5-HD abolishes ischemic preconditioning independently of monophasic action potential duration in the heart

Objective: Blocking of the K_ATP channel with either glibenclamide or 5-hydroxydecanoate (5-HD) has been shown to abolish the infarct reducing effect of ischemic preconditioning (IPC) in hearts from several species, but the results in rat and rabbit have been equivocal. In this study we investigated if 5-HD could abolish IPC in rat and rabbit and further if IPC or IPV + 5-HD were affecting action potential duration in the rabbit heart. Methods: The rat hearts were isolated and retrogradely perfused on a Langendorff perfusion apparatus with Krebs-Henseleit buffer. The rabbit experiments were performed in an in situ model. Rat and rabbit hearts were subjected to 30 min regional ischemia by ligating a coronary artery followed by 120 min (rat) or 150 min (rabbit) of reperfusion. The preconditioning protocol was one or three cycles of 5 min ischemia plus 5 min reperfusion in the rat and one cycle of 5 min ischemia plus 10 min reperfusion in the rabbit. In the rat 5-HD was added to the reservoir before ischemic preconditioning in different concentrations, and in the rabbit 5-HD was given as a bolus 5 mg/kg intraventricularly 2 min before the preconditioning ischemia. In the rabbit epicardial monophasic action potential duration at 50% repolarization (MAPD₅₀) was measured at 1, 2 and 5 min in each of the ischemic periods using a contact pressure electrode. Infarcts were measured with tetrazolium staining and risk zone volumes with fluorescent microspheres. Results: All data are presented as infarct size in % of risk zone volume (mean ± SEM). In the rat 200 μM of 5-HD abolished the protective effect of one cycle of IPC (28.6 ± 4.7 versus 8.4 ± 0.8) and 500M of 5-HD abolished three cycles of IPC (50.7 ± 7.8 versus 8.4 ± 2.0). Control was 40.9 ± 2.8. In the rabbit 5-HD abolished IPC (41.2 ± 7.2 versus 8.1 ± 3.2). Control was 53.5 ± 12.4. MAPD₅₀ were significantly more shortened compared to control at 1 and 2 min into the 30 min ischemia for the IPC and IPC+5-HD. Conclusions: We conclude that 5-HD abolishes ischemic preconditioning when given before the preconditioning ischemia in both rat and rabbit but does not abolish into ischemia induced shortening of the action potential duration in the rabbit; thus, a role for the mitochondrial K_ATP channel and not the sarcolemmal K_ATP channel in the protective mechanism behind IPC is probable. Received: 15 July 1999, Returned for 1. revision: 17 August 1999, 1. Revision received: 13 September 1999, Returned for 2. revision: 12 October 1999, 2. Revision received: 3 November 1999, Accepted: 17 November 1999     

A1 adenosine receptor overexpression decreases stunning from anoxia-reoxygenation: role of the mitochondrial K(ATP) channel

Myocardial A₁ adenosine receptor (A₁AR) overexpression protects hearts from ischemia-reperfusion injury; however, the effects during anoxia are unknown. We evaluated responses to anoxia-reoxygenation in wild-type (WT) and transgenic (Trans) hearts with ∼200-fold overexpression of A₁ARs. Langendorff perfused hearts underwent 20 min anoxia followed by 30 min reoxygenation. In WT hearts peak diastolic contracture during anoxia was 45 ± 3 mmHg, diastolic pressure remained elevated at 18 ± 3 mmHg after reoxygenation, and developed pressure recovered to 52 ± 4 % of pre-anoxia. A₁AR overexpression reduced hypoxic contracture to 29 ± 4 mmHg, and improved recovery of diastolic pressure to 8 ± 1 mmHg and developed pressure to 76 ± 3 % of pre-anoxia. Mitochondrial K_ATP blockade with 100 μM 5-hydroxydecanoate (5-HD) increased hypoxic contracture to 73 ± 6 mmHg in WT hearts, reduced post-hypoxic recoveries of both diastolic (40 ± 5 mmHg) and developed pressures (33 ± 3 %). In contrast, 5-HD had no effect on hypoxic contracture (24 ± 8 mmHg), or post-hypoxic diastolic (10 ± 2 mmHg) and developed pressures (74 ± 3 %) in Trans hearts. In summary, (i) A₁AR overexpression improves myocardial tolerance to anoxia-reoxygenation, (ii) intrinsic mitochondrial K_ATP channel activation decreases hypoxic contracture and improves functional recovery in wild-type hearts, and (iii) mitochondrial K_ATP channels do not appear to play a major role in the functional protection from anoxia afforded by A₁AR overexpression. Received: 5 February 2001, Returned for 1. revision: 21 February 2001, 1. Revision received: 20 August 2001, Returned for 2. revision: 3 September 2001, 2. Revision received: 24 October 2001, Accepted: 25 October 2001     

