated by a decrease in contractility (dp/dtmax), heart rate (HR) and coronary perfusion pressure (PP). In hearts subjected to 30 min of ischemia (without perfusion) and then reperfused with KHB, adenosine failed to decrease dp/dtmax, HR or PP. However, during infusion of PKC inhibitor H-7 (1-(5-Isoquinolinesulfonyl)-2-methylpiperazine hydrochloride) (H-7; 6 mmol/min), which commenced 10 min before ischemia and continued throughout reperfusion, adenosine produced a decrease in dp/dtmax, HR and PP, similar to that before ischemia. Infusion of the PKC activator phorbol 12,13-dibutyrate (PDBu; 2 nmol/min) but not an inactive analogue in non-ischemic hearts prevented the adenosine induced decrease in dp/dtmax. During infusion of H-7, PDBu failed to block the direct negative inotropic effect of adenosine in non-ischemic hearts. In addition, pretreatment with H-7 or indomethacin (cyclooxygenase inhibitor) significantly reduced the PGI2release following ischemia. This data suggest that PKC and PGI2regulate the direct negative inotropic effect of adenosine, which is abolished during ischemia.

Role of Sodium Channels in Ventricular Fibrillation: A Study in Nonischemic Isolated Hearts

Because the role of sodium channels in the initiation and maintenance of VF is not fully elucidated, we studied the significance of sodium channel activity in VF using sodium channel blockers. In nonischemic isolated feline hearts, the following electrophysiologic parameters were measured before and after application of tetrodotoxin (5 x 10(-7) M, n = 6) or lidocaine (1 x 10(-5) M, n = 8): (a) during pacing, epicardial conduction time; refractoriness; the fastest rate for 1:1 pacing/response capture, and all tissue resistivity, indirectly reflecting intercellular electrical resistance; (b) during 8 min of electrically induced tachyarrhythmias, all tissue resistivity; peak frequency (to measure average frequency based on fast-Fourier transformation analysis); and normalized entropy (to measure the degree of arrhythmia organization). In nonischemic isolated rabbit hearts (n = 4), three-dimensional mapping was performed before and after application of lidocaine (1 x 10(-5) M). In feline hearts, lidocaine and tetrodotoxin application resulted in: (a) more spontaneous arrhythmia termination (63-67%) than in nontreated hearts (7%); (b) transformation from mainly VF into ventricular tachycardia with increased organization; and (c) prolongation of conduction time (155-248%) (p < 0.01 for all parameters). The ventricular refractory period was slightly prolonged by tetrodotoxin in the right ventricle and exhibited rate-dependent shortening in control and with lidocaine. Tetrodotoxin and lidocaine reduced the pacing rate for 1:1 pacing/response capture, and all tissue resistivity was not significantly affected. Peak frequency was decreased by tetrodotoxin and lidocaine mainly in the left ventricle (p < 0.01). In nontreated left ventricles, peak frequency was increased over time but was attenuated by lidocaine. In isolated rabbit hearts, several simultaneous wave fronts were detected during VF in nontreated hearts and were reduced to only one or two major wavefronts after application of lidocaine. Suppression of sodium channel activity that primarily slowed conduction time and had little or no effect on ventricular refractory period and all tissue resistivity resulted in less stable and more organized arrhythmias and reduced tachyarrhythmia rate compared with nontreated hearts. These results suggest an active role for sodium channels in the maintenance of ventricular fibrillation.

Pretreatment with growth hormone-releasing peptide-2 directly protects against the diastolic dysfunction of myocardial stunning in an isolated, blood-perfused rabbit heart model.

