Regulation of the cardiac sodium pump.

In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.

AlphaB crystallin translocation and phosphorylation: signal transduction pathways and preconditioning in the isolated rat heart.

In this program of studies we have characterized in detail the translocation (assessed by Triton-insolubility) and phosphorylation (using serine-45 or -59 phosphospecific antibodies) of alphaB crystallin during myocardial ischemia [both with or without ischemic preconditioning (IPC)]. Pharmacological activators and inhibitors allowed us to characterize the signaling pathways involved in alphaB crystallin phosphorylation during ischemia. Ischemic preconditioning alone caused 30% of the heart's alphaB crystallin pool to translocate, providing a significant translocation 'head-start' in protected tissue. This enhanced translocation is coupled with increased (3-fold) alphaB crystallin phosphorylation at both serine residues. The possible role of alphaB crystallin in the protection afforded by ischemic preconditioning is supported by the signal transduction data; which showed preconditioning-induced alphaB crystallin phosphorylation can be blocked by tyrosine kinase inhibition (using genistein) and by p38 MAP kinase or PKC inhibition (using SB203580 or bisindolylmaleimide, respectively). The activation of both p38 MAP kinase and PKC are recognized requirements for the induction of preconditioning and their inhibition is known to block protection. Western immunoblotting analysis after isoelectric focusing electrophoresis, confirmed the observations made with the phosphospecific antibodies; but also showed that 27+/-4% of total cardiac crystallin was phosphorylated after 30 min of ischemia. AlphaB crystallin exists as large polymeric aggregates in cardiac tissue under basal conditions (approximately 1 MDa as determined by gel filtration chromatography). We induced phosphorylation of alphaB crystallin during aerobic perfusion by the administration of phenylephrine. However this treatment did not alter the molecular aggregate size of alphaB crystallin. It appears that alphaB crystallin molecular aggregate size is not simply regulated by phosphorylation. AlphaB crystallin may have a role to play in the myocardial protection induced by ischemic preconditioning, as both translocation and phosphorylation are both accelerated and enhanced by ischemic preconditioning.

Lipid hydroperoxide modification of proteins during myocardial ischaemia.

OBJECTIVE: Lipid hydroperoxides (LOOH) are lipid peroxidation products formed during oxidative stress. A component of their cytotoxicity is mediated by the direct modification of proteins. OBJECTIVES: (i) To assess whether ischaemia and reperfusion in the isolated rat heart generates LOOH-protein (ii) to characterise the extent, time-course and subcellular localization of any protein adducts formed. METHODS: Using a well-characterised antibody which binds to LOOH-modified proteins and densitometry of Western blots, we quantified the amounts of LOOH-protein in control aerobically perfused rat hearts and those subjected to ischaemia with and without reperfusion. RESULTS: Hearts (n=3/4 group), analysed after various periods (0, 5, 10, 20 and 30 min) of zero-flow global ischaemia, exhibited a time-dependent increase in the LOOH-mediated modification of a number of proteins. In hearts subjected to 30 min of ischaemia and then reperfused for various times (0, 5, 10, 20, 30 or 60 min) no changes in LOOH-protein content achieved during the proceeding ischaemic episode were detected. Reperfusion after short periods of ischaemia (5 or 10 min) also did not result in reperfusion-induced LOOH-protein formation. Administration of mercaptopropionylglycine (1 mM) to hearts for 5 min before the onset of 30 min ischaemia efficiently attenuated the formation of LOOH-protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in situ immunofluorescent studies which showed marked LOOH-protein immunostaining in ischaemic tissue around the sarcolemmal membrane. CONCLUSIONS: We conclude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the pathophysiology of ischaemic injury. In addition, these modifications may be initiators of oxidant-induced signal transduction pathways. These findings are consistent with an oxidant stress occurring during ischaemia which is not exacerbated or reduced during the first 60 min of reperfusion.

Effects of endothelin-1 on K(+) currents from rat ventricular myocytes.

