Mechanisms of Ischemic Preconditioning in Rat Hearts
Involvement of α1B-Adrenoceptors, Pertussis Toxin–Sensitive G Proteins, and Protein Kinase C
Background Ischemic preconditioning attenuates the effects of subsequent sustained ischemia by a mechanism involving adenosine and G proteins in several species. Adenosine is not involved in ischemic preconditioning of rat hearts, the mechanisms of which are poorly understood.
Methods and Results Reduction of isometric tension development was used as an index of the effects of ischemia in isolated, Langendorff-perfused rat hearts. Two 5-minute periods of ischemia followed by 10 minutes of reperfusion attenuated the reduction of developed tension caused by 30 minutes of ischemia and 15 minutes of reperfusion. Pretreatment with pertussis toxin (PTX), depletion of norepinephrine stores with reserpine, or blockade of α1-adrenoceptors with prazosin prevented the effects of ischemic preconditioning. Whereas α1B-receptor blockade with chloroethylclonidine blocked ischemic preconditioning, α1A-receptor blockade with 5-methylurapadil had no effect. The α-adrenergic agonist phenylephrine mimicked the effects of ischemic preconditioning in a concentration-dependent manner, and pretreatment with PTX prevented the action of maximally effective concentrations of phenylephrine. The protein kinase C activator phorbol 12-myristate 13-acetate mimicked and the protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and bisindolylmaleimide prevented ischemic preconditioning.
Conclusions Ischemic preconditioning in isolated, perfused rat hearts is caused by stimulation of α1B-adrenoceptors by endogenous catecholamines through the activation of protein kinase C via a PTX-sensitive G protein. The PTX-sensitive inhibitory protein Gi, which can be activated by adenosine, muscarinic agonists, and α1-adrenoceptor agonists, may play a central role in ischemic preconditioning mediated by protein kinase C across a broad range of species.
Brief periods of acute myocardial ischemia protect the heart against subsequent episodes of prolonged ischemia.1 This phenomenon may occur in a variety of clinical settings, including aortic cross-clamping before the establishment of extracorporeal circulation, repeated balloon inflation during angioplasty of a coronary artery lesion, and repeated angina before acute myocardial infarction. Since the first description of the protective effects of ischemic preconditioning by Murry et al in 1986,2 numerous studies have documented similar phenomena in dogs,3 rabbits,4 rats,5 pigs,6 and humans.7 8 In addition to reducing the extent of necrosis caused by subsequent prolonged ischemia,3 4 5 6 preconditioning improves the restoration of function after reperfusion and prevents ventricular arrhythmias during acute ischemia and/or after reperfusion.5 9 10 11 12
Extensive studies have been performed to elucidate the mechanism by which transient ischemia protects the heart. In rabbits,13 dogs,14 and pigs,15 activation of A1 receptors by an endogenous agonist (probably adenosine) appears to play a critical role in mediating the protective effects of preconditioning. IKATP has been implicated in preconditioning in dogs,14 16 17 and a PTX-sensitive G protein is involved in preconditioning in rabbits.18 Because adenosine activates the PTX-sensitive inhibitory G protein Gi19 and Gi can facilitate the activation of IKATP,20 Gi is a strong candidate to play an important role in mediating the protective effects of preconditioning caused by adenosine receptor activation. Compatible with this concept is the observation that muscarinic cholinergic agonists, which also activate Gi,19 mimic preconditioning in rabbits18 and dogs.21
Neither adenosine22 nor IKATP23 appears to mediate the effects of ischemic preconditioning in the rat heart. The present study was designed to address the mechanisms by which ischemic preconditioning attenuates the consequences of subsequent prolonged ischemia in the rat heart. The results indicate a central role for PTX-sensitive G proteins in the cardioprotective effects of preconditioning in the rat and raise the possibility that PTX-sensitive G proteins may constitute a common pathway mediating the protective effects of ischemic preconditioning across various species.