Brief coronary occlusion followed by reperfusion leads to reversible myocardial dysfunction (stunning) which can induce irreversible damage of other organ systems. We studied the effects of pretreatment with recombinant human GH (rhGH) and the GH-secretagogue GHRP-2 on myocardial stunning in a blood-perfused isolated rabbit heart model. In a first set of experiments, effects of bolus rhGH administration (3.5 mg/kg) (n = 5) into the aortic root of unpretreated animals were compared with those of saline (n = 6). In a second set, animals were pretreated for 14 days with SC rhGH 3.5 mg/kg x day (n = 9) or 160 microg/kg x day GHRP-2 (n = 8) in two divided doses. Body weight and plasma concentrations of rhGH, rabbit GH (rGH) and IGF-I were determined before and at the end of 14 days pretreatment. Hearts were excised and submitted to 15 min ischemia followed by 80 min reperfusion, after which postischemic recovery was compared with nonischemic hearts mounted into the same system. At study end, all hearts were snap-frozen to examine markers of apoptosis. Circulating levels of rabbit GH (rGH) remained identical in all animals. Pretreatment with rhGH for 14 days induced a 142 +/- 116% rise of serum IGF-I vs. 8 +/- 15% with GHRP-2 (P < 0.001) and increased body weight with 6.8 +/- 2.5% vs. 3.4 +/- 3.3% with GHRP-2 (P = 0.01). A bolus injection of rhGH did not alter myocardial function compared with saline allowing data from these experiments to be pooled into one ischemic control group for further analysis of the effect of pretreatment. No difference in postischemic recovery of left ventricular systolic function among the unpretreated, rhGH pretreated and GHRP-2 pretreated hearts was apparent. At the end of reperfusion, a 3-fold higher end-diastolic pressure (EDP) persisted in the unpretreated and rhGH pretreated hearts compared with the nonischemic hearts. In the GHRP-2 pretreated hearts, EDP decreased to half the pressure observed in unpretreated and rhGH pretreated hearts (all P < or = 0.02), a level which was indistinguishable from that in the non-ischemic hearts, suggesting full postischemic recovery of diastolic function. There were no signs of increased apoptosis in the experimental groups. In conclusion, 14 days pretreatment with GHRP-2, but not rhGH, protected selectively against the diastolic dysfunction of myocardial stunning in this model. This observation may open perspectives for GH-secretagogues as cardioprotective agents.

Characterization of cardioprotection mediated by AT2 receptor antagonism after ischemia-reperfusion in isolated working rat hearts.

Whether cardioprotection induced by the angiotensin II (AngII) type 2 receptor (AT(2)R) antagonist PD123,319 (PD) after ischemia-reperfusion (IR) is influenced by the concentration of PD, presence of AngII, timing of exposure, or inhibition of proton production from glucose metabolism is not known. We examined these factors in isolated working rat hearts subjected to IR injury, no treatment (control), or treatment with N(6)-cyclohexyl adenosine (CHA, 0.5 micromol/L), an adenosine A(1) receptor agonist that induces cardioprotection by decreasing protons ("positive" control). Compared with control, 1 micromol/L PD present throughout IR improved recovery of left ventricular work (73 +/- 5 vs. 40 +/- 8%) to the level with CHA (82 +/- 5%), but 0.1 micromol/L PD did not (58 +/- 6 vs. 40 +/- 8%). AngII (1 nmol/L) did not effect postischemic recovery associated with 1 micromol/L PD (73 +/- 7%) but improved that associated with 0.1 micromol/L PD (86 +/- 3%). PD (1 micromol/L), present solely during reperfusion, enhanced postischemic left ventricular recovery to 72 +/- 5%. Also, PD (1 micromol/L) did not affect glycolytic rates or proton production in nonischemic or IR hearts. PD-induced cardioprotection is 1) PD concentration-dependent, 2) AngII-sensitive, 3) mediated during reperfusion, and 4) independent of proton production, suggesting that reduction in IR injury and indirect AT(1)R stimulation might be involved.

Measurement of 1,2-diacylglycerol and ceramide in hearts subjected to ischemic preconditioning