It has been suggested that the positive inotropic effect of the vasoactive peptide hormone, endothelin-1 (ET-1), involves inhibition of cardiac K(+) currents. In order to identify the K(+) currents modulated by ET-1, the outward K(+) currents of isolated rat ventricular myocytes were investigated using whole-cell patch-clamp recording techniques. Outward currents were elicited by depolarisation to +40 mV for 200 ms from the holding potential of -60 mV. Currents activated rapidly, reaching a peak (I(pk)) of 1310 +/- 115 pA and subsequently inactivating to an outward current level of 1063 +/- 122 pA at the end of the voltage-pulse (I(late)) (n = 11). ET-1 (20 nM) reduced I(pk) by 247.6 +/- 60.7 pA (n = 11, P < 0.01) and reduced I(late) by 323.2 +/- 43.9 pA (P < 0.001). The effects of ET-1 were abolished in the presence of the nonselective ET receptor antagonist, PD 142893 (10 microM, n = 5). Outward currents were considerably reduced and the effects of ET-1 were not observed when K(+) was replaced with Cs(+) in the experimental solutions; this indicates that ET-1 modulated K(+)-selective currents. A double-pulse protocol was used to investigate the inactivation of the currents. The voltage-dependent inactivation of the currents from potentials positive to -80 mV was fitted by a Boltzmann equation revealing the existence of an inactivating transient outward component (I(to)) and a noninactivating steady-state component (I(ss)). ET-1 markedly inhibited I(ss) by 43.0 +/- 3.8% (P < 0.001, n = 7) and shifted the voltage-dependent inactivation of I(to) by +3.3 +/- 1.2 mV (P < 0.05). Although ET-1 had little effect on the onset of inactivation of the currents elicited from a conditioning potential of -70 mV, the time-independent noninactivating component of the currents was markedly inhibited. In conclusion, the predominant effect of ET-1 was to inhibit a noninactivating steady-state background K(+) current (I(ss)). These results are consistent with the hypothesis that I(ss) inhibition contributes to the inotropic effects of ET-1.

Differential centrifugation separates cardiac sarcolemmal and endosomal membranes from Langendorff-perfused rat hearts.

The application of subcellular fractionation protocols developed in soft tissues to fibrous organs such as the heart is unsuitable given the substantial differences in subcellular structure these tissues exhibit. The purpose of this study was to develop a simple method for the separation of sarcolemma and endosomes from isolated Langendorff-perfused rat hearts. Hearts were homogenized with either an Ultra-Turrax homogenizer or a hand-held glass tissue grinder. Quantitative immunoblots assessed the enrichment of the sarcolemmal proteins caveolin 3 and the sodium potassium ATPase and the endosomal proteins rab4 and GLUT4 in different membrane fractions. Application of homogenates to sucrose and Percoll density gradients failed to resolve membranes differentially enriched in sarcolemmal or endosomal marker proteins, indicating little difference in density between the sarcolemma and endosomes. However, successive spins of homogenates from a hand-held glass tissue grinder successfully separated the endosomes from the sarcolemma, indicating differences in masses between the two membrane fractions. Approximately 70% of total caveolin 3 and sodium potassium ATPase immunoreactivity was in membrane pellets up to 20,000g and approximately 85% of rab4 and GLUT4 in pellets from 20,000-100,000g. In addition, 86% of ouabain-sensitive ATPase activity (sodium potassium ATPase activity) was in membrane pellets up to 20,000g. Therefore, sarcolemmal membranes were pelleted up to 20,000g, and endosomal membranes between 20,000 and 100,000g. Regional ischemia (40 min) followed by reperfusion (60 min) caused the translocation of GLUT4 (but not rab4) from the endosomal membranes to the sarcolemma in the area of the heart subjected to ischemia.

Ischemic preconditioning: a potential role for constitutive low molecular weight stress protein translocation and phosphorylation?