Isolated Rat Heart Preparation
Male Sprague-Dawley rats, each weighing 300 to 400 g, were decapitated. The chest was immediately opened, and the aorta was isolated, partially incised, cannulated, and perfused in situ with a 37°C oxygenated (95% O2/5% CO2) Krebs-Ringer solution containing (mmol/L): NaCl 118.3, KCl 4.7, MgSO4 1.2, KH2PO4 1.22, CaCl2 1.3, NaHCO3 25, and glucose 15; heparin 16 IU/mL; pH 7.4 at 37°C. The perfused hearts were then excised from the chest and mounted on a modified Langendorff apparatus. The total duration of myocardial ischemia was limited to less than 45 seconds. The perfusate was filtered by a 45-μm filter and maintained at 37°C by a circulating water bath system. Myocardial temperature was maintained in a heart chamber within a jacket heated with water at 37°C. The perfusion pressure was maintained at 80 cm H2O. The hearts were allowed to beat spontaneously, and a Grass polygraph recorder (model 7D, Grass Instruments) was used to monitor contractile force and the ECG. A pair of silver needle electrodes in the right atrium and left ventricular free wall were used to obtain an ECG signal. Hearts were equilibrated for 20 minutes before any intervention. Ischemia was achieved by clamping the aortic perfusion catheter so that coronary flow was reduced to zero (no-flow ischemia).
Sets of rats were studied in parallel, with one nonpreconditioned heart and one preconditioned heart studied on each experimental day. Experimental interventions were applied to both hearts to compare recovery of developed tension in both preconditioned and nonpreconditioned hearts exposed to the same intervention. The general experimental protocols employed are illustrated in Fig 1⇓ and described in detail below.
Preconditioning was performed with two 5-minute periods of no-flow ischemia, followed by 10 minutes of reperfusion for each period of no-flow ischemia. Changes in left ventricular developed tension were used as an index of ventricular dysfunction caused by myocardial ischemia. Preconditioning (with ischemia or with drugs dissolved in the Krebs buffer) was applied for 5 minutes on two occasions separated by a 10-minute interval. Ten minutes after the second preconditioning period, hearts were subjected to 30 minutes of sustained ischemia, followed by 15 minutes of reperfusion. Tension generation was measured under control conditions and at the end of each reperfusion period. When blockers of α1-receptors and PKC were used, they were added to the perfusate for the 10-minute intervals preceding each ischemic period (asterisks in Fig 1⇑).
Measurement of Tension Development
The heart was preloaded with an initial resting tension of 1.0 g. Cardiac contractile force was estimated by monitoring isometric tension with the use of a hook attached to the apex of the heart and a force-displacement transducer (model FT 03, Grass Instruments). The signal from the force transducer was measured with a Grass 7P1 preamplifier, which was coupled to a 7DA driver amplifier.
Drugs and Solutions
Phenylephrine, prazosin, propranolol, reserpine, and PTX were purchased from Sigma Chemical Co. The α1A-selective antagonist 5-MU and the α1B-specific antagonist CEC were purchased from Research Biochemicals, Inc. The PKC activator PMA, also referred to as 12-O tetradecanoyl phorbol 13 acetate or TPA, and the protein kinase inhibitor H-7 were purchased from ICN Biochemicals. The highly selective PKC inhibitor bisindolylmaleimide hydrochloride24 and the non–PKC stimulating phorbol ester 4α-PDD were purchased from Calbiochem-Novabiochem International. PMA was prepared as a 0.8 mmol/L stock solution in DMSO and added in small quantities to the Krebs buffer to achieve the desired concentration. Prazosin was dissolved in absolute ethanol to produce a 2-mmol/L stock solution. Stock solutions of the other compounds were prepared in distilled water and added in small quantities to produce the final concentrations needed.
PMA was studied at a concentration of 0.1 μmol/L, which has been shown to potently enhance PKC activity in the perfused beating rat heart.25 H-7 was studied at a concentration of 6 μmol/L, which causes significant inhibition of PKC.26 Bisindolylmaleimide was studied at a concentration of 30 nmol/L, over twice the Ki for PKC (14 nmol/L).24 PTX was administered as a dose of 25 μg/kg IP 48 hours before study, according to a previously described regimen.18 Reserpine was administered as a 0.5 mg/kg IP dose 24 hours before study to deplete endogenous norepinephrine stores.27
Group data are expressed as mean±SEM. ANOVA with a range test (the least significant difference test) was used for comparison between group means. Fisher’s exact test was applied to contingency data. A two-tailed P value of ≤.05 was taken to indicate statistical significance.
