Cardiac Preconditioning Research Articles

Protein Kinase C Isoform Diversity in Preconditioning

Protein kinase C (PKC) appears to be a common intracellular effector and signal collector during cardiac preconditioning; however, it remains unknown whether agonists that activate different PKC isoforms are also linked to select aspects of myocardial protection. Using agonists that are known to activate unique combinations of PKC isoforms, we interogated the relationship between isoform activation and the different aspects (pH, function, and viability) of endogenous myocardial protection. To study this, isolated rat hearts were subjected to ischemia–;reperfusion (I/R) (20 min/40 min), without (control = Ctrl) or with receptor-dependent [phenylephrine (PE), 50 μM;adenosine (ADO), 125 μM] or -independent [phorbol myristate acetate (PMA), 100 nM] activation of PKC. Function, pH, and viability were assessed by rate pressure product (%RPP) and coronary flow (CF; ml/min), by31P NMR, and by CF creatine kinase (CK; U/liter) leak, respectively. PMA, which activates PKCδ but not η, resulted in intracellular pH (pHi) and viability protection, but did not protect against postischemic myocardial stunning. ADO, which activates PKCη but not δ, protects against stunning, but not acidosis or necrosis. PE, which activates PKCδ and η, provided global myocardial protection against necrosis, acidosis, and stunning. Different PKC isoforms may be linked to distinct aspects of myocardial protection. Targeted activation of PKC isoforms may allow precise mechanistic application of preconditioning-like myocardial protection.

Regular alcohol consumption mimics cardiac preconditioning by protecting against ischemia-reperfusion injury.

Epidemiologic studies indicate that long-term alcohol consumption decreases the incidence of coronary disease and may improve outcome after myocardial infarction. Attenuation of ischemia-reperfusion injury after myocardial infarction improves survival. This study investigates the possibility that alcohol consumption can improve survival after myocardial infarction by reducing ischemia-reperfusion injury. Hearts were isolated from guinea pigs after drinking ethanol for 3-12 weeks and subjected to global ischemia and reperfusion. Hearts from animals drinking ethanol showed improved functional recovery and decreased myocyte damage when compared with controls. Adenosine A1 receptor blockade abolished the protection provided by ethanol consumption. These findings indicate that long-term alcohol consumption reduces myocardial ischemia-reperfusion injury and that adenosine A1 receptors are required for this protective effect of ethanol. This cardioprotective effect of long-term alcohol consumption mimics preconditioning and may, in part, account for the beneficial effect of moderate drinking on cardiac health.

Enhanced cardiac preconditioning in the isolated heart of the transgenic ((mREN-2) 27) hypertensive rat.

Cardiac preconditioning represents an important cardioprotective mechanism which limits myocardial ischaemic damage. The aim of this investigation was to assess the impact of preconditioning in hypertension, which is a major risk factor for ischaemic heart disease. Hearts isolated from male transgenic ((mREN-2)27) hypertensive (TGH) rats, and their normotensive controls (Hannover Sprague-Dawley; SD rats), were perfused at constant flow using the Langendorff technique, and were subjected to either ischaemic preconditioning (3 x 4 min ischaemia) or continuous perfusion. Global ischaemia was then induced for 30 min, followed by 60 min of reperfusion, during which time mechanical performance was assessed (left ventricular developed pressure (LVDP), heart rate, and end-diastolic pressure). In the absence of preconditioning, mechanical performance was substantially depressed on reperfusion, and there was no difference between TGH hearts and SD hearts (area under the curve (AUC) for the LVDP (LVDP0-60) plot against time for reperfusion = 356 +/- 115 and 296 +/- 206 mmHg.min, respectively). Cardiac preconditioning caused significant protection in both groups, but this was significantly (P < 0.05) greater (3-fold) in the TGH hearts (AUC for LVDP0-60 = 3349 +/- 610 mmHg.min) compared to the SD hearts (AUC for LVDP0-60 = 1153 +/- 527 mmHg.min). In both groups, preconditioning induced significant protection of diastolic function. The enhanced effects of preconditioning on mechanical performance in the TGH hearts were unaffected by the angiotensin AT1-receptor antagonist, losartan (3 microM). However, losartan did partially reverse the beneficial effects of preconditioning on post-ischaemic diastolic function in the TGH hearts. The results of the present investigation clearly show that cardiac preconditioning is substantially enhanced in hearts from TGH rats. Furthermore, the beneficial effects of preconditioning on diastolic function, but not mechanical performance, in TGH hearts is partially mediated by AT1-receptors.