A role of PKC in the improvement of energy metabolism in preconditioned heart

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function. Received: 29 January 1999, Returned for 1. revision: 26 February 1999, 1. Revision received: 27 April 1999, Returned for 2. revision: 18 May 1999, 2. Revision received: 12 July 1999, Returned for 3. revision: 26 July 1999, 3. Revision received: 25 October 1999, Accepted: 3 November 1999     

Coronary blood flow control is impaired at rest and during exercise in conscious diabetic dogs

This study tested whether diabetes mellitus impairs coronary blood flow control sufficiently to alter the balance between myocardial oxygen delivery and metabolism. Dogs (n = 7) were instrumented with catheters in the aorta and coronary sinus, and with a flow transducer on the circumflex coronary artery. Coronary blood flow, myocardial oxygen consumption (MVO₂), heart rate and aortic pressure were measured at rest and during treadmill exercise before and after induction of diabetes with alloxan monohydrate (40 – 60 mg/kg). Arterial plasma glucose concentration increased from 4.6 ± 0.2 mM in non-diabetic, control dogs to 20.2 ± 2.3 mM one week after alloxan injection. In non-diabetic control dogs, exercise increased MVO₂ 3.1-fold, coronary blood flow 2.7-fold, and heart rate 2.4-fold. Coronary venous PO₂ decreased from 19.4 ± 0.6 mmHg at rest to 14.7 ± 0.7 mmHg during exercise. Diabetes significantly attenuated exercise coronary hyperemia and reduced coronary venous PO₂ at rest (15.6 ± 0.5 mmHg) and during exercise (12.6 ± 0.8 mmHg). Diabetes also significantly reduced myocardial oxygen delivery at each level of exercise. Acute hyperglycemia alone did not alter exercise-induced coronary vasodilation or reduce coronary venous PO₂. These findings demonstrate that experimental diabetes attenuates functional coronary hyperemia and impairs the balance between coronary blood flow and myocardial metabolism. However, this deleterious effect is not related to acute hyperglycemia but to the chronic disease process of diabetes mellitus. Received: 19 July 2001, Returned for 1. revision: 20 August 2001, 1. Revision received: 17 October 2001, Returned for 2. revision: 19 October 2001, 2. Revision received: 2 November 2001, Accepted: 5 November 2001     

Xanthine oxidase contributes to preconditioning's preservation of left ventricular developed pressure in isolated rat heart: developed pressure may not be an appropriate end-point for studies of preconditioning

Studies of preconditioning frequently use the isolated rat heart model in which recovery of post-ischemic function is the end-point. However, function following an episode of ischemia/reperfusion represents a composite of both stunning, which is related to free radical production and is not attenuated by preconditioning, and tissue salvage, the primary effect of preconditioning. Brief ischemia/reperfusion is also known to diminish adenosine release during subsequent ischemia by a mechanism independent of preconditioning's anti-infarct effect. Reduced purine release would diminish generation of free radicals by xanthine oxidase in rat heart and thus produce less stunning. In this paradigm preserved post-ischemic function in rat heart might look similar to salvage by preconditioning, but its mechanism would be quite different and not be relevant to the xanthine oxidase-deficient human heart. This hypothesis was tested in isolated rat hearts. Control or ischemically preconditioned hearts were subjected to 30 min of global ischemia and 60 min of reperfusion, either in the presence or absence of 25 μmol/l allopurinol, an inhibitor of xanthine oxidase. In non-preconditioned hearts allopurinol increased left ventricular developed pressure after 60 min of reperfusion from 26 ± 5 mmHg in control hearts to 47 ± 7 mmHg, whereas developed pressure in preconditioned hearts following reperfusion was 59 ± 5 mmHg and was unaffected by allopurinol. Developed pressure in non-preconditioned hearts treated with allopurinol was midway between that for untreated control and preconditioned hearts suggesting that at least 50 % of the recovery of developed pressure in preconditioned hearts may be related to free radical-induced stunning. In xanthine oxidase-deficient rabbit hearts, return of function was not different between non-preconditioned and preconditioned hearts. Therefore, post-ischemic developed pressure in the rat is significantly affected by purine-dependent stunning, and, hence, may be an unreliable marker of tissue salvage and also a poor index of what might be cardioprotective in man. Received: 2 May 2001, Returned for 1. revision: 25 May 2001, 1. Revision received: 13 June 2001, Returned for 2. revision: 18 June 2001, 2. Revision received: 11 July 2001, Accepted: 18 July 2001     