Cardioprotective Effect of Preconditioning Occlusions
Fig 2⇓ shows the degree of reduction in tension produced by brief and longer ischemic periods. In nonpreconditioned hearts, 30 minutes of ischemia followed by 15 minutes of reperfusion (30′ Is in Fig 2⇓) caused a >40% reduction in tension generation compared with preischemic values. In hearts subjected to preconditioning ischemia, the first 5-minute ischemic period followed by 10 minutes of reperfusion (5′ I1 in Fig 2⇓) caused an ≈23% reduction in tension. The second ischemic period (5′ I2 in Fig 2⇓) was followed by a significantly smaller reduction in tension. After a 30-minute period of ischemia, preconditioned hearts (hatched bars) showed a substantially and significantly lesser reduction in developed tension than nonpreconditioned hearts. These results show that a 5-minute period of no-flow ischemia is sufficient to reduce the consequences of a subsequent 5-minute ischemic period and that two 5-minute episodes of ischemia substantially reduce the consequences of a subsequent prolonged ischemic period compared with prolonged ischemia without ischemic preconditioning.
A second effect of preconditioning that was noted was a reduction in the prevalence of ventricular tachycardia and ventricular fibrillation. Among hearts not subjected to preconditioning ischemia, over 80% experienced ventricular tachycardia or ventricular fibrillation during 30 minutes of ischemia followed by 15 minutes of reperfusion. As shown in Fig 3⇓, less than 20% of hearts subjected to preconditioning ischemia developed ventricular tachycardia or fibrillation (P<.001 compared with control).
Effects of Pretreatment With PTX
To evaluate the potential role of PTX-sensitive G proteins, the protocols shown in Fig 1⇑ were applied to hearts pretreated with PTX. A single 5-minute period of ischemia followed by 10 minutes of reperfusion was associated with a 34±4% reduction in tension relative to preischemic values. A subsequent period of 5 minutes of ischemia followed by 10 minutes of reperfusion produced a very similar reduction in tension (35±4%, P=NS). In these preconditioned hearts pretreated with PTX, 30 minutes of ischemia followed by 15 minutes of reperfusion caused a 46±2% reduction in developed tension. Nonpreconditioned hearts pretreated with PTX and exposed to 30 minutes of ischemia followed by 15 minutes of reperfusion showed a very similar reduction (48±2%) in developed tension. Fig 3⇑ shows that in PTX-pretreated hearts there was no significant difference in the incidence of ventricular tachyarrhythmias in preconditioned compared with nonpreconditioned hearts. These results indicate that pretreatment with PTX abolishes the protective effect of preconditioning ischemia against reductions in developed tension and the induction of ventricular arrhythmias caused by subsequent ischemic episodes.
These results strongly suggest that a PTX-sensitive G protein mediates the protective effects of preconditioning ischemia in this isolated rat heart model. PTX-sensitive G proteins are important in mediating preconditioning in the rabbit heart, presumably in relation to activation of Gi by stimulation of adenosine A1-receptors.18 In the rat heart, however, evidence has been presented against a role for adenosine receptor stimulation in the protective effects of ischemic preconditioning.22 We therefore turned our attention to the potential role of endogenous catecholamines in preconditioning the rat heart, because α-adrenergic stimulation can also lead to effects mediated by inhibitory G proteins.19
Evidence for a Role of Stimulation of α1B-Adrenoceptors by Endogenous Catecholamines in Ischemic Preconditioning
Reserpine was used to deplete endogenous norepinephrine stores, and the protocols shown in Fig 1⇑ were applied. Thirty minutes of ischemia (without preconditioning) followed by 15 minutes of reperfusion caused a 35±2% reduction in developed tension in the hearts of rats pretreated with reserpine 24 hours before study (n=6 hearts). Reserpine-pretreated hearts subjected to the preconditioning protocol shown in Fig 1⇑ had a very similar reduction (37±2%; n=6; P=NS) in developed tension after a 30-minute period of ischemia. Furthermore, both the first and second 5-minute periods of preconditioning ischemia were followed by very similar reductions in developed tension (21±3% and 22±3%, respectively), with values that resemble those produced by a single 5-minute ischemic episode in control hearts (Fig 2⇑). These results indicate that pretreatment with reserpine, which depletes endogenous norepinephrine stores, prevents the protective effects of preconditioning ischemia in this model.
