Reduced Creatine Kinase Release Research Articles

Lidocaine and structure-related mexiletine induce similar contractility-enhancing effects in ischaemia–reperfusion injured equine intestinal smooth muscle in vitro

Postoperative ileus (POI) is a severe complication following small intestinal surgery in horses. It was hypothesised that prokinetic effects of lidocaine, the most commonly chosen drug for treatment of POI, resulted from drug integration into smooth muscle (SM) cell membranes, thereby modulating cell membrane properties. This would probably depend on the structural and lipophilic characteristics of lidocaine. To assess the influence of molecular structure and lipophilicity on prokinetic effects in vitro, the current study compared the effects of lidocaine with four structure-related drugs, namely, mexiletine, bupivacaine, tetracaine and procaine.The response to cumulative drug administration and reversibility of effects were tested by measuring isometric contractile performance of equine jejunal circular SM strips, challenged by a standardised, artificial in vivo ischaemia–reperfusion injury. A second set of SM strips were incubated with the different drugs to determine changes in creatine kinase (CK) release. All drugs caused a drug-specific increase in contractility, although only lidocaine and mexiletine induced similar concentration-dependent curve progressions, significantly reduced CK release, and featured shorter recovery times of tissue contractility after washing, compared to bupivacaine and tetracaine. In was concluded that the structural and lipophilic similarity of mexiletine and lidocaine were responsible for the similar effects of these drugs on SM contractility and cell membrane permeability, which supported the hypothesis that prokinetic effects of lidocaine are based on interactions with SM cell membranes modulated by these features.

Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor of autophagy, Tat-Atg5(K130R). Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5(K130R). Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia.

Temporary blockade of contractility during reperfusion elicits a cardioprotective effect of the p38 MAP kinase inhibitor SB-203580

p38 MAP kinase activation is known to be deleterious not only to mitochondria but also to contractile function. Therefore, p38 MAP kinase inhibition therapy represents a promising approach in preventing reperfusion injury in the heart. However, reversal of p38 MAP kinase-mediated contractile dysfunction may disrupt the fragile sarcolemma of ischemic-reperfused myocytes. We, therefore, hypothesized that the beneficial effect of p38 MAP kinase inhibition during reperfusion can be enhanced when contractility is simultaneously blocked. Isolated and perfused rat hearts were paced at 330 rpm and subjected to 20 min of ischemia followed by reperfusion. p38 MAP kinase was activated after ischemia and early during reperfusion (<30 min). Treatment with the p38 MAP kinase inhibitor SB-203580 (10 microM) for 30 min during reperfusion, but not the c-Jun NH(2)-terminal kinase inhibitor SP-600125 (10 microM), improved contractility but increased creatine kinase release and infarct size. Cotreatment with SB-203580 and the contractile blocker 2,3-butanedione monoxime (BDM, 20 mM) or the ultra-short-acting beta-blocker esmorol (0.15 mM) for the first 30 min during reperfusion significantly reduced creatine kinase release and infarct size. In vitro mitochondrial ATP generation and myocardial ATP content were significantly increased in the heart cotreated with SB-203580 and BDM during reperfusion. Dystrophin was translocated from the sarcolemma during ischemia and reperfusion. SB-203580 increased accumulation of Evans blue dye in myocytes depleted of sarcolemmal dystrophin during reperfusion, whereas cotreatment with BDM facilitated restoration of sarcolemmal dystrophin and mitigated sarcolemmal damage after withdrawal of BDM. These results suggest that treatment with SB-203580 during reperfusion aggravates myocyte necrosis but concomitant blockade of contractile force unmasks cardioprotective effects of SB-203580.

