Substrate-free Hypoxia Research Articles

Adenosine protects against hypoxic injury at hypothermia in guinea pig papillary muscles

Objectives. To investigate the protective effects of adenosine against hypoxic injury at hypothermia; both magnitude and mechanisms. Design. Receptor versus non-receptor dependent mechanisms in cardioprotection by adenosine were examined in guinea pig papillary muscles exposed to glucose free hypoxia at 24°C. Contractile force amplitude (CFA) and action potential duration (APD) during increasing concentrations of adenosine at 37°C, 30°C and 24°C normoxia were also examined. Results. CFA was significantly improved after adenosine treatment during hypothermic hypoxia compared to control (80.7±17.4% vs 40.5±10.7%, p<0.001). Adenosine receptor antagonist SPT did not antagonize (64.6±21.1%), and adenosine receptor agonists (APNEA+NECA) could not mimic the cardioprotection (53.8±9.3%). MitoKCa blocker paxilline antagonized the cardioprotection (40.0±7.7%). During normoxic conditions hypothermia-induced increase in CFA was significantly decreased by adenosine (0.12–12 mM) whereas the increase in action potential duration was potentiated. Conclusion. Adenosine (1.2 mM) had marked cardioprotective effect in hypothermic substrate free hypoxia. Possible mechanisms are non-receptor dependent and related to mitoKCa channels. The cardiodepressive effect at hypothermia may contribute to cardioplegia.

Substrate dependence of the postischemic cardiomyocyte recovery: Dissociation between functional, metabolic and injury markers

Defining the substrate that influences the most favourably the myocardial post-ischemic recovery is subject of debates, due to dissociation between functional and biochemical benefits. Hence, we studied the effects of either glucose or different fatty acids on the functional and metabolic recovery of post-ischemic cardiomyocytes in a substrate-free hypoxia model of simulated ischemia-reperfusion. Rat cardiomyocytes were submitted to a 2.5 h simulated ischemia followed by a 2 h reoxygenation without substrate (control), or with either glucose, octanoic acid, oleic acid, or elaidic acid. During simulated ischemia, electromechanical function gradually disappeared while the cellular viability and mitochondrial function declined. During control simulated reperfusion, cardiomyocytes recovered near normal function but a significant reduction in the action potential amplitude and rate persisted. The addition of glucose or oleic acid during simulated reperfusion promoted a faster, better and sustain functional recovery. Amongst the fatty acids, the functional recovery was slower with elaidic and octanoic acids as compared with oleic acid. The mitochondrial function was better improved during simulated reperfusion with glucose than with the tested fatty acids, among which elaidic acid was the less unfavourable. Paradoxically, the addition of whichever substrate during simulated reperfusion tended to worsen the cellular viability. Thus, cardiomyocytes recovery strongly relies on the characteristics of the substrate supplied at the onset of simulated reperfusion: glucidic or lipidic nature, chain-length, insaturation degree. Moreover, these data suggest that defining the appropriateness of a given substrate for the post-ischemic cardiomyocyte recovery is closely related to the functional and the biological endpoints in consideration.

Physiological and metabolic actions of mycophenolate mofetil on cultured newborn rat cardiomyocytes in normoxia and in simulated ischemia.

Mycophenolate mofetil (MMF) is a new immunosuppressive drug used to reduce acute rejection after heart transplantation. As with other immunosuppressive drugs, MMF therapy is associated with several adverse effects. However, the direct effects of MMF on myocardial tissue has not been yet evaluated. The aim of the work was thus to evaluate the effects of MMF on isolated cardiomyocytes (CM) in normal conditions and in an in vitro model of simulated ischemia (SI; substrate-free hypoxia) and reperfusion (R; reoxygenation). Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded using conventional microelectrodes and the cell contractions were monitored with a photoelectric device. In basal conditions, MMF (10(-6) and 10(-5) M) exerted no significant effects on the survival and on the electrical and contractile activities of CM in culture, even during long-term exposure (up to 48 h). SI per se led to a gradual decrease and then an abortion of the spontaneous automaticity and electromechanical activity of CM. Pretreating CM with either 10(-6) or 10(-5) M MMF was able to reduce the SI-induced cell dysfunctions. The presence of MMF at these concentrations did not hamper the post-SI functional recovery of CM during reoxygenation. At 10(-5) M, MMF applied during reoxygenation only permitted a better recovery of CM. However, the mitochondrial function after reoxygenation, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) test, was not significantly influenced by the addition of MMF before as well as after ischemia. Conversely, MMF was able to reduce in this model the postischemic rise in xanthine and hypoxanthine. These data from CM-enriched model show that MMF: (i) had no cytotoxic effect, (ii) displayed a cytoprotective effect during SI, and (iii) exerted its beneficial effect at least partly through the decrease in the xanthine oxidase-dependent free radical production.

Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes.

Cytoskeleton damage, particularly microtubule (MT) alterations, may play an important role in the pathogenesis of ischemia-induced myocardial injury. However, this disorganization has been scarcely confirmed in the cellular context. We evaluated MT network disassembly in myoblast cell line H9c2 and in neonatal rat cardiomyocytes in an in vitro substrate-free hypoxia model of simulated ischemia (SI). After different duration of SI from 30 up to 180 min, the cells were fixed and the microtubule network was revealed by immunocytochemistry. The microtubule alterations were quantified using a house-developed image analysis program. Additionally, the tubulin fraction were extracted and quantified by Western blotting. The cell respiration, the release of cellular LDH and the cell viability were evaluated at the same periods. An early MT disassembly was observed after 60 min of SI. The decrease in MT fluorescence intensity at 60 and 90 min was correlated with a microtubule disassembly. Conversely, SI-induced significant LDH release (35%) and decrease in cell viability (34%) occurred after 120 min only. These results suggest that the simulated ischemia-induced changes in MT network should not be considered as an ultrastructural hallmark of the cell injury and could rather be an early ultrastructural correlate of the cellular reaction to the metabolic challenge.

Specific electromechanical responses of cardiomyocytes to individual and combined components of ischemia.

The main factors of myocardial ischemia are hypoxia, substrate deprivation, acidosis, and high extracellular potassium concentration ([K+]e), but the influence of each of these factors has not yet been evaluated in a cardiomyocyte (CM) culture system. Electromechanical responses to the individual and combined components of ischemia were studied in CM cultured from newborn rat ventricles. Action potentials (APs) were recorded using glass microelectrodes and contractions were monitored photometrically. Glucose-free hypoxia initially reduced AP duration, amplitude, and rate and altered excitation-contraction coupling, but AP upstroke velocity (Vmax) remained unaffected. Early afterdepolarizations appeared, leading to bursts of high-rate triggered impulses before the complete arrest of electromechanical activity after 120 min. Acidosis reduced Vmax whereas AP amplitude and rate were moderately decreased. Combining acidosis and substrate-free hypoxia also decreased Vmax but attenuated the effects of substrate-free hypoxia on APs and delayed the cessation of the electrical activity (180 min). Raising [K+]e reduced the maximal diastolic potential and Vmax. Total ischemia (substrate deletion, hypoxia, acidosis, and high [K+]e) decreased AP amplitude and Vmax without changing AP duration. Moreover, delayed afterdepolarizations appeared, initiating triggered activity. Ultimately, 120 min of total ischemia blocked APs and contractions. To conclude, glucose-free hypoxia caused severe functional defects, acidosis delayed the changes induced by substrate-free hypoxia, and total ischemia induced specific dysfunctions differing from those caused by the former conditions. Heart-cell cultures thus represent a valuable tool to scrutinize the individual and combined components of ischemia on CMs.