An accumulation of recent evidence suggests that the mechanism in ischemic preconditioning (IPC) may involve the activation of protein kinase C (PKC) regulatory pathway. In this study, we examined whether the content of 1,2-diacylglycerol (1,2-DAG) and ceramide, which are intracellular second messengers regulating PKC activity, change during IPC in isolated perfused rat hearts, and whether the observed change in 1,2-DAG is accompanied with alteration in its fatty acid composition. Hearts subjected to IPC, consisting of 5-min transient global ischemia followed by 5-min reperfusion, presented a significant functional recovery during subsequent 40-min reperfusion following 40-min global ischemia compared with non-preconditioned hearts. An increase in 1,2-DAG content was observed in hearts subjected to 5-min transient ischemia compared with non-ischemic control hearts, however this was not seen in hearts harvested after 5-min reperfusion following 5-min ischemia. While fatty acid composition in 1,2-DAG was virtually unchanged in hearts subjected to 5-min ischemia, saturated 1,2-DAG decreased and monounsaturated/polyunsaturated 1,2-DAG increased in hearts reperfused for 5-min following 5-min ischemia compared with the non-ischemic control hearts. Ceramide mass did not change significantly, suggesting that the contribution of ceramide may be small in IPC. These data are in concert with the hypothesis that 1,2-DAG is a second messenger in IPC and the changes in fatty acid composition of 1,2-DAG may add new insight concerning signal transduction pathway in IPC.

The role of the L-arginine/nitric oxide pathway in myocardial ischaemic and reperfusion injury.

Myocardial ischaemia followed by reperfusion (I/R) is associated with impaired endothelial function including diminished release and/or effects of nitric oxide (NO) which may contribute to the development of I/R injury. The aim of the present study was to investigate the role of the L-arginine/NO pathway in myocardial I/R injury. In isolated rat hearts subjected to global ischaemia followed by reperfusion L-arginine and the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), but not D-arginine, significantly enhanced the recoveries of mycardial performance and coronary flow, and reduced the area of no-reflow and creatine kinase outflow. The NO synthase inhibitor NG-nitro-L-arginine (L-NNA) abolished the protective effects of L-arginine. Endothelium-dependent vasodilatation after I/R was preserved in L-arginine treated but not in vehicle hearts. Following I/R Ca2+-dependent NO synthase activity was reduced by 90% in comparison with non-ischaemic hearts. L-arginine but not D-arginine significantly increased NO synthase activity. In anaesthetized pigs, L-arginine given by local coronary venous retroinfusion reduced myocardial infarct size induced by 45 min of coronary artery ligation and 4 h of reperfusion to 35% of the area at risk from 76% in controls. The protective effect of L-arginine was blocked by L-NNA. Acetylcholine-induced coronary vasodilatation following I/R was attenuated in controls but not in L-arginine treated pigs. It is concluded that L-arginine or the NO donor SNAP reduces I/R-induced myocardial and endothelial injury. The protective effect of L-arginine seems to be mediated through maintained production of NO by preserving the function of Ca2+-dependent NO synthase in the heart.

Trace Amounts of Albumin Protect Against Ischemia and Reperfusion Injury in Isolated Rat Hearts

Albumin is used to provide colloid osmotic pressure in some resuscitation and organ preservation protocols. These solutions are expensive and carry the risks of using high concentrations of blood products. Used as a carrier of drugs and substrates, the concentration of albumin present in perfusates may be considerably lower in experimental ischemia. The present study examined if trace amounts of albumin (0.0004%) reduce injury from ischemia and reperfusion in isolated rat hearts. Hearts were perfused by the Langendorff technique (60 mmHg) with an intraventricular balloon. Zero-flow ischemia (20 min, 37°C) was followed by reperfusion (35 min, 37°C). Recovery of contractile function during reperfusion was significantly improved by the presence of fatty acid-free bovine serum albumin (BSA) (22 290±1280 mmHg/min, pressure–rate product) or rat serum albumin (RSA) (21 095±2836 mmHg/min) compared with Krebs–Henseleit buffer with no albumin (KHB) (9660±2324 mmHg/min). Release of lactate dehydrogenase activity, formation of tissue edema and accumulation of tissue malonyldialdehyde were significantly reduced in hearts receiving BSA or RSA compared with KHB alone. These parameters were not altered by the presence of albumin in non-ischemic control hearts or in the pre-ischemic values of the hearts subjected to ischemia and reperfusion. Development of ischemic contracture with an extended period of ischemia (27 min) was not altered by the presence of BSA, suggesting that protection observed with albumin occurred during reperfusion, rather than during ischemia. Reperfusion following 45 min of ischemia with bovine serum albumin resulted in similar myocardial injury to hearts that were reperfused following 20 min of ischemia without bovine serum albumin. Thus, trace amounts of albumin provide significant reduction in myocardial injury from ishemia and reperfusion, probably via antioxidant mechanisms.