Preconditioning preserves energy metabolism in prolonged low-flow ischemia

Background: A reduction in coronary flow leads to a parallel decrease in contractile function. Thus, a flow/function balance is established in the myocardium under certain circumstances avoiding the development of a necrosis (referred to as “hibernating” myocardium). The impact of a preconditioning period on this critical balance was examined. Methods: In 116 isovolumetrically beating rat hearts, 3 h of hypoperfusion with 15% of control coronary flow were performed followed by 1 h reperfusion; 40 hearts served as controls. As a preconditioning period, in half of the rat hearts a 5 min no-flow ischemia followed by 10 min reperfusion was performed, preceding the prolonged hypoperfusion. Results: Systolic function was identically reduced in both groups after 3 h hypoperfusion (LVP: 39±2 mmHg, 40±2 mmHg vs. controls 90±3 mmHg; p<0.01). Without preconditioning hypoperfusion resulted in a marked initial decrease in function. This period was followed by an adaptation to a higher steady state level of function compared with non-preconditioned hyperperfused hearts (p<0.05). After preconditioning hypoperfusion directly resulted in this level of contraction. Contractile reserve was reduced (p<0.01) identically in both hypoperfusion groups. Adenine nucleotides were decreased (p<0.01) after 3 h hypoperfusion to 2.1±0.2 μmol/gww vs. controls (4.7±0.2 μmol/gww). After initial preconditioning adenine nucleotides were better preserved (3.2±0.2 μmol/gww) going ahead with a creatine phosphate overshoot of 126% (p<0.01). After reperfusion, systolic function and contractile reserve were identical in both groups. Conclusion: A period of preceding no-flow ischemia followed by reperfusion modifies functional adaptation to hypoperfusion and preserves high energy phosphates. Therefore, the metabolic balance during hypoperfusion is improved by preconditioning, although no impact on contractile reserve or the functional status of reperfused myocardium after low-flow ischemia can be seen. Received: 12 March 1998, Returned for 1. revision: 25 March 1998, 1. Revision received: 12 May 1998, Returned for 2. revision: 2 June 1998, 2. Revision received: 13 July 1998, Accepted: 21 July 1998     

Fasting limits the increase in intracellular calcium during ischemia in isolated rat hearts

Introduction Fasting has been shown to limit ischemic injury and improve functional activity after global ischemia. Because calcium overload is considered a mechanism of ischemic injury, we hypothesized that fasting would limit the accumulation of intracellular calcium [Ca]_i during ischemia, potentially due to reduced accumulation of intracellular sodium [Na]_i. Methods To address this hypothesis, hearts isolated from rats fed either a normal diet or fasted for 24 hours underwent 20 min of global ischemia at 37°. In addition to functional parameters, [Na]_i and [Ca]_i were measured using ²³Na and ¹⁹F spectroscopy using thulium-DOTP⁻⁵ and 5F-BAPTA, respectively. In vitro measurement of sarcoplasmic reticulum calcium uptake and release, as well as activity of the sarcolemmal Na-Ca exchanger, was performed in hearts from fed and fasted animals under baseline and ischemic conditions. Results Hearts from fasted animals showed greater recovery of developed pressure (37 ± 9 vs. 11 ± 6 cm H₂O, p < 0.05) and less contracture (end-diastolic pressure 25 ± 2 vs. 47 ± 2 cm H₂O, p < 0.05) by the end of the reperfusion period. [Na]_i was similar in the 2 groups during the first half of the ischemic period, albeit with a higher concentration of [Na]_i in hearts from fed compared to fasted animals at reperfusion. Fasting markedly limited calcium accumulation during ischemia, with end-ischemic calcium being 419 ± 46 nM in the hearts from fasted animals and 858 ± 140 nM in the hearts from fed animals (p < 0.01). There was no significant effect of fasting on calcium uptake or release by the SR, nor on sarcolemmal Na-Ca exchange activity. Conclusions Fasting for 24 hours improves functional recovery and markedly limits [Ca]_i accumulation during ischemia and early reperfusion. The mechanism for this phenomenon remains to be elucidated. Received: 7 December 2000, Returned for revision: 22 December 2000, Revision received: 5 February 2001, Accepted: 8 February 2001     

Lack of nuclear apoptosis in cardiomyocytes and increased endothelin-1 levels in a rat heart model of myocardial stunning