We have investigated whether translocation of constitutive low molecular weight stress proteins (alphaB-crystallin and HSP27) to the myofilament/cytoskeletal compartment occurs during ischemic preconditioning and assessed if this is causally associated with cardioprotection. Triton-insoluble preparations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little alphaB-crystallin (96 +/- 43 and 43 +/- 36 units respectively) or HSP27 (177 +/- 32 and 101 +/- 26 units respectively). Three preconditioning cycles of (5 min ischemia + 5 min reperfusion) increased the Triton-insoluble crystallin to 864 +/- 61 units (P<0.05) and HSP27 to 1353 +/- 53 units (P<0.05). Two hours of aerobic perfusion following the preconditioning protocol resulted the return of alphaB-crystallin and HSP27 to near control levels (189 +/- 14 units and 252 +/- 24 units, respectively). Stress protein translocation, comparable to that achieved by the IPC protocol was induced by aerobic perfusion with hypercarbic (pH 6.8) perfusion. Thus, three cycles of 5 min hypercarbia + 5 min normocarbia increased alphaB-crystallin to 628 +/- 30 units (P<0.05) and HSP27 to 1353 +/- 53 units. In parallel functional studies, the recovery of LVDP after 35 min ischemia and 60 min of reperfusion was 43 +/- 7% in the ischemic control group, 61 +/- 3% (P<0.05) in the preconditioned group and 42 +/- 6% in the hypercarbic group. Thus, translocation of alphaB-crystallin and/or is not of-itself sufficient to induce cardioprotection. Using a phospho-specific antibody, we have demonstrated that preconditioning not only translocates alphaB-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to aerobic controls (1616 +/- 402 v 166 +/- 28 units respectively). In contrast, hypercarbia while eliciting a comparable translocation, failed to alter the phosphorylation state of alphaB-crystallin. Preconditioning-induced phosphorylation was significantly attenuated by 50 microM genistein (by 61%), 10 microM SB203580 (by 91%) and 10 microM bisindolylmaleimide (by 68%), but not by 10 microM PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and translocation of constitutive low molecular weight stress proteins, particularly alphaB-crystallin.

L-type calcium current of isolated rat cardiac myocytes in experimental uraemia.

BACKGROUND: End-stage renal failure is associated with a low-output cardiomyopathy, left ventricular hypertrophy and increased QTc dispersion. Cardiac dysfunction is prevalent in patients at the beginning of dialysis and is an important predictor of mortality. Ca(2+) influx through voltage-gated L-type Ca(2+) channels plays a key role in the excitation-contraction coupling of cardiac myocytes. The purpose of this study was to examine the effect of subtotal nephrectomy (SNx) in the rat on both cardiac L-type Ca(2+) currents and action potential duration. METHODS: Wistar rats underwent two-stage SNx; control rats (C) underwent bilateral renal decapsulation. Animals were sacrificed after 8 weeks, and ventricular myocytes were isolated. SNx rats showed a 2-fold increase in plasma urea and creatinine compared with C rats. Whole-cell patch clamp techniques were used to examine L-type Ca(2+) channel currents in isolated cardiac myocytes at 37 degrees C. In separate experiments, the epicardial monophasic action potentials of isolated perfused whole hearts from C and SNx rats were recorded. RESULTS: The amplitude and current-voltage relationships of the L-type Ca(2+) current were not significantly different in myocytes from C (n=11) and SNx (n=8) rats. However, the rate of inactivation of the Ca(2+) current was increased by approximately 15-25% (P<0. 05) in myocytes from SNx rats. The action potential duration (APD(33)) at the apex of the left ventricle was approximately 20% shorter (P<0.01) in hearts from SNx rats as compared with controls. CONCLUSIONS: Renal failure is associated with rapid inactivation of cardiac ventricular myocyte L-type Ca(2+) currents, which may reduce Ca(2+) influx and contribute to shortening of the action potential duration.

Long-term myocardial preservation: effects of hyperkalemia, sodium channel, and Na/K/2Cl cotransport inhibition on extracellular potassium accumulation during hypothermic storage.