Prolonged preservation of the blood-perfused canine heart with glycolysis-promoting solution

Background. Prolonged ischemia and inadequate myocardial preservation remain significant perioperative risk factors in cardiac transplantation. Long-term preservation techniques that have been effective in small rodent hearts have not been as effective in larger animal models or in clinical studies. We developed a cardioplegia solution formulated to promote high-energy phosphate production from glycolysis and determined its efficacy in a blood perfused canine heart model subjected to 24 hours of ischemia. Methods. Hearts harvested from adult dogs (n = 6 per group) were flushed with a histidine-buffered cardioplegia solution containing glucose or University of Wisconsin solution. The hearts were maintained at 4°C for 24 hours then reperfused with autologous blood. After reperfusion, left ventricular pressures were measured with an intracavitary balloon at varying balloon volumes and compared with control nonischemic hearts. Predicted stroke volume and ejection fraction were calculated at an end-systolic pressure of 70 mm Hg and end-diastolic pressure of 15 mm Hg. Results. Developed pressure was better preserved in the hearts that received histidine-buffered solution (93 ± 9 versus 38 ± 7 mm Hg, p < 0.05), along with a higher end-diastolic volume at 15 mm Hg (31 ± 3 versus 22 ± 2 mL histidine-buffered versus University of Wisconsin solutions, respectively, p < 0.05). Stroke volume and ejection fraction were also higher in the histidine group (17 ± 2.5 versus 2.3 ± 1.2 mL and 50% ± 3.5% versus 9% ± 4.5%, respectively) in the presence of dobutamine. Conclusions. The highly buffered glycolysis-promoting cardioplegia solution provided effective preservation of the blood perfused canine heart with superior recovery of pump performance after 24 hours of hypothermic ischemia compared with University of Wisconsin solution in this model.

The effects of levosimendan on the left ventricular function and protein phosphorylation in post-ischemic guinea pig hearts.

The widely accepted theories for the decreased function in the stunned myocardium relate to Ca2+ desensitization and free radical-mediated tissue damage of the myofilaments. The aim of the present study was to examine whether the depressed contractile function and Ca2+ responsiveness of the stunned myocardium may be restored by a new Ca2+ sensitizer (levosimendan), which has been shown to improve the Ca2+ response of the myofilaments. The effects of levosimendan on the left ventricular function and the in vivo protein phosphorylation were examined in both the non-ischemic and the stunned myocardium. Myocardial stunning was induced in Langendorff-perfused guinea pig hearts by suspending the circulation for 8 min, followed by a 20-min reperfusion period. Perfusion of post-ischemic guinea pig hearts with levosimendan (0.03-0.48 microM, 6 min) was associated with dose- and time-dependent increases in both dP/dtmax (contractility) and dP/dtmin (speed of relaxation). When the effectiveness of levosimendan was compared in non-ischemic and post-ischemic hearts, no significant differences were noted in the relative stimulatory effects on contractility and relaxation, at any given time point (time-response curve) or concentration (dose-response curve). Perfusion of the guinea pig hearts with a high (0.3 microM) levosimendan concentration did not reveal any qualitative or quantitative difference in the phosphodiesterase inhibitory potential of the compound (elevation of tissue cyclic AMP levels and characteristics of protein phosphorylation) between the non-ischemic and the post-ischemic myocardium. However, when isoproterenol was administered to induce maximal in vivo phosphorylation of cardiac phosphoproteins, an attenuation of the 32P-incorporation into troponin I was noted in the post-ischemic hearts. The decrease in isoproterenol-induced 32P-incorporation into troponin I was associated with similar alterations in the tissue level of this protein. We conclude that the Ca2+ sensitizer levosimendan exerts dose- and time-dependent positive inotropic and lusitropic effects on the post-ischemic myocardium, lending support to the hypothesis tha Ca2+ desensitization of the myofibrils is involved in myocardial stunning.