Objective. Reperfusion injury may affect the cardiac NO and endothelin production. We investigated whether 20 min of total ischemia followed by 40 min of reperfusion can induce apoptosis in a Langendorff model of retrogradely perfused rat hearts (37°C; paced at 300/’), and we attempted to correlate these findings with measured tissue NO and ET-1 levels. Methods. An apoptosis detection system was utilized which catalytically incorporates fluorescein-12-dUTP at the 3’-OH DNA ends using the principle of the TUNEL assay, with direct visualization of the labeled DNA. ET-1 was measured by radioimmunoassay and NO3/NO2 by ion pairing HPLC on C18 reverse phase columns. Results. None of the postischemic (n = 6) nor of the control perfused (90 min, n = 6) hearts showed signs of apoptosis, while those exposed to longer ischemia (40 min) and reperfusion (2 h) confirmed the presence of apoptotic cells. Myocardial ET-1 concentrations were 4.8±1.0 versus 8.3±2.5 pg/100 mg (control vs. ischemic hearts, respectively; mean ±SD; p < 0.05). Myocardial NO contents showed no differences. Conclusion. These data suggest that the time window of apoptosis with detectable DNA fragmentation exceeds 20 min of global total ischemia and 40 min of reperfusion, a model frequently used for inducing myocardial stunning. While NO was not increased in postischemic hearts, increased ET-1 levels indirectly argue for a role of ET-1 as inducer of apoptosis, but only at a later stage of reperfusion. Received: 17 August 1999, Returned for revision: 16 September 1999, Revision received: 6 January 2000, Accepted: 8 February 2000     

Endogenous nitric oxide regulates right coronary blood flow during acute pulmonary hypertension in conscious dogs

This study investigated the role of nitric oxide (NO) in the control of right coronary (RC) blood flow at rest and during acute pulmonary hypertension. Experiments were performed in seven chronically instrumented, conscious dogs. NO synthesis was inhibited by systemic administration of N^ω-nitro-L-arginine (LNA, 35 mg/kg). Inflation of a balloon in the main pulmonary artery raised right ventricular (RV) peak systolic pressure from 34 ± 2 to 47 ± 3 mmHg before LNA and from 37 ± 2 to 47 ± 3 mmHg after LNA, but did not affect mean systemic arterial pressure. RV O₂ consumption (MVO₂) increased from 4.4 ± 0.7 to 6.1 ± 0.7 ml/min/100 g. 82 % of the elevated RV MVO₂ was provided by RC blood flow, which increased from 46 ± 7 to 61 ± 8 ml/min/100 g. After LNA, resting RV MVO₂ and RC flow fell. RC venous PO₂ fell, but RV lactate uptake was not altered. During pulmonary hypertension, the increase in RC blood flow was blunted by LNA, so that only 66 % of the elevated RV MVO₂ was supplied by increased RC flow. Analysis of O₂ supply variables as functions of RV MVO₂ further demonstrated a significant role of NO in regulating RC flow at rest and during moderate pulmonary hypertension. Conclusions NO is required for the RC hyperemic response to acute pulmonary hypertension as well as for normal resting RC blood flow. After blockade of NO synthesis, RV O₂ supply at rest and during pulmonary hypertension was sustained by increased RV O₂ extraction. Received: 2 April 2002, Returned for 1. revision: 17 April 2002, 1. Revision received: 13 May 2002, Returned for 2. revision: 29 May 2002, 2. Revision received: 5 June 2002, Accepted: 10 June 2002     

Preconditioning-induced cardioprotection and release of the second messenger inositol (1,4,5)-trisphosphate are both abolished by neomycin in rabbit heart

The mechanisms responsible for infarct size reduction with preconditioning remain controversial. Our aim was to determine whether release of the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P₃) during the preconditioning stimulus may play a role. To test this concept, Langendorff-perfused rabbit hearts underwent sham perfusion, 5 min of coronary artery occlusion (CO), or 5 min of CO+infusion of neomycin, an agent which inhibits formation of Ins(1,4,5)P₃. Direct quantitation (by competitive binding assay) revealed a 2-fold increase in Ins(1,4,5)P₃ content with brief ischemia vs shams (0.69±0.14 vs 0.34±0.05 pmol/mg tissue; p<.05) that was blocked by neomycin (0.15±0.04 pmol/mg). Infarct size (by tetrazolium staining) was assessed in additional hearts that underwent 30 min of sustained CO and 2 h of reperfusion. As expected, two 5-min episodes of preconditioning ischemia reduced infarct size versus controls (30±6% versus 63±3% of the myocardium at risk; p<.01). In contrast, infarct size was comparable (54–56% of the risk region) in neomycin-treated control and preconditioned hearts. These results demonstrate that myocardial Ins(1,4,5)P₃ content is increased in response to brief preconditioning ischemia and are consistent with the concept that Ins(1,4,5)P₃ may be a potential mediator of infarct size reduction with preconditioning in isolated rabbit heart. Received: 12 February 1998, Returned for revision: 1 April 1998, Revision received: 1 June 1998, Accepted: 12 August 1998     