Characterization of Human Proximal Tubular Cells after Hypoxic Preconditioning: Constitutive and Hypoxia-Induced Expression of Heat Shock Proteins HSP70 (A, B, and C), HSC70, and HSP90

In animal models of cardiac or cerebral ischemic preconditioning, induction of heat shock proteins (HSPs), especially HSP70, correlates with protection from subsequent injury. The extent of HSP70 induction after stress correlates inversely with initial HSP70 levels. Primate cells, unlike nonprimate cells, express high basal levels of HSP70; thus, primate cells may respond differently to preconditioning than nonprimate cells. We have demonstrated that exposing cultured human proximal tubular epithelial cells (PTEC) to 12 h of hypoxia followed by a 24-h recovery period (hypoxic preconditioning) induces resistance to subsequent hypoxic injury. Herein, we characterize the expression of HSP70, HSP90, and heat shock cognate-70 (HSC70) in PTEC under basal conditions and after hypoxic preconditioning. By Northern blot analysis, we demonstrate that hypoxic preconditioning of PTEC increases mRNA for HSP70 > HSP90 > HSC70. With reverse transcription and polymerase chain reaction, mRNA transcripts from three different HSP70 genes (HSP70 A, B, and C) were detected in unstressed PTEC. Transcripts from these genes were also detected in freshly isolated human renal cortex, indicating that all three genes are expressedin vivo.By Western blot analysis, we demonstrate that PTEC express high basal levels of HSP70, HSC70, and HSP90. Hypoxic preconditioning did not lead to a significant increase in protein content of any of these HSPs, despite increased mRNA levels. This suggests that HSP accumulation cannot account for the development of cytoresistance after hypoxic preconditioning in PTEC. However, high basal expression of HSP70 in human PTEC may contribute to their innate resistance for hypoxia.

Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors.

Preconditioning with brief ischemia before a sustained period of ischemia reduces infarct size in the perfused heart. A cultured chick ventricular myocyte model was developed to investigate the role of adenosine receptor subtypes in cardiac preconditioning. Brief hypoxic exposure, termed preconditioning hypoxia, prior to prolonged hypoxia, protected myocytes against injury induced by the prolonged hypoxia. Activation of the adenosine A1 receptor with CCPA or the A3 receptor with C1-IB-MECA can replace preconditioning hypoxia and simulate preconditioning, with a maximal effect at 100 nM. While activation of the A2a receptor by 1 microM CGS21680 could not mimic preconditioning, its stimulation during preconditioning hypoxia, however, attenuated the protection against hypoxia-induced injury. Blockade of A2a receptors with the selective antagonist CSC (1 microM) during preconditioning hypoxia enhanced the protective effect of preconditioning. Nifedipine, which blocked the A2a receptor-mediated calcium entry, abolished the A2a agonist-induced attenuation of preconditioning. Isoproterenol, forskolin, and BayK 8644, which stimulated calcium entry, also attenuated preconditioning. Nifedipine blocked the increase in calcium uptake by these agents as well as their attenuating effect on preconditioning. The present study provides the first evidence that the adenosine A3 receptor is present on ventricular myocytes and can mediate simulation of preconditioning. The data demonstrate, for the first time, that activation of the A2a receptor antagonizes the preconditioning effect of adenosine, with increased calcium entry during the preconditioning stimuli as a novel mechanism.

Cardiac preconditioning with calcium: Clinically accessible myocardial protection