Glucose Utilization and Glycogen Turnover are Accelerated in Hypertrophied Rat Hearts During Severe Low-flow Ischemia

We undertook this study to determine if the metabolism of exogenous glucose and glycogen in hypertrophied hearts differed from that in normal hearts during severe ischemia. Thus, rates of glycolysis (3H2O production) and oxidation (14CO2production) from exogenous glucose and glycogen were measured in isolated working control (n=13) and hypertrophied (n=12) hearts from sham-operated and aortic-banded rats during 40 min of severe low-flow ischemia. Hearts, in which glycogen was prelabelled with [5-3H]- or [14C]-glucose, were paced and perfused with Krebs–Henseleit solution containing 1.2 mmpalmitate, 5.5 mm[5-3H]- or [14C]-glucose (different from the isotope used to label glycogen), 0.5 mmlactate and 100μU/ml insulin during ischemia. Rates of glycolysis from exogenous glucose (3301±122v2467±167 nmol/min/g dry wt, mean±s.e.mP<0.05) and glucose from glycogen (808±27v725±21 nmol/min/g dry wt,P<0.05) were accelerated in hypertrophied hearts compared to control hearts. However, rates of oxidation of exogenous glucose and glucose from glycogen were not significantly different between the two groups. As observed in normoxic non-ischemic hearts, glucose from glycogen was preferentially oxidized compared to exogenous glucose. Additionally, rates of glycogen synthesis (167±7v140±9 nmol/min/g dry wt,P<0.05) were increased in hypertrophied hearts compared to control hearts during severe low-flow ischemia indicating that glycogen turnover (i.e. simultaneous synthesis and degradation) was accelerated in the hypertrophied heart. Thus, we demonstrate that glucose utilization and glycogen turnover are accelerated in the hypertrophied heart during severe low-flow ischemia as compared to the normal heart.

Differential depression of myocardial function and metabolism by lactate and H+.

The effects of both high blood H+ concentration ([H+]) and high blood lactate concentration ([lactate]) under ischemia-reperfusion conditions are receiving attention, but little is known about their effects in nonischemic hearts. Isolated rat hearts were Langendorff perfused at constant flow with media at two pH values (7.4 and 7.0) and two [lactate] (0 and 20 mM) in various sequences (n = 6/group). Coronary flow and arterial O2 content were kept constant at levels that allowed hearts to function without O2 supply limitation. We measured contractility, O2 uptake, diastolic pressure, and at the end of the protocol, tissue [lactate] and pH. Perfusion with high [lactate] raised tissue [lactate] from 5.5 +/- 0.1 to 17.5 +/- 2.6 micromol/heart (P < 0.0001), whereas decreasing the pH of the medium decreased tissue pH from 6.94 +/- 0.02 to 6.81 +/- 0.06 (P = 0.002). Heart rate was not affected by high [lactate] but was reversibly depressed by high [H+] (P = 0.004). Developed pressure declined by 20% in response to high [lactate], high [H+], and high [lactate] + high [H+] (P = 0.002). After the high-[lactate] challenge was withdrawn, pressure continued to decline. In contrast, withdrawing the high [H+] challenge allowed partial recovery. The behavior of diastolic pressure mirrored that of developed pressure. Although unaffected by high [lactate], the O2 uptake was reversibly depressed by high [H+]. This suggests higher O2 cost per contraction in the presence of high [lactate]. We conclude that for similar acute contractility depression, high [lactate] induces irreversible damage, likely at some point in the pathway of O2 utilization. In contrast, the effect of high [H+] appears reversible. These differential behaviors may have implications for heart function during heavy exercise and ischemia-reperfusion events.

Intracoronary administration of adenosine triphosphate increases coronary blood flow and attenuates the severity of myocardial ischemic injury in dogs.

ATP generates nitric oxide (NO) via activation of P2y receptors, and is degraded to adenosine. This study was undertaken to examine whether ATP causes coronary hyperemic flow via purinoceptors-, NO- and adenosine-dependent mechanisms, and attenuates the severity of contractile and metabolic dysfunction in the ischemic myocardium. In the non-ischemic canine hearts, the infusions of ATP into the coronary artery dose-dependently increased coronary blood flow. The levels of adenosine and end-product of NO in coronary venous blood over the arterial blood also increased. This hyperemic flow was partially attenuated by either 8-sulfophenyltheophylline (8SPT) or L(omega)-nitro arginine methyl ester (L-NAME), and completely blocked by the treatment with 8SPT, L-NAME and suramin (SRM). During myocardial ischemia, exogenous ATP increased coronary blood flow, and attenuated myocardial metabolic and contractile dysfunction, which was completely blunted by the treatment with 8SPT, L-NAME and SRM. We conclude that exogenous ATP increases coronary blood flow in the non-ischemic and ischemic myocardium mainly via either NO- or adenosine-dependent mechanisms.