Protection of ischemic rat heart by dantrolene, an antagonist of the sarcoplasmic reticulum calcium release channel

Cytosolic Ca²⁺ overload plays a major role in the development of irreversible injury during myocardial ischemia. Such overload is due at least in part to the release of Ca²⁺ from the sarcoplasmic reticulum. Therefore, we investigated whether dantrolene, a blocker of the sarcoplasmic reticulum Ca²⁺ release channel, may protect from ischemic injury. In binding experiments, we determined the effect of dantrolene on [³H]-ryanodine binding in rat cardiac tissue. In perfusion experiments, isolated rat hearts were perfused for 20 min in the working mode, in the presence of 0–45 μM dantrolene. The hearts were then subjected to 30 min of global ischemia and 120 min of retrograde reperfusion. Tissue injury was evaluated on the basis of triphenyltetrazolium chloride (TTC) staining and LDH release. The binding experiments showed that dantrolene displaced 4 nM [³H]-ryanodine with IC₅₀ of 34 μM. In the perfusion experiments, tissue necrosis (i.e., TTC-negative tissue) averaged 28.3±1.6% of the ventricular mass under control conditions. Dantrolene was protective at micromolar concentrations: tissue necrosis decreased to 21.4±1.0% and 8.4±1.4% with 1 μM and 45 μM dantrolene, respectively (P < 0.05 and P < 0.01). Similar results were obtained with regard to LDH release. At low concentrations (up to 4 μM), dantrolene did not produce any significant hemodynamic effect, except for a slight increase in coronary flow, whereas at higher concentration a negative inotropic effect was apparent. In conclusion, dantrolene reduced ischemic injury even at concentrations that did not affect contractile performance. Modulation of sarcoplasmic reticulum Ca²⁺ release might represent a new cardioprotective strategy. Received: 28 June 1999, Returned for revision: 9 September 1999, 1. Revision received: 27 September 1999, Accepted: 29 September 1999     

The effects of levosimendan on the left ventricular function and protein phosphorylation in post-isschemic 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 μM, 6 min) was associated with dose- and time-dependent increases in both dP/dt_max (contractility) and dP/dt_min (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 μM) 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 adminstered to induce maximal in vivo phosphorylation of cardiac phosphorproteins, 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 postischemic myocardium, lending support to the hypothesis tha Ca²⁺ desensitization of the myofibrils is involved in myocardial stunning. Received: 20 July 1998, Returned for 1. revision: 27 August 1998, 1. Revision received: 6 January 1999, Returned for 2. revision: 5 February 1999, 2. Revision received: 25 February 1999, Accepted: 3 March 1999     

Nifedipine limits infarct size via NO-dependent mechanisms in dogs

Objectives Amlodipine increases NO levels in coronary vessels and aorta via bradykinin-dependent mechanisms in vitro. We have previously reported that nifedipine increases cardiac NO levels in the ischemic canine hearts, suggesting that nifedipine may also have protective effects against ischemia and reperfusion injury, because the enhancement of NO production limits infarct size. We tested whether nifedipine limits infarct size via NO-dependent mechanisms. Methods In open chest dogs, the left anterior descending coronary artery was perfused with blood through a bypass tube and occluded for 90 min followed by 6 hours of reperfusion. Infarct size was assessed at 6 hours of reperfusion. Nifedipine of 3 or 6 μg/kg/min was infused into the bypass tube between 10 min prior to the onset of ischemia and 60 min of reperfusion. Results Neither systemic blood pressure nor heart rate changed during infusion of nifedipine. Infarct size was reduced by the administration of nifedipine (3 or 6 μg/kg/min) compared with the untreated condition (25.6 plusmn; 2.6 and 19.1 ± 3.5 vs. 43.4 ± 5.6 %, respectively), which was completely blunted by L-NAME (45.0 ± 3.6 and 45.4 ± 4.2 vs. 47.9 ± 3.9 % in the nifedipine (3 or 6 μg/kg/min) with L-NAME groups vs. the L-NAME group). Myeloperoxidase activity of the myocardium increased after 6 hours of reperfusion, which was attenuated by nifedipine. The limitation of infarct size and the attenuation in myeloperoxidase activity were completely blunted by L-NAME. There were no significant differences in collateral blood flow at 45 min of ischemia between each group. Conclusions We conclude that the Ca channel blocker, nifedipine, limits infarct size via NO-dependent mechanisms. Received: 21 September 2000, Returned for 1. revision: 9 October 2000, 1. Revision received: 17 January 2001, Returned for 2. revision: 5 February 2001, 2. Revision received: 13 February 2001, Accepted: 14 February 2001     