Assessment of the cytoprotective role of adenosine in an in vitro cellular model of myocardial ischemia

This work aimed to detect functional adenosine receptors in isolated rat cardiomyocytes and to study the influence of stimulation of these receptors in an in vitro model of ischemia. Cultures of cardiomyocytes were prepared from newborn rat ventricles. The contractions were photometrically monitored. In this preparation, adenosine induced a positive chronotropic response. This effect was reproduced by CGS 21680 (2-(4-[2-carboxyethyl]-phen-ethyl-amino) adenosine-5′ N-ethylunosamide), a specific adenosine A 2 receptor agonist, and antagonized by DMPX (3,7-dimethyl-1-propargylxanthine), an adenosine A 2 receptor antagonist. However, R-PIA ( R- N 6-(2-phenylisopropyl)-adenosine; a specific adenosine A 1 receptor agonist) induced a negative chronotropic effect that was abolished by its corresponding adenosine A 1 antagonist DPCPX (1,3-dipropyl-8-cyclo-pentyl-adenosine). Substrate-free hypoxia, as simulation of ischemia, induced a progressive decrease and then arrest of spontaneous cell contractions. The spontaneous rhythmic contractile activity was restored during reoxygenation following simulated ischemia. Adenosine A 1 receptor stimulation with R-PIA induced a decrease of hypoxia-induced damage. This effect was antagonized by DPCPX, an adenosine A 1 receptor antagonist. Conversely, the cells treated with CGS 21680 did not display complete recovery after reoxygenation. In addition, this effect was abolished by DMPX, since the cells recovered normal function after reoxygenation. To conclude, it appeared that cardiomyocytes possess both functional adenosine A 1 and A 2 receptors and that only the activation of adenosine A 1 receptor had a cytoprotective effect against simulated ischemia-induced cardiac cell injury.

Effects of a selective inhibitor of Na+/Ca2+ exchange, KB-R7943, on reoxygenation-induced injuries in guinea pig papillary muscles.

The effects of a novel agent that is reported to selectively block Ca2+ influx by Na+/Ca2+ exchange (NCX), KB-R7943, on the reoxygenation-induced arrhythmias and the recovery of developed tension after reoxygenation, were investigated in guinea pig papillary muscles. KB-R7943 dose-dependently suppressed the contracture tension during low-sodium (21.9 mM) perfusion (23+/-8% of steady-state developed tension at 10 microM vs. 56+/-11% in control; n = 6, p<0.05), but did not change action potential and contractile parameters. During the reoxygenation period after 60-min substrate-free hypoxia, KB-R7943 (10 microM) significantly decreased the incidence of arrhythmias (44 vs. 100% in control; n = 9, p <0.05) and shortened the duration of arrhythmias (16+/-11 vs. 72+/-14 s; p<0.01). KB-R7943 (10 microM) significantly enhanced the recovery of developed tension after reoxygenation (83+/-4 vs. 69+/-3% in control; p<0.05). We conclude that KB-R7943 (10 microM) selectively inhibits the reverse mode of NCX, and that it attenuates reoxygenation-induced arrhythmic activity and prevents contractile dysfunction in guinea pig papillary muscles. These results suggest that Ca2+ influx by NCX may play a key role in reoxygenation injury.

Protection by hypoxic preconditioning against hypoxia-reoxygenation injury in guinea-pig papillary muscles.

Developed tension in guinea-pig papillary muscles is depressed by prolonged hypoxia; subsequent reoxygenation leads to a partial recovery that stabilizes after an early period of arrhythmia. We have investigated whether hypoxic preconditioning in these muscles (1) improves the recovery of developed tension, (2) protects against arrhythmia, and (3) causes other significant electromechanical changes. Papillary muscles stimulated at 1 Hz were superfused with oxygenated Krebs solution for 60 min and either preconditioned (5 min of 3 Hz pacing substrate-free hypoxic conditions, 10 min of normoxic recovery) or equilibrated for an extra 15 min. Muscles were subsequently challenged with substrate-free hypoxia (1 Hz), and reoxygenated (1 Hz) for 60 min. Contractile performance, action potential parameters, and indicators of arrhythmic activity were measured in 10 preconditioned and 10 non-preconditioned muscles. Developed tension in preconditioned muscles declined to the same level (10-15% control) as in non-preconditioned muscles after 60 min hypoxia. A notable difference was that developed tension in the preconditioned muscles failed to rebound during mid-hypoxia, a hallmark feature in non-preconditioned muscles. The action potential duration and overshoot collapsed at a significantly faster rate in hypoxic preconditioned muscles. Action potential recovery during reoxygenation was similar in the two groups of muscles, but recovery of developed tension was significantly stronger in preconditioned (76.7 +/- 5.4%) than in non-preconditioned (42.9 +/- 1.7%) muscles (P < 0.001). Reoxygenation provoked arrhythmic activity in all muscles, but the summed average duration was shorter (5.5 +/- 1.0 vs. 9.4 +/- 1.5 min) (P < 0.05) in the preconditioned muscles. Hypoxic preconditioning can significantly enhance post-hypoxia recovery of developed tension, and significantly attenuate arrhythmic activity, in guinea-pig papillary muscles.