OBJECTIVES: We previously demonstrated improved myocardial preservation with polarized (tetrodotoxin-induced), compared with depolarized (hyperkalemia-induced), arrest and hypothermic storage. This study was undertaken to determine whether polarized arrest reduced ionic imbalance during ischemic storage and whether this was influenced by Na+/K +/2Cl- cotransport inhibition. METHODS: We used the isolated crystalloid perfused working rat heart preparation (1) to measure extracellular K+ accumulation (using a K+-sensitive intramyocardial electrode) during ischemic (control), depolarized (K+ 16 mmol/L), and polarized (tetrodotoxin, 22 micromol/L) arrest and hypothermic (7.5 degrees C) storage (5 hours), (2) to determine dose-dependent (0.1, 1.0, 10 and 100 micromol/L) effects of the Na +/K+/2Cl- cotransport inhibitor, furosemide, on extracellular K+ accumulation during polarized arrest and 7.5 degrees C storage, and (3) to correlate extracellular K+ accumulation to postischemic recovery of cardiac function. RESULTS: Characteristic triphasic profiles of extracellular K+ accumulation were observed in control and depolarized arrested hearts; a significantly attenuated profile with polarized arrested hearts demonstrated reduced extracellular K+ accumulation, correlating with higher postischemic function (recovery of aortic flow was 54% +/-4% [P =.01] compared with 39% +/-3% and 32% +/-3% in depolarized and control hearts, respectively). Furosemide (0.1, 1.0, 10, and 100 micromol/L) modified extracellular K+ accumulation by -18%, -38%, -0.2%, and +9%, respectively, after 30 minutes and by -4%, -27%, +31%, and +42%, respectively, after 5 hours of polarized storage. Recovery of aortic flow was 53% +/-4% (polarized arrest alone), 56% +/-8%, 70% +/-2% (P =.04 vs control), 69% +/-4% (P =.04 vs control), and 65% +/-3% ( P =. 04 vs control), respectively. CONCLUSIONS: Polarized arrest was associated with a reduced ionic imbalance (demonstrated by reduced extracellular K+ accumulation) and improved recovery of cardiac function. Further attenuation of extracellular K + accumulation (by furosemide) resulted in additional recovery.

Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart.

4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.

Ischemic preconditioning in immature myocardium.

BACKGROUND: Protection by ischemic preconditioning (PC) has not been studied extensively in immature hearts. We studied the developmental differences in the ability of the rat heart to precondition with ischemia. METHODS AND RESULTS: Hearts from 4-, 7-, 14-, and 21-day-old and adult (approximately 50-day-old) rats were aerobically perfused in Langendorff mode before a PC stimulus of either (1) 3-minute ischemia (I), 3-minute reperfusion (R), 5-minute I, 5-minute R, or (2) 4 cycles of 5-minute I and 5-minute R, before a prolonged I (chosen to give 30% to 40% recovery) and 60-minute R. LVDP recovery was expressed as percent of baseline reading (after 20-minute aerobic perfusion). Protection was seen after protocol 1 in 14- and 21-day-old and adult hearts (45 +/- 5%, 53 +/- 7%, and 58 +/- 5% versus 30 +/- 4%, 29 +/- 3%, and 32 +/- 2% in controls, respectively) but not in 4- and 7-day (neonatal) hearts; neonatal hearts were also not protected in protocol 2. To determine whether this inability of neonatal hearts to precondition was due to insufficient duration of the PC cycle, they were subjected to increased I durations up to 20 minutes before 5-minute R, prolonged I (90 minutes and 60-minute R) (protocol 3); protection was not seen. To determine whether the inability to precondition was due to an excessively prolonged ischemic duration, neonatal hearts were subjected to only 45 minutes of prolonged I (protocol 4); again, PC protection was not evident. CONCLUSIONS: Protection by PC develops after 7 days; the inability of neonatal hearts (< 7 days old) to precondition is not due to insufficient stimulus or extended ischemia.

Hypothermic preservation of isolated rat lungs in modified bicarbonate buffer, EuroCollins solution or St Thomas' Hospital cardioplegic solution.