The potential role of α1-adrenoceptor stimulation by endogenous norepinephrine was evaluated by studying the effects of prazosin (1 μmol/L) included in the perfusate for 10 minutes before and during the 10-minute reperfusion periods following each episode of preconditioning ischemia in five hearts. In the presence of prazosin, the first 5-minute ischemic period followed by 10 minutes of reperfusion resulted in a 16±5% reduction in developed tension. A second period of 5 minutes of ischemia was followed by virtually the same reduction (17±3%). A subsequent 30-minute period of ischemia followed by 15 minutes of reperfusion reduced tension by 42±3%, a value virtually identical to the result (40±1% reduction) obtained in four nonpreconditioned hearts that received prazosin for 10 minutes before the same duration of ischemia and reperfusion. These results indicate that in the presence of prazosin, brief periods of ischemia had no protective effect to attenuate the reduction of tension produced by subsequent ischemic periods. Taken together, the results of experiments with reserpine and prazosin suggest that the endogenous release of catecholamines mediates the effects of preconditioning by stimulating α1-adrenergic receptors.
To explore further the α1-adrenoceptor system involved, we exposed hearts to the selective α1A-receptor antagonist 5-MU or the irreversible α1B-receptor antagonist CEC.28 CEC (100 μmol/L) was administered in the superfusate for 10 minutes before the first ischemic period in both preconditioned and nonpreconditioned hearts and other procedures applied as shown in Fig 1⇑. 5-MU (0.1 μmol/L) was administered in the superfusate at all times indicated by asterisks in Fig 1⇑. Concentrations of these antagonists were selected on the basis of previous work demonstrating high degrees of selective receptor blockade at these concentrations.28 As shown in Fig 4⇓, the α1A-antagonist 5-MU had no apparent effect on ischemic preconditioning, whereas the latter was completely blocked by the α1B-antagonist CEC.
To evaluate further the potential protective effect of α1-adrenergic stimulation, we studied the ability of 5-minute infusions of phenylephrine, a selective α-adrenergic agonist, to mimic the effects of preconditioning. The protocol in Fig 1⇑ was employed, but instead of preconditioning with two 5-minute periods of ischemia, phenylephrine was infused for 5 minutes, followed by 10 minutes of perfusion with phenylephrine-free solution, followed by an additional 5 minutes of phenylephrine, 10 minutes of perfusion with phenylephrine-free solution, and then 30 minutes of ischemia and 15 minutes of reperfusion in the absence of phenylephrine. Fig 5⇓ illustrates the effects of preconditioning with phenylephrine on the reduction of tension produced by 30 minutes of ischemia followed by 15 minutes of reperfusion. In each experiment, one phenylephrine concentration was infused during both preconditioning periods. Phenylephrine caused a concentration-dependent attenuation in the reduction of tension caused by 30 minutes of ischemia followed by 15 minutes of reperfusion. The threshold dose for a statistically significant effect was 1 μmol/L, and a maximum effect was observed at 100 μmol/L. To determine whether the effects of phenylephrine were mediated by a PTX-sensitive G protein, we pretreated rats with PTX 48 hours before study and then observed the effects of preconditioning with a maximal concentration of phenylephrine (100 μmol/L) in the hearts of rats exposed to PTX. As shown in Fig 5⇓, pretreatment with PTX prevented the protective effects of phenylephrine. The reduction in tension caused by ischemia after preconditioning with 100 μmol/L phenylephrine was significantly greater in the hearts of rats pretreated with PTX compared with rats not so pretreated (P<.01) and was not significantly different in hearts pretreated with PTX compared with nonpreconditioned control hearts not exposed to phenylephrine. To exclude a β-adrenergically mediated effect of phenylephrine, hearts were preconditioned with 100 μmol/L phenylephrine in the presence of 1 μmol/L propranolol. As shown in Fig 5⇓, 100 μmol/L phenylephrine continued to produce a substantial attenuation in the reduction of tension produced by subsequent ischemia despite the presence of the β-blocker. These results indicate that the α-agonist phenylephrine is able to mimic the effects of ischemic preconditioning in the isolated rat heart and that these preconditioning effects of phenylephrine are mediated by α1B-receptors and dependent on the presence of a PTX-sensitive G protein.