Influence of phospholipid long chain polyunsaturated fatty acid composition on neonatal rat cardiomyocyte function in physiological conditions and during glucose-free hypoxia-reoxygenation.

There is evidence that dietary polyunsaturated fatty acids (PUFA) may protect against cardiovascular diseases, but the involvement of the cardiac muscle cell in this beneficial action remain largely unknown. The present study compared the respective influence of n-3 and n-6 PUFA on the function of cultured neonatal rat cardiomyocytes (CM). Cells were grown for 4 days in media enriched either n-3 (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) or n-6 (arachidonic acid, AA) PUFA. The PUFA n-6/n-3 ratio in the phospholipids was close to 1 and 20 in the n-3 and n-6 cells, respectively. The transmembrane potentials were recorded using microelectrodes and the contractions were monitored with a photoelectric device. In physiological conditions, the increase of n-6 PUFA level in the phospholipids resulted in a significant decrease in the maximal rate of initial depolarization (-16%). In opposition, the action potential amplitude and duration were not altered, and the cell contraction outline was not affected. Ischemia was simulated in vitro using a substrate-free, hypoxia-reoxygenation procedure in a specially designed gas-flow chamber. The progressive loss of electrical activity induced by the substrate-free, hypoxic treatment was affected by the n-6/n-3 ratio, since the n-6 rich CM displayed a slower depression of the AP amplitude and duration parameters. Conversely, the recovery of the resting potential (MDP) during reoxygenation was faster in n-3 CM, whereas the recovery of the contraction parameters was unaffected by the fatty acid composition of the cells. These results suggested that, in physiological conditions, the modification of long chain PUFA balance in the phospholipids of cardiac muscle cells may modulate the initial AP upstroke, which is governed by sodium channels. Moreover, the presence of n-3 PUFA appeared to accelerate the electrical depression during substrate-free hypoxia but in turn to allow a faster recovery upon reoxygenation.

Arrhythmia and electrical heterogeneity during prolonged hypoxia in guinea pig papillary muscles

To investigate the mechanism of late arrhythmias in myocardial ischemia, effects of prolonged substrate-free hypoxia or hypoxia with glycolytic inhibition on action potentials of guinea pig papillary muscles were studied. During substrate-free hypoxia, the action potential duration (APD) shortened until 120-210 min, and there was recovery of the APDs thereafter. Surface mapping after prolonged hypoxia showed various APDs in single muscles. Hypoxia with glycolytic inhibition (20 mM 2-deoxyglucose) caused the recovery of APDs after 90 min. The double-microelectrode method confirmed different APDs and action potential configurations (electrical heterogeneity) in these conditions. There were triggered activities as a result of delayed afterdepolarizations, an indication of Ca2+ overload after prolonged hypoxia. There was also conduction delay between two recording sites. It was shown, for the first time, that triggered activities, recovery of APDs, electrical heterogeneity, and conduction delay appear after prolonged hypoxia. It is suggested that electrical heterogeneity and conduction delay caused by cell-to-cell uncoupling may predispose cardiac tissue to reentrant arrhythmias during ischemia.