Antioxidant properties of acetaminophen and cardioprotection

The acute administration of acetaminophen to isolated, perfused guinea pig hearts appears to have cardioprotective effects against the injury/mechanical dysfunction caused by global, low-flow, myocardial ischemia and reperfusion. In the current study we selected ischemia/reperfusion and administration of sodium pentobarbital as perturbations of the electrical stability of the myocardium. We investigated their ability to induce ventricular arrhythmias and changes in the characteristics of monophasic action potentials in the absence and presence of acetaminophen (0.35 mmol/l). The numbers of ventricular premature beats and ventricular salvos encountered in the presence of pentobarbital were significantly (P < 0.05) reduced by acetaminophen. The combined frequency of these arrhythmias was 0.14 ± 0.06/min vs 0.03 ± 0.01/min (P < 0.05) in the absence and presence of acetaminophen, respectively. The incidence of ventricular salvos increased steadily in vehicle-treated hearts after administration of pentobarbital. No such trend was seen with acetaminophen. After 10 min of global, low-flow myocardial ischemia, MAP50 and MAP90 (monophasic action potentials at 50 and 90 % repolarization, respectively) decreased without acetaminophen (e.g. MAP50, 31 ± 4 ms) but did not change during the same time interval with acetaminophen (e.g. MAP50, 57 ± 6 ms)(P < 0.05). During ischemia and reperfusion, acetaminophen attenuated the release of hydroxyl radicals and peroxynitrite. Collectively these data reveal cardioprotective, antioxidant behavior of acetaminophen. Under selected conditions (e.g. those causing release of free radicals and other oxidants) such behavior might also prevent ventricular arrhythmias. Received: 30 October 2000, Returned for 1. revision: 20 November 2000, 1. Revision received: 12 December 2000, Returned for 2. revision: 2 January 2001, 2. Revision received: 14 January 2001, Accepted: 31 January 2001     

Critical timing of mitochondrial K(ATP) channel opening for enhancement of myocardial tolerance against infarction

Objective The present study was designed to assess the relationship between the timing of a mitoK_ATP channel opener, diazoxide, and its infarct size-limiting effect. Methods In isolated rabbit hearts, infarction was induced by 30 min of global ischemia and 2 h of reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of the left ventricle (%IS/LV). Diazoxide, a mitoK_ATP channel selective opener, and/or 5-hydroxydecanoate (5-HD), a mitoKATP channel blocker, were infused before or after the onset of ischemia. When these agents were infused during the ischemic period, they were dissolved in a hypoxic buffer at concentrations 10-fold higher than those in the pre-ischemic period, and the infusion rate was set at 2 % of the pre-ischemic coronary flow. Results In untreated controls, %IS/LV was 53.2 ± 4.1 (SE). Pretreatment with diazoxide (100 μM) with a 10-min washout period reduced %IS/LV to 7.8 ± 2.4 and this protection was abolished by co-infusion of 5-HD (50 μM). Pre-ischemic infusion of diazoxide without a washout period reduced %IS/LV to 7.3 ± 1.4, and infusion of diazoxide from 10 min after the onset of ischemia also limited %IS/LV to 14.9 ± 4.6. However, diazoxide infusion from 25 min after the onset of ischemia failed to reduce infarct size (%IS/LV = 54.5 ± 7.2). Furthermore, pretreatment with 5-HD (50 μM) also completely abolished the protection afforded by early post-ischemic diazoxide infusion (%IS/LV = 48.3 ± 6.5). Neither infusion of 5-HD nor the anoxic vehicle alone during ischemia modified %IS/LV. Conclusion These findings suggest that opening of mitoK_ATP channels before ischemia and during early ischemia, but not that upon reperfusion, is important for enhancement of myocardial tolerance against infarction. Received: 6 November 2000, Returned for revision: 21 November 2000, Revision received: 24 January 2001, Accepted: 25 January 2001     

Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart

Because tyrosine kinase blockade prevents protection by ischemic preconditioning (PC) in several species, activation of tyrosine kinase appears to be critical for cardioprotection. The tyrosine kinase's identity, however, is unknown. The present study tested whether activation of a receptor tyrosine kinase, the insulin receptor, could mimic PC and if the mechanism of protection was similar to that of PC. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarct size in control hearts was 32.6 ± 2.3 %. A 5-min infusion of insulin (5 mU/ml) followed by a 10-min washout period prior to ischemia significantly reduced infarction to 14.7 ± 2.1 % (P < 0.05). The tyrosine kinase inhibitor genistein (50 μM) given around the insulin infusion blocked protection (28.9 ± 2.8 %). However, when present during the onset of ischemia, genistein had no effect on protection triggered by insulin (14.0 ± 2.4 %; P < 0.05). Inhibition of either PKC by polymyxin B (50 μM) or K_ATP channels by 5-hydroxydecanoate (100 μM) also failed to prevent protection by insulin (17.5 ± 3.2 % and 17.6 ± 3.0 %, respectively). However, the reduction in infarct size by insulin was significantly attenuated by wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (P13K, 28.3 ± 2.2 %). Insulin was still able to protect the heart when given only during the reperfusion period (13.2 ± 3.4 %). PC reduced infarction to 12.8 ± 2.0 % (P < 0.05). In conclusion, activation of the insulin receptor reduces infarct size in the rabbit heart even when instituted upon reperfusion. However, the mechanism of protection is quite different from that of PC and involves activation of P13K but not PKC or K_ATP channels. Received: 12 November 1998, Returned for revision: 25 November 1998, Revision received: 8 December 1998, Accepted: 10 December 1998     

Brief preconditioning ischemia alters diacylglycerol content and composition in rabbit heart

In order to give further insight into the potential role of PKC in beneficial effects of ischemic preconditioning, we have characterized the production of diacylglycerol, the endogenous activator of PKC, and its molecular species composition in ischemic control and preconditioned hearts. Preconditioning was induced by 1 cycle of 5 min of ischemia followed by 5 min of reperfusion. In control and preconditioned groups, hearts were harvested under deep anesthesia at baseline (preischemia) and at 2,5 and 10 min into the sustained coronary artery occlusion, i.e., preceding myocyte death. Diacylglycerol content and fatty acid composition were analysed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. Myocardial diacylglycerol content was increased at 2 min into the sustained ischemia in the control group (481 ± 34 vs 292 ± 64 ng.mg⁻¹ at baseline; p < 0.05), but was comparable to the baseline value at 5 and 10 min. In the preconditioned group, diacylglycerol production remained unchanged throughout the 10-min test ischemia (317 ± 17 at 2 min vs 312 ± 38 ng.mg⁻¹ at baseline; p = NS). A detailed analysis of the molecular species composition at the time of 2 min revealed a reduced contribution of phosphatidy-linositol to diacylglycerol production in preconditioned myocardium (global correlation coefficient 0.57 vs 0.66 in control myocardium) with a trend toward an enrichment of diacylglycerol composition with some species originating from phosphatidylcholine. Thus, our study revealed that brief preconditioning ischemia: (1) prevents the increase of diacylglycerol content in the early minutes of the sustained ischemia, and (2) emphasizes the contribution of phosphatidylcholine in diacylglycerol formation to the detriment of that of phosphatidylinositol. Received: 12 October 1999, Returned for 1.revision: 3 November 1999, 1.Revision received: 6 January 2000, Returned for 2.revision: 14 February 2000, 2.Revision received: 21 April 2000, Accepted: 9 May 2000     

23Na NMR demonstrates prolonged increase of intracellular sodium following transient regional ischemia in the in situ pig heart

This study tests the hypothesis that Na⁺ _i increases during regional ischemia in the in situ pig heart. An extracorporeal shunt was created between the carotid artery and the left anterior descending artery of 14 open chest pigs. ²³Na and ³¹P NMR spectroscopy measured myocardial Na⁺ _i and high energy phosphates (HEPs). The protocol consisted of three 40 min periods: pre-ischemia (shunt pressure, 76±23 mmHg (S.D.)), ischemia (shunt pressure, 25±7 mmHg), and post-ischemia (shunt pressure, 53±11 mmHg). The pre-ischemia Na⁺ _i concentration was 6.7±4.2 mM. Phosphocreatine (PCr) was 15.3±0.5 mM, ATP 9.4±0.4 mM, inorganic phosphate (Pi) 1.5±0.2 mM, and pH_i 7.16±0.09. At the end of ischemia Na⁺ _i had increased to 10.5±2.8 mM (p<0.0002); PCr decreased to 5.9±2.1 mM (p<0.0002); ATP was 6.5±0.5 mM (p<0.003); Pi had increased to 6.3±1.0 mM (p<0.0002), and pH_i was 6.41±0.06 (p<0.0002). During the first 10 min of the reperfusion, Na⁺ _i increased further to 12.4±2.8 mM (p<0.025), whereas HEPs all returned to pre-ischemic values. Na⁺ _i increases during regional ischemia in the in situ pig heart, suggesting reduced Na⁺/K⁺ ATPase activity. While ATP probably does not limit Na⁺/K⁺ ATPase activity, increases in Pi and decreases in pH_i may reduce Na⁺/K⁺ ATPase activity. Additional Na⁺ _i increases during reperfusion suggest either augmented Na⁺ influx or decreased Na⁺ efflux. Received: 25 May 1998, Returned for revision: 22 June 1998, Revision received: 20 August 1998, Accepted: 15 September 1998     