Role of PKC in Ischemic Preconditioning in the Rat
The activation of PKC is a potential mechanism of action of PTX-sensitive G proteins.19 To examine the possible role of this second messenger system in transducing the protective effects of α-adrenergic preconditioning, we used stimulators and blockers of PKC (Fig 6⇓). When the phorbol ester PMA was administered at a concentration of 0.1 μmol/L during two 5-minute drug preconditioning periods, a subsequent 30-minute ischemic period followed by 15 minutes of reperfusion resulted in an approximately 24% reduction in developed tension. This value was substantially less than the reduction of tension produced by ischemia in nonpreconditioned hearts and not significantly different from that observed after ischemic preconditioning. In contrast, when the same procedure was repeated with an equimolar concentration (0.1 μmol/L) of a phorbol ester devoid of PKC-stimulating activity, 4α-PDD, the impairment in tension development was the same as in nonpreconditioned control hearts.
In an additional set of hearts, the ischemic preconditioning protocol was applied with the protein kinase inhibitor H-7 (6 μmol/L) or the highly-selective PKC inhibitor bisindolylmaleimide (30 nmol/L) administered for 10 minutes before preconditioning and during the reperfusion periods after each preconditioning ischemic interval. Ischemic preconditioning was studied in five hearts for each drug, and five nonpreconditioned hearts that received H-7 or bisindolylmaleimide for 10 minutes before ischemia were studied as controls. As shown in Fig 6B⇑, the reduction of tension after the prolonged ischemic period was identical for preconditioned and nonpreconditioned hearts in the presence of H-7 or bisindolylmaleimide. The results in Fig 6⇑ suggest that PKC stimulation with phorbol ester mimics ischemic preconditioning, and inhibition of PKC with H-7 or bisindolylmaleimide prevents the protective effect of ischemic preconditioning.
We have found that pretreatment with PTX blocks the protective effects of preconditioning in isolated rat hearts, as does depletion of norepinephrine stores with reserpine or blockade of α1-adrenoceptors. Blockade of α1A-receptors with 5-MU does not affect ischemic preconditioning, whereas blockade of α1B-receptors with CEC prevents ischemic preconditioning. Phenylephrine mimics the effects of preconditioning in a concentration-related way, and this action of phenylephrine is prevented by pretreatment with PTX. The phorbol ester PMA mimics preconditioning at concentrations that activate PKC,25 whereas the non–PKC activating phorbol ester 4α-PDD does not mimic preconditioning, and doses of H-726 and bisindolylmaleimide24 that inhibit PKC block ischemic preconditioning. These results suggest that ischemic preconditioning in the rat heart is due to the stimulation of α1B-adrenoceptors by the release of endogenous catecholamines, resulting in the activation of a PTX-sensitive G protein that enhances PKC activity.
Comparison With Other Studies of Ischemic Preconditioning in Rat Hearts
Previous studies have suggested that neither adenosine22 nor IKATP23 participates in the protective effects of ischemic preconditioning in rat hearts. In a recent publication, Banerjee et al29 presented evidence for a role of endogenous norepinephrine in preconditioning rat hearts. They found that either norepinephrine or phenylephrine (0.95 μmol/min, or about 56 μmol/L) simulated ischemic preconditioning, and that reserpine, phentolamine, and BE-2254 (a selective α1-adrenoceptor antagonist) block ischemic preconditioning. These results are consistent with our findings.
There are conflicting reports regarding the role of PTX-sensitive G proteins in ischemic preconditioning in the rat. Piacentini et al30 reported that pretreatment with PTX abolished the antiarrhythmic effect of ischemic preconditioning in Langendorff-perfused rat hearts, whereas Lawson et al31 did not observe an effect of pretreatment with PTX on the antiarrhythmic action of ischemic preconditioning in rats. Liu and Downey32 found that pretreatment with PTX did not significantly influence the effects of ischemic preconditioning on infarct size and arrhythmias in rats subjected to 30 minutes of ischemia followed by 120 minutes of reperfusion. The reasons for these discrepancies are unclear but may be related to the details of experimental models studied. We found that pretreatment with PTX prevented the actions of ischemic preconditioning on two separate indexes (ventricular tachyarrhythmias and recovery of developed tension) and that PTX also prevented the protective effects of preconditioning by phenylephrine in an additional series of studies.