Arrhythmia and electrical heterogeneity during prolonged hypoxia in guinea pig papillary muscles

To investigate the mechanism of late arrhythmias in myocardial ischemia, effects of prolonged substrate-free hypoxia or hypoxia with glycolytic inhibition on action potentials of guinea pig papillary muscles were studied. During substrate-free hypoxia, the action potential duration (APD) shortened until 120-210 min, and there was recovery of the APDs thereafter. Surface mapping after prolonged hypoxia showed various APDs in single muscles. Hypoxia with glycolytic inhibition (20 mM 2-deoxyglucose) caused the recovery of APDs after 90 min. The double-microelectrode method confirmed different APDs and action potential configurations (electrical heterogeneity) in these conditions. There were triggered activities as a result of delayed afterdepolarizations, an indication of Ca2+ overload after prolonged hypoxia. There was also conduction delay between two recording sites. It was shown, for the first time, that triggered activities, recovery of APDs, electrical heterogeneity, and conduction delay appear after prolonged hypoxia. It is suggested that electrical heterogeneity and conduction delay caused by cell-to-cell uncoupling may predispose cardiac tissue to reentrant arrhythmias during ischemia.

Electrical heterogeneity and conduction block in reoxygenated guinea pig papillary muscles.

To investigate the changes in electrical activity after reoxygenation, guinea pig papillary muscles were reoxygenated with 5 mM glucose solution after various durations (30-120 min) of substrate-free hypoxia. Action potential duration recovered to the control level on reoxygenation following 30-120 min hypoxia. The recovery of developed tension or resting tension was incomplete after reoxygenation following longer periods of hypoxia. There was a high incidence of arrhythmias on reoxygenation after 60 min hypoxia, which have been shown to be triggered activities due to delayed afterdepolarizations. In some muscles reoxygenated after prolonged hypoxia (90-120 min), there was electrical heterogeneity which was shown by variable action potential durations and configurations. There was also conduction block around the microelectrode site in muscle. It was suggested that the electrical heterogeneity and conduction block could predispose to reentry, and that triggered activities and reentry could be involved in the genesis of arrhythmias on reperfusion.

Stimulation of mitochondrial oxygen consumption in isolated cardiomyocytes after hypoxia-reoxygenation.

An increase in mitochondrial matrix free calcium has been shown to occur during oxygen and substrate deprivation of the perfused heart which predisposes to calcium overload and inhibition of mitochondrial function on reoxygenation. In the current study we have assessed the effect of substrate free hypoxia on mitochondrial oxygen consumption and ATP synthesis in isolated rat cardiomyocytes. Myocytes were subjected to 40 min of substrate-free hypoxia and the oxygen consumption measured together with the effects on ATP and PCr synthesis. After hypoxia myocytes showed a fall in ATP to 10% of the control value. Within 5 sec of reoxygenation the ATP level recovered to a new steady state level of 30% of the original value. The rate of oxygen consumption of hypoxic/reoxygenated cells was 3-4 fold higher than that of cells maintained under normoxic controls but in the presence of oligomycin the difference was only 1.5-fold higher, indicating a greater requirement for mitochondrial synthesis of ATP following reoxygenation. Reoxygenation in the absence of extracellular Ca2+ resulted in a lower rate of oxygen consumption (50% of the rate measured in the presence of 1 mM-Ca2+) but did not affect the steady state concentration of ATP attained 5 min after reoxygenation. These results support the idea that the increased O2 consumption of myocytes following hypoxia/reoxygenation is due to an increased demand for ATP synthesis by mitochondria and is a response to the NA+ and Ca2+ loading of the cells which occurs under these conditions. This increased demand is likely to result in a greater generation of free radicals such as superoxide by the respiratory chain which could impair cellular function over the long term.

Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein genes in myogenic cells

Cells respond to sub-lethal hear stress by preferential synthesis and accumulation of several members of functionally and compartmentally distinct families of heat shock (or stress) proteins (such as hsp70, hsp90, hsp60 and hsp27). Some of these have been implicated in the development of thermotolerance and resistance to other environmental stresses. The aim of this investigation was to determine the ability of hsp70, hsp90 or hsp60 to individually protect embryonal rat heart-derived H9c2 myocytes against both (i) heat stress and (ii) substrate-free hypoxia. When H9c2 cells were subjected to a sub-lethal stress (43 degrees C for 30 min) they were shown to have elevated levels of hsp70, hsp90 and hsp60 which was maximal between 14-24 h and was associated with increased survival at 24 h against a subsequent lethal heat stress (47 degrees C for 2 h) (20.4 +/- 2.6% v 7.3 +/- 1.8%; P < 0.002). H9c2 myocytes were transfected with a plasmid containing human hsp70i, hsp90 beta or hsp60 expressed under the control of the constitutively active human beta-actin promoter or with control vector alone (containing no hsp gene). Stable colonies of primary transfectants selected for neomycin resistance showed different degrees of over-expression of hsp70i, hsp90 beta or hsp60 expression as determined by Western blotting using specific monoclonal antisera. Cells constitutively expressing high levels of hsp70i showed significantly higher survival against lethal heat stress compared to cells transfected with vector alone (39.2 +/- 6.5% v 4.5 +/- 1.0%; P < 0.0001) and also against 20 h of substrate-free hypoxia (86.4 +/- 3.0% v 3.5 +/- 0.7%; P < 0.0001). Cells constitutively expressing high levels of hsp90 beta also showed significantly higher survival against heat stress (21.5 +/- 3.0% v 9.8 +/- 3.7%; P < 0.05) but were not resistant to 20 hours of substrate-free hypoxia (0.5 +/- 1.0% v 2.0 +/- 1.7%). Cells transfected with the hsp60 plasmid showed no increased survival against either heat stress or substrate-free hypoxia. These results demonstrate that transfection of H9c2 cells with either hsp70i, hsp90 beta or hsp60 genes confers different patterns of protection against heat stress and substrate-free hypoxia, indicating functional differences between stress proteins in their ability to protect against divergent stresses.

Role for ATP-sensitive K + channel in the development of A-V block during hypoxia

The role of the ATP-sensitive K+ channel (IK,ATP) in the development of A-V block during hypoxic interventions was studied using Langendorff perfused rabbit hearts, multicellular rabbit A-V nodal preparations and single guinea-pig ventricular myocytes. With the Langendorff perfused hearts, hypoxic perfusion (PO2 not equal to 40 mmHg) for 30 min caused slowing of the sinus rate and prolongation of the A-H interval without the appearance of blocked beats. Substrate-free hypoxic perfusion induced second or third degree A-V block in 8/10 hearts and substrate-free hypoxic perfusion plus 2-deoxyglucose (5 mM) produced third degree A-V block in 6/6 preparations. Addition of 50 mM glucose in the perfusate restored A-V conduction during hypoxic intervention. Application of glibenclamide, a specific blocker of IK,ATP, prevented the development of second or third degree A-V block during substrate-free hypoxia (n = 6), whereas pinacidil, an opener of IK,ATP, caused development of third degree A-V block during hypoxia (n = 9). Application of theophylline (100 microM) or 8-phenyl-theophylline (10 microM) did not prevent the development of advanced degrees of A-V block during hypoxic interventions. In multicellular A-V nodal preparations, 10 microM glibenclamide prevented the shortening of action potential duration and block development without marked effects on the maximum upstroke velocity of action potentials during hypoxic intervention. In isolated ventricular myocytes studied by the patchclamp method, the substrate-free hypoxia plus 2-deoxyglucose induced the openings of IK,ATP in 9/25 preparations with cell-attached patches, while 2/20 patches exhibited sporadic channel openings in the normoxic condition during comparable observation period. These results suggest that openings of the ATP-sensitive K+ channels play a role in the development and aggravation of advanced A-V block during hypoxic interventions.

Can Ischemic Preconditioning Protect Against Hypoxia-induced Damage? Studies of Contractile Function in Isolated Perfused Rat Hearts