Cardiac preconditioning is mediated by protein kinase C. Although endogenous calcium is a potent stimulus of protein kinase C, it remains unknown whether preischemic administration of exogenous calcium can induce protein kinase C–mediated myocardial protection against ischemia-reperfusion injury. To study this, calcium chloride was administered retrogradely through the aorta at a rate 5 nmol/min for 2 minutes to isolated perfused rat hearts 10 minutes before a 20-minute ischemia and 40-minute reperfusion insult. Calcium-mediated cardioadaptation was then linked to protein kinase C by means of the protein kinase C inhibitor chelerythrine (20 μmol · L -1 · 2 min -1). To determine whether exogenous calcium administration induces protein kinase C translocation and activation, immunohistochemical staining for the calcium-dependent protein kinase C isoform α was performed on adjacent 5 μm myocardial sections with and without calcium chloride treatment. Results indicated that preischemic calcium chloride administration improved myocardial functional recovery, as determined by enhanced developed pressure, improved coronary flow, reduced end-diastolic pressure, and decreased creatine kinase leakage during reperfusion. Beneficial effects of calcium chloride were eliminated by concurrent protein kinase C inhibition. Immunohistochemical staining for the α isoform of protein kinase C demonstrated that calcium chloride induces translocation of this isoform from the cytoplasm to the sarcolemma, indicating that exogenous calcium administration activates this isoform. These results suggest that calcium chloride, a safe and routinely administered agent, can induce protein kinase C–mediated cardiac preconditioning. Calcium-induced cardioadaptation to ischemia-reperfusion injury may be promising as a clinically feasible therapy before planned ischemic events such as cardiac allograft preservation and elective cardiac operations. (J T HORAC C ARDIOVASC S URG 1996;112:778-86)

The obligate role of protein kinase C in mediating clinically accessible cardiac preconditioning

Cardiac preconditioning is an adaptation of cardiomyocytes that promotes tolerance to a subsequent ischemic insult. Adenosine receptor signaling is proposed as a mediator of preconditioning, but its mechanism of protection remains unknown. We hypothesized that protection against hypoxia-reoxygenation (H/R) injury could be conferred in a rat ventricle by adenosine-mediated protein kinase C (PKC) activation and that adenosine-mediated cardioprotection could be extended to human ventricular muscle. Isolated rat and human ventricular muscle (VM) strips were subjected to 30 minutes of hypoxia and 60 minutes of reoxygenation (H/R control). The VM was pretreated with 125 mumol/L adenosine, an adenosine antagonist ((p-Sulfophenyl) theophylline [SPT] 50 mumol/L) and adenosine (adenosine + SPT), or with a PKC inhibitor (chelerythrine, 10 mumol/L) and adenosine (adenosine + chelerythrine) before H/R Developed force (DF) and tissue creatine kinase (CK) activity were assessed at end reoxygenation. Human trabeculae were obtained from diseased explanted hearts at cardiac transplantation and were also subjected to H/R injury. Human VM was pretreated with adenosine (125 mumol/L) before H/R injury. Results are expressed as mean +/- standard error of mean. In the rat, adenosine pretreatment conferred protection of DF against H/R injury (adenosine, 62% +/- 6%; H/R control, 27% +/- 2%, p < 0.05). Adenosine + SPT or adenosine + chelerythrine eliminated the functional recovery conferred by adenosine. This recovery of contractile function was associated with greater tissue CK activity (adenosine, 415 +/- 40 units/gm; H/R control, 78 +/- 13 units/gm, p < 0.05). The protective effects of adenosine against H/R were present in the human ventricle and with recovery of DF in adenosine (66% +/- 5%) and H/R control (24% +/- 4%), p < 0.05. Adenosine, a clinically accessible agonist, induces protection against H/R injury through a PKC-mediated mechanism in the rat ventricle. Further, the protection conferred by adenosine against H/R extends to the human ventricle.

Regulation and functional significance of phospholipase D in myocardium.

There is now clear evidence that receptor-dependent phospholipase D is present in myocardium. This novel signal transduction pathway provides an alternative source of 1,2-diacylglycerol, which activates isoforms of protein kinase C. The members of the protein kinase C family respond differently to various combinations of Ca2+, phosphatidylserine, molecular species of 1,2-diacylglycerol and other membrane phospholipid metabolites including free fatty acids. Protein kinase C isozymes are responsible for phosphorylation of specific cardiac substrate proteins that may be involved in regulation of cardiac contractility, hypertrophic growth, gene expression, ischemic preconditioning and electrophysiological changes. The initial product of phospholipase D, phosphatidic acid, may also have a second messenger role. As in other tissues, the question how the activity of phospholipase D is controlled by agonists in myocardium is controversial. Agonists, such as endothelin-1, atrial natriuretic factor and angiotensin II that are shown to activate phospholipase D, also potently stimulate phospholipase C-beta in myocardium. PMA stimulation of protein kinase C inactivates phospholipase C and strongly activates phospholipase D and this is probably a major mechanism by which agonists that promote phosphatidyl-4,5-bisphosphate hydrolysis secondary activate phosphatidylcholine-hydrolysis. On the other hand, one group has postulated that formation of phosphatidic acid secondary activates phosphatidyl-4,5-bisphosphate hydrolysis in cardiomyocytes. Whether GTP-binding proteins directly control phospholipase D is not clearly established in myocardium. Phospholipase D activation may also be mediated by an increase in cytosolic free Ca2+ or by tyrosine-phosphorylation.