Fucoidin reduces coronary microvascular leukocyte accumulation early in reperfusion

Background. Leukocytes rapidly accumulate in the heart early in reperfusion after ischemia, contributing to reperfusion injury. The purpose of this study was to determine whether treatment with the selectin blocker fucoidin (FCN) would attenuate early leukocyte retention in coronary venules and capillaries during low flow reperfusion.Methods. Isolated rat hearts subjected to 30 minutes of 37°C, no-flow ischemia were initially reperfused with blood containing labeled leukocytes, followed by reperfusion with a Krebs red cell solution. The deposition of leukocytes in coronary capillaries and venules was observed using intravital microscopy. Three groups were studied: nonischemic control hearts, untreated postischemic hearts reperfused at low flow, and postischemic hearts reperfused at low flow, where both the hearts and the blood reperfusate were pretreated with FCN (0.36 mg/mL blood).Results. In the ischemia-reperfusion group, we observed a rapid and significant increase in leukocyte accumulation in both capillaries and venules. Treatment with FCN significantly reduced the leukocyte accumulation in both capillaries and venules (p < 0.05). In addition, FCN significantly reduced the persistence of leukostasis in both capillaries and venules, indicating that FCN affected a transient adhesion process.Conclusions. These results suggest that the selectin family of leukocyte–endothelial cell adhesion proteins mediates the initial retention of leukocytes in both coronary capillaries and venules during reperfusion. Selectin blockade may be effective in reducing the contribution of leukocytes to early reperfusion injury.

Augmented myocardial ischaemia by nicotine--mechanisms and their possible significance.

1. To study the effect of nicotine on the severity of experimental myocardial ischaemia, Langendorff hearts of rabbits (n=7-12 per group) were subjected to 2 h of low-flow ischaemia followed by 1 h of reperfusion. 2. Infusion of nicotine (100 ng ml(-1)) caused only minor changes in non-ischaemic conditions but a significant (P<0.05) increase in end-diastolic pressure (LVEDP), loss of creatine kinase (CK) and troponin (TnT) as well as increase in noradrenaline (NA) overflow in reperfused ischaemic hearts. 3. RT PCR was done on total RNA for mRNA expression of the constitutive (COX-1) and inducible cyclooxygenase (COX-2). There was no COX-2 in non-ischaemic hearts but a significant expression in ischaemia (n=5) which was further increased by nicotine. These data were confirmed at the protein level by Western blotting and additionally shown that COX-1 remained unchanged. 4. There was a marked increase in prostacyclin (PGI2) and a 2 fold increase in NA overflow which were both stimulated by nicotine. 5. The aggravating effects of nicotine on myocardial ischaemia (CK release) as well as the expression of COX-2 mRNA were prevented by pretreatment with the beta-blocker pindolol (1 microM). 6. The data demonstrate marked deleterious actions of nicotine in reperfused ischaemic hearts. These actions are probably related to the increase in catecholamine overflow, are beta-receptor-mediated and involve enhanced gene expression of COX-2.

Regression of cellular hypertrophy after left ventricular assist device support.