Relation to Preconditioning Mechanisms in Other Species
Ischemic preconditioning in the rat does not depend on adenosine activation of A1-receptors,22 in contrast to ischemic preconditioning in rabbits,13 dogs,14 and pigs.15 Rather, the evidence presented in the present study, as well as in previously published work,29 points toward norepinephrine as the endogenous mediator of ischemic preconditioning in rat hearts. On the other hand, there are some interesting similarities between the mechanisms described in the present study and those mediating preconditioning in rabbits. As in rabbits,18 ischemic preconditioning in rat hearts in the present study was blocked by pretreatment with PTX. Furthermore, a recent study indicates that pretreatment with reserpine prevents ischemic preconditioning in rabbit hearts,33 suggesting a role for endogenous catecholamines in preconditioning in the rabbit. Thornton et al34 showed that α1-receptor activation caused by the release of endogenous norepinephrine by tyramine can precondition the rabbit heart. This effect was blocked by an adenosine receptor antagonist, suggesting that the stimulation of α1-receptors may act in rabbits by enhancing adenosine release or by modulating the actions of background adenosine receptor stimulation. Further studies are warranted to evaluate the potential role of endogenous norepinephrine in ischemic preconditioning in other species and to determine the potential interrelations between norepinephrine and adenosine action, because preventing the action of either seems to inhibit preconditioning in the rabbit. Given the importance of ischemic preconditioning and its similar behavior across a broad range of species, it would not be surprising if underlying mechanisms shared common elements among various species.
Signal Transduction and Mechanisms of Ischemic Preconditioning
α1-Adrenergic receptor stimulation has been shown to cause hydrolysis of phosphoinositides in adult rat ventricular myocytes35 and to lead to increased production of 1,4,5-inositol triphosphate.36 These actions depend on the participation of a PTX-sensitive G protein.36 In addition to producing inositol triphosphate, the hydrolysis of phosphatidylinositol 4,5-biphosphate liberates DAG, which activates PKC.37 Phorbol esters activate PKC by binding to a cell surface receptor for DAG.38 The present study provides substantial evidence for involvement of this system in ischemic preconditioning in the rat heart. The role of endogenous norepinephrine is suggested by the inhibition of preconditioning produced by reserpine, and α1-receptor involvement is indicated by the blocking action of the selective antagonist prazosin. Furthermore, our studies with CEC and 5-MU indicate that the receptor subtype involved is an α1B-receptor. The importance of PTX-sensitive G proteins is indicated by the inhibiting effect of PTX on both ischemic preconditioning and preconditioning produced by phenylephrine. Involvement of PKC is suggested by the ability of PMA to mimic, and of the protein kinase inhibitors H-7 and bisindolylmaleimide to block, ischemic preconditioning.
There is evidence that Gi levels and activity are reduced by acute myocardial ischemia.39 40 The activation of Gi before an ischemic insult, via either adenosine or norepinephrine released during a preceding brief ischemic period, could result in the initiation of cardioprotective mechanisms that would otherwise be suppressed by Gi inhibition during subsequent ischemia. Even in the rat, in which endogenous adenosine does not appear to mediate ischemic preconditioning, activation of Gi by cyclohexyladenosine (as well as carbamylcholine) protects the heart against subsequent ischemia.13 The mechanisms by which Gi activation and consequent PKC stimulation protect against ischemia require further elaboration, and much more work remains to be done to fully elucidate the signal transduction pathways and physiological mediators of endogenous cardioprotective mechanisms.
Selected Abbreviations and Acronyms
|IKATP||=||adenosine triphosphate–sensitive potassium current|
|PKC||=||protein kinase C|
This study was supported in part by the Medical Research Council of Canada, the Quebec Heart Foundation, and the Fonds de Recherche de l’Institut de Cardiologie de Montréal. The authors thank Josie Buluran, Dr Daya Varma, and Dr Raymond Cartier for help in developing experimental procedures and Mary Morello and Luce Bégin for excellent secretarial assistance.
- Received March 25, 1994.
- Revision received April 17, 1995.
- Accepted May 18, 1995.
- Copyright © 1995 by American Heart Association
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