Role of bradykinin in cardiac functional protection after global ischemia-reperfusion in rat heart.

We have reported that cardiac preconditioning against ischemia-reperfusion (IR) can be induced by transient ischemia (TI) and alpha 1-adrenoreceptor stimulation, both mediated by protein kinase C (PKC) (Mitchell, M., X. Meng, C. Parker, E. Brew, A. Harken, and A. Banerjee. Circ. Res. 76: 73-81, 1995). Our study objective was to explore the mechanism of endogenous preconditioning and address the role of PKC activation in bradykinin-mediated cardiac functional protection. Isolated rat heart was used to assess the effects of exogenous bradykinin, TI, selective B2-receptor blocker, and PKC antagonism on cardiac functional recovery after a global IR injury. Final recovery of developed pressure was improved in hearts treated with bradykinin and TI compared with controls. Bradykinin- and TI-mediated preconditioning was eliminated with coinfusion of the B2-receptor antagonist. Further evaluation of bradykinin-mediated preconditioning revealed that PKC blockade also eliminated functional protection. Immunofluorescent stains of bradykinin-treated hearts demonstrated translocation and activation of specific PKC isoforms in the preconditioned heart. We conclude that TI-mediated preconditioning involves intrinsic cardiac bradykinin receptor stimulation. Bradykinin, through the B2 receptor, initiates a series of intracellular events culminating in the activation of PKC.

Cardiac Preconditioning Protects against Irreversible Injury Rather Than Attenuating Stunning

The purpose of this experiment was to determine if cardiac preconditioning (PC) mediates protection by attenuating stunning or preventing irreversible injury. Inherent in the definition of myocardial stunning is the ability to respond to catechol stimulation after ischemia/reperfusion (I/R). Irreversibly injured myocardium cannot respond to catechols. We hypothesized that α 1-adrenergic-stimulated PC is mediated through a functional protection against reversible injury. We investigated this hypothesis in the isolated, buffer-perfused rat heart subjected to global ischemia (20 min, 37.5°C) and reperfusion (40 min). The PC group received an α 1-adrenergic stimulus (norepinephrine, 0.5-1.0 μ M, 2 min) 10 min prior to ischemia. Control hearts were perfused normoxically for 80 min. Developed pressure (DP) and heart rate were recorded continuously. To determine maximal myocellular function, all hearts received a β-adrenergic pathway stimulus (forskolin (FSK), 100 μ M bolus) at end reperfusion. The ability to improve DP in response to FSK was indicative of reversible dysfunction (stunning). Failure to attain the maximal DP established in normoxic controls was utilized as a measure of irreversible dysfunction. Recovery was assessed as a percentage of initial DP. The results suggest that (1) PC protects against an I/R injury (recovery: I/R, 50.1%; PC + I/R, 76.0%; P < 0.05); (2) all groups exhibit reversible dysfunction (all increased DP in response to FSK); (3) when maximally stimulated, I/R hearts are unable to develop pressures similar to those of normoxic controls, suggesting irreversible injury; and (4) PC hearts, however, attained similar maximal pressures compared to controls. We conclude that α 1-adrenergic PC improves postischemic cardiac function by preventing irreversible injury. Furthermore, α 1-adrenergic PC improves the postischemic response to inotropic stimulation by 36.7%.