Although multiple studies have shown that the left ventricular assist device (LVAD) improves distorted cardiac geometry, the pathological mechanisms of the "reverse remodeling" of the heart are unknown. Our goal was to determine the effects of LVAD support on cardiac myocyte size and shape. Isolated myocytes were obtained at cardiac transplantation from 30 failing hearts (12 ischemic, 18 nonischemic) without LVAD support, 10 failing hearts that received LVAD support for 75+/-15 days, and 6 nonfailing hearts. Cardiac myocyte volume, length, width, and thickness were determined by use of previously validated techniques. Isolated myocytes from myopathic hearts exhibited increased volume, length, width, and length-to-thickness ratio compared with normal myocytes (P<0.05). However, there were no differences in any parameter between myocytes from ischemic and nonischemic cardiomyopathic hearts. Long-term LVAD support resulted in a 28% reduction in myocyte volume, 20% reduction in cell length, 20% reduction in cell width, and 32% reduction in cell length-to-thickness ratio (P<0.05). In contrast, LVAD support was associated with no change in cell thickness. These cellular changes were associated with reductions in left ventricular dilation and left ventricular mass measured echocardiographically in 6 of 10 LVAD-supported patients. These studies suggest that the regression of cellular hypertrophy is a major contributor to the "reverse remodeling" of the heart after LVAD implantation. The favorable alterations in geometry that occur in parallel fashion at both the organ and cellular levels may contribute to reduced wall stress and improved mechanical performance after LVAD support.

ADMINISTRATION OF FRUCTOSE 1,6-DIPHOSPHATE DURING EARLY REPERFUSION SIGNIFICANTLY IMPROVES RECOVERY OF CONTRACTILE FUNCTION IN THE POSTISCHEMIC HEART

Objectives: Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. Methods: Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37° C followed by reperfusion ( n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31–nuclear magnetic resonance spectroscopy. Results: In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 ± 2 vs 70 ± 4 mm Hg, p < 0.05), lower diastolic pressures (7 ± 1 vs 12 ± 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. Conclusions: We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion. (J Thorac Cardiovasc Surg 1998;116:335-43)

Basal metabolism does not account for high O2 consumption in stunned myocardium.

Myocardial O2 consumption (MVo2) in stunned myocardium is relatively high compared with the reduced ventricular function. The mechanism of this "oxygen paradox" could occur at different levels: basal metabolism, excitation-contraction coupling, and energy production. In one previously reported series on 12 isolated, blood-perfused rabbit hearts, left ventricular systolic and diastolic function in stunned myocardium were significantly decreased compared with control, whereas total MVo2 was not. The MVo2 for the unloaded contraction was overproportionately high for the decreased function in stunned myocardium, and contractile efficiency was clearly deteriorated. To assess whether the basal metabolism specifically is elevated in stunned myocardium, a second series (n = 14) with a similar protocol was performed in this study. Basal MVo2 after KCl arrest (0.5 +/- 0.3 ml.min-1.100 g-1) was significantly lower than that measured after KCl arrest (1.2 +/- 0.5 ml.min-1.100 g-1) in an additional series on nonischemic hearts (n = 8). Our conclusion is that basal MVo2 in stunned myocardium is not elevated. Thus this O2-consuming portion of total MVo2 is not responsible for the inefficiency in stunned myocardium. Instead, a "metabolic stunning" occurs at the level of both excitation-contraction coupling and force development by the contractile apparatus.

Effects of dichloroacetate on mechanical recovery and oxidation of physiologic substrates after ischemia and reperfusion in the isolated heart.

The effects of dichloroacetate (DCA) on fatty acid oxidation and flux through pyruvate dehydrogenase (PDH) were studied in ischemic, reperfused myocardium supplied with glucose, long-chain fatty acids, lactate, pyruvate, and acetoacetate. The oxidation rates of all substrates were determined by combined 13C nuclear magnetic resonance (NMR) spectroscopy and oxygen-consumption measurements, and PDH flux was assessed by lactate plus pyruvate oxidation. In nonischemic control hearts, DCA increased PDH flux more than eightfold (from 0.68 +/- 0.28 to 5.81 +/- 1.16 micromol/min/g dry weight; n = 8 each group; p < 0.05) and significantly inhibited the oxidation of acetoacetate and fatty acids. DCA also improved mechanical recovery after 30 min of ischemia plus 30 min of reperfusion but did not significantly increase PDH flux measured at the end of the reperfusion period (1.35 +/- 0.42 micromol/min/g dry weight) compared with untreated ischemic hearts (0.87 +/- 0.28 micromol/min/g dry weight; n = 8 each group; p = NS). Although DCA had a modest effect on functional recovery in the reperfused myocardium, this beneficial effect was not associated with either marked stimulation of PDH flux or inhibition of fatty acid oxidation.