Ischemic preconditioning in the rat significantly improves functional recovery following global ischemia by undefined mechanisms. It has been suggested that preconditioning protects by altering the tissue metabolic milieu during ischemia, either by increasing ischemic tissue accumulation of a beneficial substance (e.g. adenosine), or inhibiting tissue accumulation of a malefic component (e.g. protons). If this is the case, we hypothesized that no protection should be afforded by preconditioning against a prolonged period of hypoxia, since the continued coronary flow would prevent the accumulation of any metabolic products in the myocardium. To test this hypothesis, isolated buffer-perfused rat hearts were preconditioned by 5 min of ischemia + 5 min of reperfusion and then subjected to 30 min of ischemia, or 25 min of substrate-free hypoxia, or 60 or 90 min of hypoxia with substrate. Function was re-assessed after reperfusion/reoxygenation for a further 30 min and compared to non-preconditioned controls. Ischemic preconditioning improved functional recovery following 30 min of global ischemia (% recovery of developed pressure (LVDP) in control v preconditioned hearts was 31±4 v 66±6%; P<0.05). Importantly, this protection was achieved almost entirely via a better preservation of diastolic function (end diastolic pressure=78±3 mmHg in control and 40±5 mmHg in preconditioned hearts following 30 min of reperfusion; P<0.05). However, no preconditioning-induced protection was observed following either substrate-free hypoxia or hypoxia with substrate (% recovery of LVDP in control v preconditioned hearts was 31±4 v 34±4% after 25 min of substrate-free hypoxia, 48±3 v 53±6% after 60 min of hypoxia+substrate and 25±5 v 30±6% after 90 min of hypoxia+substrate respectively). Furthermore, no protection by preconditioning against hypoxia-induced diastolic dysfunction was observed. We conclude that preconditioning protects against ischemic injury, but not hypoxic injury. Although hypoxia-induced injury may differ from that induced by ischemia, the results are consistent with the hypothesis that the continued presence of flow with hypoxia abolishes the protective effect of preconditioning. Furthermore, the results support the concept that preconditioning of the ischemic myocardium requires the accumulation of a factor in the ischemic myocardium, either to exert the preconditioning protective effect, or as a factor of injury against which preconditioning affords protection.

Stable High Level Expression of a Transfected Human HSP70 Gene Protects a Heart-Derived Muscle Cell Line Against Thermal Stress

Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischemia has been shown to protect against subsequent myocardial ischemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from other suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocytes cell line which retains certain features of cardiac cells (as opposed to non-myocytes cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human) β-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control: pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i. It was found that cells constitutively expressing HSP70i were significantly protected (P ≦ 0.01) against lethal heat stress (47°C for 2 h) compared to cells transfected with the control plasmid. The degree of protection was slightly greater than that obtained by "preconditioning" control (untransfected) H9c2 cells with a mild-heat stress (43°C for 30 min followed by 24 h recovery) which was associated with elevation of endogenous HSP70i. These results demonstrate that stable, constitutive over-expression of an inducible isoform of HSP70 can confer a significant degree of thermotolerance to heart-derived muscle cells.

Metabolic regulation of cardiac ATP-sensitive K+ channels

Activation of ATP-sensitive K+ (KATP) channels has been implicated as a cause of increased cellular K+ efflux and action potential duration (APD) shortening during myocardial ischemia, hypoxia, and selective glycolytic inhibition, since selective KATP channel antagonists partially or completely block increased cellular K+ efflux and APD shortening under these conditions. During substrate-free hypoxia or myocardial ischemia in intact rabbit ventricle, unidirectional K+ efflux rate during systole approximately doubled and APD decreased by approximately 40% after 10 minutes. In patch-clamped guinea pig ventricular myocytes, similar changes could be produced by activation of < 0.5% of the maximal KATP channel conductance. Furthermore, from studying the desensitizing effects of ADPi on the ATP sensitivity of KATP channels in excised inside-out patches, it was estimated that the rapid changes in the cytosolic ATP/ADP ratio during ischemia and hypoxia were of sufficient magnitude to activate KATP channels to this degree. During selective glycolytic inhibition, however, the global cytosolic ATP/ADP ratio in intact heart remained normal despite an increase in cellular K+ efflux comparable to ischemia and hypoxia. In patch-clamped saponin-permeabilized ventricular myocytes, KATP channels were preferentially suppressed by glycolytic ATP production compared to ATP generated by mitochondria or by the creatinine kinase reaction, and functional glycolytic enzymes were found to be associated with KATP channels in excised membrane patches. We hypothesize that sarcolemma-associated glycolytic enzymes may be important in maintaining a high local cytosolic ATP/ADP ratio in the vicinity of KATP channels, where sarcolemmal ATPases are tending to depress the local ATP/ADP ratio.