Ischemic myocardial cell protection conferred by the opening of ATP-sensitive potassium channels

The responses of the cardiac myocyte to a potentially injurious ischemic stress are multiple. The opening of the ATP-sensitive K+ channels may constitute one such response. These channels are present in the plasmalemma at very elevated density and have a large unitary conductance. Consequently, the opening of a small fraction (0.01-0.1%) of these channels during ischemia can help to drive the myocyte into an "emergency" state, in which its syncytial functions become rapidly downregulated and strategies appropriate to preserving cell viability are implemented. Thus, ATP-sensitive K+ channels in cardiac myocytes would appear to be an efficient and apparently redundant natural means of defense against metabolic stress. These channels can undergo physiologic modulation, as occurs during cardiac ischemic preconditioning in several species, including humans. The term cardioprotection refers to an endogenous cardioprotective strategy, whereby the myocardium slows its energy demands, produces fewer toxic glycolytic products, and exhibits reduced injury following a potentially lethal ischemic stress. Openers of cardiac ATP-sensitive K+ channels, a class of drugs that includes, in particular, aprikalim and nicorandil, also afford cardioprotection by reducing the functional and biochemical damage produced by ischemia. Hence, these compounds can improve the recovery of cardiac contractility, reduce the extracellular leakage of intracellular enzymes, delay the loss of ATP, and preserve the cell ultrastructure in isolated heart preparations subjected to transient ischemic conditions. Furthermore, when segmental contractility has been strongly depressed by a stunning insult, nicorandil and aprikalim can accelerate recovery at the reperfusion. Finally, nicorandil and aprikalim decrease substantially the size of the necrotic region that results from a prolonged ischemic insult followed by reperfusion. All of these desirable effects of K(+)-channel openers can be abolished by blockers of ATP-sensitive K+ channels, such as glibenclamide. The fundamental mechanism of the myocyte viability protection conferred by K(+)-channel openers is not yet clear. It may exploit some of the same pathways that mediate cardiac ischemic preconditioning. If this suggestion holds true, drugs opening cardiac ATP-sensitive K+ channels would mimic, exploit, or intensify those cardioprotective means that are naturally available to the cardiac myocyte for overcoming metabolic stress.

Cardiac Preconditioning

Cardiac preconditioning is a phenomenon by which a brief exposure to ischemia renders the heart more tolerant of a subsequent sustained ischemic insult. Understanding the mechanism involved may allow pharmacologic access to this protective state before cardiopulmonary bypass surgery and transplantation. We discuss herein the preconditioning phenomenon, the potential mechanisms involved, and the therapeutic implications of cardiac preconditioning.

Cardiac preconditioning does not require myocardial stunning

Efforts to minimize the deleterious effects of intraoperative myocardial ischemia-reperfusion (I/R) injury have been primarily directed at optimizing cardioplegic solutions and altering reperfusion conditions. Classically, myocardial I/R has been associated with cardiac mechanical dysfunction (“stunning”). Recently, we reported an α 1-adrenergic receptor-mediated mechanism of paradoxical myocardial protection against I/R insult induced by a prior episode of transient ischemia, a phenomenon known as “ischemic preconditioning”. Myocardial stunning resulting from transient ischemia has previously been associated with ischemic preconditioning, prompting intuitively negative bias against the clinical application of this phenomenon. The purpose of this study was to determine whether transient ischemia of insufficient duration to cause prolonged mechanical dysfunction (stunning) can induce favorable cardiac preconditioning. Isolated-perfused rat hearts were allowed to equilibrate for 8 minutes and were then subjected to either 2 minutes of global, normothermic transient ischemia or 2 minutes of 50 μmol/L phenylephrine infusion. A stabilization period of perfusion lasting 10 minutes after the termination of transient ischemia or phenylephrine infusion was followed by a standard I/R challenge (20 minutes of global, normothermic ischemia; 40 minutes of reperfusion). Ventricular function (measured as developed pressure in millimeters of mercury) recovered rapidly after transient ischemia such that no impairment was present before the subsequent standard I/R challenge. Phenylephrine treatment was associated with no residual inotropy before I/R challenge. Control hearts were subjected only to the standard I/R challenge after an initial 20-minute equilibration period. After reperfusion control hearts exhibited 54.4% recovery of initial left ventricular developed pressure. Transient ischemia- and phenylephrine-preconditioned hearts recovered 84.4% ( p < 0.01) and 82.4% ( p < 0.01), respectively. We conclude that the benefits of myocardial preconditioning do not require the deleterious effects of myocardial stunning.