OBJECTIVES: Inadequate preservation solutions limit lung storage times and, consequently, transplant programs. To address this problem we established an isolated, ventilated and perfused rat lung preparation. Here we report the effects of hypothermic storage in EuroCollins solution, St Thomas' Hospital cardioplegic solution and a modified bicarbonate buffer solution. METHODS: Lungs from male Wistar rats (230-330 g) were perfused via the pulmonary artery with modified bicarbonate buffer (37 degrees C, 15 ml/min, constant flow) and ventilated by positive pressure (tidal volume:1.6-1.8 ml, 80 breaths/min). Vascular resistance (pulmonary artery pressure:perfusate flow ratio) and airways compliance (tidal volume:tracheal pressure ratio) were measured. After a control perfusion period (20 min), lungs were flushed with, then immersed in, bicarbonate buffer (4 degrees C) for varying periods (0-24 h). After storage, lung function was assessed during 20 min reperfusion. Having established a suitable period for study, storage in EuroCollins, St Thomas' Hospital cardioplegic solution or bicarbonate buffer were compared. RESULTS: Pulmonary compliance (ml/cmH2O) was significantly (P < 0.05) reduced in lungs stored for 6 h in modified bicarbonate buffer (0.026 +/- 0.008), EuroCollins solution (0.013 +/- 0.002) or St Thomas' Hospital solution (0.025 +/- 0.005) compared to unstored lungs (0.068 +/- 0.007). Vascular resistance, (1.32 +/- 0.13 cmH2O/ml per min) in unstored lungs, was similar in lungs stored in St Thomas' Hospital solution but increased significantly in lungs stored in modified bicarbonate buffer (3.22 +/- 0.78 cmH2O/ml per min) or EuroCollins solution (4.66 +/- 0.57 cmH2O/ml per min). CONCLUSIONS: Hypothermic storage of rat lungs for 6 h in modified bicarbonate buffer or St Thomas' Hospital solution causes less increase in vascular resistance on reperfusion than EuroCollins solution.

Developmental differences in superoxide production in isolated guinea-pig hearts during reperfusion.

The production of free radicals on reperfusion has been implicated as an important factor governing post-ischemic recovery of cardiac function. Although the response of the heart to ischemia and reperfusion is known to change during cardiac development, it is not known if different rates of free radical production play a role in these altered responses. The aim of this investigation was to determine if the production of the superoxide anion (O2-) on reperfusion differs in the immature and mature heart. Immature hearts, obtained from 3-day premature guinea pigs (delivered by cesarean section) were compared with those from adults (7 weeks old). Using the isolated Langendorff preparation. O2- production was measured during reperfusion following ischemic durations [0 (aerobic control), 15, 20, 30, and 60 min, n = 6/group] by the reduction of succinylated ferricytochrome c in the perfusate. Both immature and mature hearts exhibited bell-shaped relationship between ischemic duration and peak O2- production on reperfusion: (13.4 +/- 5.9; 22.2 +/- 5.4; 23.0 +/- 7.8; 59.3 +/- 16.2; 33.7 +/- 15.1; 32.6 +/- 8.5 nmol/min/g wet weight in the immature heart and 15.7 +/- 1.9; 55.0 +/- 30.2; 82.8 +/- 14.0; 78.8 +/- 33.8; 40.6 +/- 16.4; 45.4 +/- 13.1 nmol/min/g wet weight in the mature heart after 0; 15; 20; 30; 45 and 60 min of ischemia, respectively). A similar relationship was also demonstrated with O2- production over the 20-min reperfusion period: (134.0 +/- 57.1; 106.5 +/- 46.2; 199.3 +/- 50.6; 362.0 +/- 99.5; 375.0 +/- 60.9; 221.0 +/- 73.0 nmol/20 min/g wet weight in the immature heart and 97.8 +/- 54; 282.0 +/- 139.0; 933.3 +/- 210.3; 964.0 +/- 374.0; 443.0 +/- 106.0; 352.0 +/- 1551.0 nmol/20 min/g wet weight in the mature heart after 0, 15, 20, 30, 45 and 60 min of ischemia, respectively). Mature hearts consistently produced more O2- than immature hearts on reperfusion, while there was no significant difference in their capacity to produce O2- during aerobic perfusion. We conclude that the immature heart may be at less risk from the free radical component of reperfusion injury than the mature heart.

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle.

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.

Comparison of polarized and depolarized arrest in the isolated rat heart for long-term preservation.