Effect of perfusion pressure at reoxygenation on reflow and function in isolated rat hearts.

The effect of coronary perfusion pressure during reoxygenation on recovery of endocardial flow, arrhythmogenesis, and mechanical function was investigated in the isolated rat heart. Hearts were subjected to 30 min of substrate-free hypoxia followed by 30 min reoxygenation at either 80 or 150 cmH2O perfusion pressure. No flow areas were quantified by 0.3% phthalocyanine blue injection after 30 min of hypoxia, 30 min reoxygenation at 80 cmH2O, or 30 min reoxygenation at 150 cmH2O. After hypoxia, 31 +/- 2% of the myocardium was unperfused. After 80 cmH2O reoxygenation, 13 +/- 4% of the heart remained unperfused. Ten of 12 (83%) 80-cmH2O hearts were in sustained fibrillation after 10 min of reoxygenation. Reoxygenation at 150 cmH2O resulted in complete reperfusion of the myocardium. Fibrillation was absent in all hearts reoxygenated at this higher pressure. Functional recovery after 30 min reoxygenation (% of normoxic heart rate x left ventricular developed pressure) was significantly (P less than 0.05) higher in 150 cmH2O vs. 80 cmH2O (60 +/- 5 vs. 42 +/- 8%). Elevating perfusion pressure upon reoxygenation appears to counter the vascular compression caused by contracture and leads to a more rapid and homogeneous restoration of coronary flow during the transition from the hypoxic to the normoxic state.

ATP-sensitive K+ channels and cellular K+ loss in hypoxic and ischaemic mammalian ventricle.

1. The contribution of ATP-sensitive K+ (K+ATP) channels to the rapid increase in cellular K+ efflux and shortening of action potential duration (APD) during early myocardial ischaemia and hypoxia remains controversial, because for the first 10 min of ischaemia or hypoxia in intact hearts cytosolic [ATP] remains about two orders of magnitude greater than the [ATP] causing half-maximal blockade of K+ATP channels in excised membrane patches. The purpose of this study was to investigate this apparent discrepancy. 2. During substrate-free hypoxia, total, diastolic and systolic unidirectional K+ efflux rates increased by 43, 26 and 103% respectively after 8.3 min in isolated arterially perfused rabbit interventricular septa loaded with 42K+. APD shortened by 39%. From the Goldman-Hodgkin-Katz equation, the relative increases in systolic and diastolic K+ efflux rates were consistent with activation of a voltage-independent K+ conductance. 3. During total global ischaemia, [K+]o measured with intramyocardial valinomycin K(+)-sensitive electrodes increased at a maximal rate of 0.68 mM min-1, which could be explained by a less than 26% increase in unidirectional K+ efflux rate (assuming no change in K+ influx), less than the increase during hypoxia. APD shortened by 23% over 10 min. 4. During hypoxia and ischaemia, cytosolic [ATP] decreased by about one-third from 6.8 +/- 0.5 to 4.3 +/- 0.3 and 4.6 +/- 0.4 mM respectively, and free cytosolic [ADP] increased from 15 to 95 and approximately 63 microM respectively. 5. To estimate the percentage of activation of current through K+ATP channels (IK,ATP) necessary to double the systolic K+ efflux rate (comparable to the increase during hypoxia), K+ efflux during a single simulated action potential was measured by blocking non-K+ currents under control conditions and after IK,ATP was fully activated by metabolic inhibitors. Activation of 0.41 +/- 0.07% of maximal IK,ATP was sufficient to double the systolic K+ efflux rate. The equivalent amount of constant hyperpolarizing current also shortened the APD in the isolated myocytes by 41 +/- 5%, compared to the 39% APD shortening observed during hypoxia in the intact heart. 6. The degree of activation of IK,ATP expected to occur during hypoxia and ischaemia was estimated by characterizing the ATP sensitivity of K+ATP channels in the presence of 2 mM-free Mgi2+ and 0, 10, 100 and 300 microM-ADPi in inside-out membrane patches excised from guinea-pig ventricular myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)