Glucose uptake increases relative to oxygen consumption during short-term hibernation

Background: Although glucose uptake is increased in chronically hyperfused, viable myocardium, the dynamic changes in glucose uptake relative to oxygen consumption in “short-term” models of hibernation have not been fully explored. Methods: 14 anesthetized swine were instrumented with an hydraulic occluder and flow probe on the proximal LAD artery. Blood flow was reduced ∼30% for 1 hour. Myocardial blood flow and uptake of oxygen, free fatty acids, glucose and lactate were determined in the LAD region at baseline and at 10, 30 and 60 minutes of ischemia. Transmural biopsies for ATP and creatine phosphate (CP) were obtained in the LAD region prior to and at 15 and 45 minutes of ischemia. In 5 animals, glycogen was assayed at baseline and at the end of 60 minutes of ischemia. Results: In the LAD region, myocardial oxygen consumption was reduced from 2.06 ± 0.16 μmol/min/gram to 1.46 ± 0.13 μmol/min/gram (P < 0.05). By 15 minutes of ischemia, transmural creatine phosphate fell from 7.48 ± 0.76 μmol/g-wet weight at baseline to 6.19 ± 0.32 μmol/g-wet weight (P < 0.05) but normalized by 45 minutes of ischemia (7.39 ± 0.56 μmol/g-wet weight; NS). Between 10 and 60 minutes of constant flow reduction, glucose uptake as a percentage of MVO₂ increased from 3 ± 2% to 10 ± 2% (P < 0.05) while lactate uptake increased from −9± 9% to −1± 2% (P < 0.05). Glycogen decreased from 27.8 ± 3.7 at baseline to 16.9 ± 1.2 μmol/g-wet weight at end-ischemia. Conclusions: In this model of short-term hibernation, glucose and lactate uptake increase relative to oxygen consumption during sustained ischemia, and temporally coinicide with the recovery of bioenergetics. The findings are consistent with the notion that glycolytically derived ATP is important for the maintainance of energy supply during sustained ischemia. Received: 26 May 1999, Returned for revision: 1 June 1999, Revision received: 22 June 1999, Accepted: 22 June 1999     

NHE1-inhibitor cariporide prevents the transient reperfusion-induced shortening of the monophasic action potential after coronary ischemia in pigs

During myocardial ischemia intracellular acid load increases as a consequence of anaerobic metabolism. Exchange of excessive protons for sodium via the sodium proton exchanger type 1 (NHE1) is supposed to cause intracellular sodium accumulation. The NHE1 inhibitor cariporide has been shown to inhibit ischemia and reperfusion-induced ventricular fibrillation (VF) but the mechanisms are not fully understood. During early reperfusion transient shortening of the action potential has been reported, which renders the heart susceptible to reentrant arrhythmias. In anesthetized pigs subjected to 10 min of left circumflex coronary artery (LCX) occlusion and reperfusion we have investigated whether NHE1 is involved in reperfusion-induced shortening of the monophasic action potential (MAP) taken with an epicardial probe over the ischemic area. In control pigs (n = 7) a moderate decrease in the duration of the MAP at 50% repolarization (MAPD₅₀) occurred during ischemia reaching 78.8 ± 5.0% of the pre-ischemic duration at 5 min (p < 0.01) and 87.3 ± 7.6% after 10 min. An additional, transient but marked shortening occurred during the first 2 min of reperfusion, which fully recovered after 4 min. At 50 sec of reperfusion MAPD₅₀ fell to 53.1 ± 8.2% of the pre-ischemic value corresponding to 90.1 ± 20.2 msec of reperfusion-induced shortening. Cariporide, 3 mg/kg i. v. 5 min before occlusion (n = 6), totally prevented reperfusion-induced MAP shortening while heaving no effect on MAPD₅₀ during ischemia. In conclusion, our data suggest that the immediate, transient, but strong action potential shortening during early reperfusion after 10 min of coronary ischemia is due to the activity of the NHE1. Received: 16 August 2000, Returned for 1. revision: 8 September 2000, 1. Revision received: 21 September 2000, Returned for 2. revision: 27 September 2000, 2. Revision received: 2 November 2000, Accepted: 7 November 2000