BACKGROUND: Hypothermic hyperkalemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalemia-induced depolarization of the resting membrane potential (Em) may predispose the heart to Na+ and Ca2+ loading via voltage-dependent "window currents," thereby exacerbating injury and limiting the safe storage duration. Alternatively, maintaining the resting Em with a polarizing solution may reduce ionic movements and improve postischemic recovery; we investigated this concept with the reversible sodium channel blocker tetrodotoxin (TTX) to determine (1) whether polarized arrest was more efficacious than depolarized arrest during hypothermic long-term myocardial preservation and (2) whether TTX induces and maintains polarized arrest. METHODS AND RESULTS: The isolated crystalloid-perfused working rat heart preparation was used in this study. Preliminary studies determined an optimal TTX concentration of 22 micromol/L and an optimal storage temperature of 7.5 degrees C. To compare depolarized and polarized arrest, hearts were arrested with either Krebs-Henseleit (KH) buffer (control), KH buffer containing 16 mmol/L K+, or KH buffer containing 22 micromol/L TTX and then stored at 7.5 degrees C for 5 hours. Postischemic recovery of aortic flow was 13+/-4%, 38+/-2%, and 48+/-3%* (*P<.05 versus control and 16 mmol/L K+), respectively. When conventional 3 mol/L KCl-filled intracellular microelectrodes were used, Em gradually depolarized during control unprotected ischemia to approximately -55 mV before reperfusion, whereas arrest with 16 mmol/L K+ caused rapid depolarization to approximately -50 mV, where it remained throughout the 5-hour storage period. In contrast, in 22 micromol/L TTX-arrested hearts, Em remained more polarized, at approximately -70 mV, for the entire ischemic period. CONCLUSIONS: Blockade of cardiac sodium channels by TTX during ischemia maintained polarized arrest, which was more protective than depolarized arrest, possibly because of reduced ionic imbalance.

Myocardial stunning: do we know the mechanism?

In response to the challenge of the title of this article, it is clear that we do know the principal players involved in myocardial stunning. We know that: approximately 80-85% of stunning can be attributed to free radicals the principal targets for free radical damage are the proteins involved in E-C coupling the action potential and the Ca transient remain largely intact in the stunned myocardium and thus the principal lesion in stunned hearts is at the level of the myofilaments myofilament damage can be induced either directly by free radicals or indirectly via Ca overload-induced activation of proteases which degrade myofilament proteins Ca overload and free radicals are likely to interact to promote stunning the 15-20% of stunning which is not directly attributable to free radicals is likely to reflect damage mediated by Ca overload this free radical-independent Ca overload is principally mediated via the activation of Na/Ca exchange secondary to the activation of Na/H exchange a principal player in reperfusion-induced Ca overload is the recovery of intracellular Na which may be compromised by both damage to the Na/K pump and the physical translocation of the pump away from the sarcolemmal membrane.

Electrophysiological characteristics of repetitive ischemic preconditioning in the pig heart.

S-T segment changes have been cited as evidence for preconditioning in the human heart during repeated angioplasty inflations. Opening of preformed collaterals, however, could explain these observations. We have measured the profile of S-T segment and monophasic action potential (MAP) changes in a species with low collateralization. Open-chested pigs were subjected to two cycles of 8-min LAD occlusion and 8-min reperfusion prior to 60-min ischemia and 2-h reperfusion. Two epicardial ECGs and MAP were continuously recorded from the ischemic zone and one ECG from the normal zone. Flow was measured using Xenon washout. Infarct (IS) and risk zone (RZ) sizes were assessed after reperfusion in a subset of six pigs and confirmed profound protection with preconditioning (IS/RZ = 14 +/- 9% v 42 +/- 3% in controls, P < 0.05). S-T segment elevation was smaller early in the 2nd or 3rd (0-3 min) ischemic cycles than in the 1st. In contrast, in the 1st ischemic cycle, MAP duration after 3 min was reduced to 90 +/- 2% control and this was further reduced in the 2nd and 3rd ischemic episodes to 74 +/- 4% and 77 +/- 3% respectively. Thus, preconditioning increased APD shortening while simultaneously decreasing S-T segment elevation during the early minutes of ischemia. It therefore seems unlikely that the ability of preconditioning to limit S-T segment changes is related to limitations in APD shortening. All electrophysiological differences were lost later during ischemia. Collateral flow during the three ischemic cycles was 4.8 +/- 3.7, 5.8 +/- 2.3 and 5.6 +/- 2.9% (n = 5/grp, ns) respectively. Thus, in the absence of a significant increase in collateral flow. S-T segment and MAP changes provide an index of preconditioning but only during the first few minutes of occlusion. S-T segment changes observed during PTCA may therefore reflect genuine preconditioning in man although the contribution of ischemia-induced increases in collateral flow cannot be ignored.

Ventricular arrhythmias induced by ischaemia-reperfusion are unaffected by myocardial glutathione depletion.

Reduced glutathione (GSH) is a major myocardial antioxidant. Since reperfusion phenomena such as ventricular fibrillation (VF) are associated with oxygen free radical production during ischaemia, myocardial GSH depletion might be expected to increase susceptibility to such phenomena. This possibility was tested in isolated rat hearts using diethylmaleate (DEM) or L-buthionine-SR-sulfoximine (BSO) to deplete myocardial GSH. High dose DEM (860 mg/kg) depleted myocardial GSH from a control mean of 7.64 +/- 0.73 to 3.18 +/- 0.56, low dose DEM (215 mg/kg) to 4.29 +/- 0.53 nmol/mg protein and BSO (4 mmol/kg) from a control mean of 6.94 +/- 0.54 to 2.18 +/- 0.14 nmol/mg protein. Hearts were perfused in the Langendorff mode at 37 degrees C with bicarbonate buffer (K+ = 4.3 mM). Regional ischaemia was induced for 5, 8.5, 10, 20 or 40 min (DEM groups: n = 10/treatment/time point) or 8.5 min only (BSO groups: n = 10/treatment) then hearts were reperfused for 5 min. Reperfusion VF incidence showed a classical "bell-shaped" curve, but there was no difference in VF incidence, VF time-to-onset, arrhythmia duration and "arrhythmia scores" between GSH-depleted and control hearts. Depleting myocardial GSH is not proarrhythmic for reperfusion-induced arrhythmias. It would appear GSH is not significantly involved in protecting against the oxidant stress of reperfusion, or conversely that the reserve of this redox system is so high only severe depletion might show an effect.

The role of the rate of vascular collapse in ischemia-induced acute contractile failure and decreased diastolic stiffness.

We investigated the contribution of the rate of vascular collapse to the early contractile failure and decreased diastolic stiffness induced by ischemia. Isolated rat hearts (n = 8/group), perfused at 37 degrees C with blood through a roller pump and paced at 320 beats/min, were subjected to global ischemia either by switching off the roller pump (slow vascular collapse-group 1) or by reversing the direction of the roller pump for 5 s prior to switch-off (rapid vascular collapse-group 2). In group 1, residual coronary pressure declined progressively over the first 20 s of ischemia whereas in group 2 the pressure had fallen to zero within 2 s. The profile of ischemia-induced contractile failure was however, similar in both groups. Thus, after 2 s of ischemia, when residual perfusion pressure had declined by only 10% in group 1 (60.0 +/- 0.0 to 54.9 +/- 0.8 mmHg) but was virtually non-existent in group 2 (60.0 +/- 0.0 to 0.4 +/- 12.7 mmHg), left ventricular developed pressure had fallen to a similar extent in both groups (86 +/- 2% and 84 +/- 3%, respectively). Curve-fitting analysis for individual hearts showed that the profile of contractile failure was described by a double exponential process that was not significantly affected by the rapid vascular collapse. Left ventricular end-diastolic pressure in group 1 hearts progressively declined over the first 20 s of ischemia, the profile paralleling that of the dissipation of perfusion pressure. In contrast, in group 2 hearts, left ventricular end-diastolic pressure rose rapidly and leaked at 5 s, a period that coincided with the reversed direction of the perfusion pump. Similarly, in a separate study, the analysis of ventricular diastolic stiffness (n = 6/group) showed a rapid decline during the first 20 s of ischemia: this decline could be inhibited by the use of rapid vascular collapse. In additional experiments, hearts (n = 8/group) were paced at 220, 320 or 420 beats/min and ischemia was induced by reversing (5 s) and then stopping the perfusion pump. Myocardial oxygen consumption increased in parallel with heart rate and was matched by commensurate increases in the rate of contractile failure. Curve-fitting analysis showed that slow stimulation rates (220 beats/ min) significantly delayed contractile failure during the first 60 s of ischemia (first time constant = 14.5 +/- 4.1 s compared with 8.1 +/- 1.1 s at 320 beats/min and 6.3 +/- 1.1 s at 420 beats/min; P < 0.05 in both instances). In conclusion, vascular collapse associated with ischemia may contribute to the initial decrease in ventricular diastolic stiffness; however, it does not play a major role in determining the rate of acute contractile failure. Metabolic processes as reflected by oxygen consumption do however, appear to be important.