Reduced creatine kinase release with statin use at the time of myocardial infarction

Background: Statin pre-treatment has been shown to reduce myocardial infarct size in animal models. We evaluated peak creatine kinase levels in humans based on concomitant or very early statin initiation following myocardial infarction. Methods: We identified 66 consecutive patients who received a statin within 24 h of admission to our coronary care unit for myocardial infarction. Each statin patient was matched with three patients who had not received statin therapy ( n=198). Statin patients were subgrouped into those receiving statin therapy at the time of infarction ( n=44) and those initiated on statin therapy within 24 h of infarction ( n=22). Peak total creatine kinase concentrations were compared between groups. A linear regression model was developed to test for differences in peak creatine kinase after adjusting for differences between groups. Results: Patients receiving statin therapy within 24 h of admission had significantly smaller median peak creatine kinase concentrations compared to those not receiving a statin (416 IU/l [258, 992] vs. 699 IU/l [339, 1728]; p=0.020). Subgroup analysis revealed that the lower peak creatine kinase concentrations within the statin group were a result of lower creatine kinase concentrations in those patients on a statin at the time of myocardial infarction (399 IU/l [255, 869] vs. 678 IU/l [276, 1870]; p<0.05). This difference retained statistical significance after adjustment for differences between groups. Conclusion: Statin therapy at the time of myocardial infarction is associated with lower peak creatine kinase concentrations. This suggests that statins may exhibit protective effects in the setting of myocardial ischemia and/or infarction in humans.

Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt.

C57BL/6 mice were fed 18% ethanol (vol/vol) in drinking water for 12 wk. Isovolumic hearts were subjected to 20 min of ischemia and 30 min of reperfusion on a Langendorff apparatus. There were no differences in baseline hemodynamic function between hearts from ethanol (EtOH)-fed mice and controls. However, prior alcohol consumption doubled recovery of left ventricular developed pressure (68 +/- 8 vs. 33 +/- 8 mmHg for controls; n = 10, P < 0.05) and reduced creatine kinase release by half (0.26 +/- 0.04 vs. 0.51 +/- 0.08 U x min(-1) x g wet wt(-1) for controls; n = 10, P < 0.05). EtOH feeding doubled expression of activated protein kinase C epsilon (PKC)epsilon (n = 6, P < 0.05); whereas PKC inhibition blocked protection during ischemia-reperfusion. EtOH feeding also increased expression of Akt three- to fivefold (n = 6, P < 0.05), whereas PKC inhibition prevented increases in Akt kinase activity. We conclude that signaling pathways involving PKC-epsilon are critical for sustained EtOH-mediated cardioprotection and that Akt may be a downstream effector of resistance to myocardial reperfusion injury.

Very low dose of the Na+/Ca2+ exchange inhibitor, KB-R7943, protects ischemic reperfused aged Fischer 344 rat hearts: considerable strain difference in the sensitivity to KB-R7943

Decreased ischemic tolerance in the aged myocardium is associated with accelerated intracellular Ca(2+) overload. However, few drugs have been shown to attenuate reperfusion injury in aged hearts. Because the Na(+)/Ca(2+) exchanger (NCX1) has been reported to play an important role in Ca(2+) overload during reperfusion, we investigated whether KB-R7943 (KB-R), a novel inhibitor of the reverse mode of Na(+)/Ca(2+) exchange, can protect aged rat hearts against reperfusion injury. In the pilot study, isolated hearts from young Sprague-Dawley (SD) rats (12 weeks old), and young (12 weeks old) and aged (78 weeks old) Fischer 344 (F) rats were subjected to 25 min of global ischemia followed by 10 min of reperfusion with various concentrations of KB-R (0, 1 nM, 0.1 microM, 10 microM) and additional 20 min of reperfusion without agent. In subsequent studies with the same protocol in aged F rats, we added a protective dose of KB-R (1 nM) during the initial 10 min of reperfusion. In addition, we compared the amount of NCX1 and the sensitivity of the reverse mode of Na(+)/Ca(2+) exchange to KB-R under extracellular Na(+)-free condition in F rats with young SD rats. In the pilot study, protective effects were elicited with 1 nM of KB-R in both young and aged F rats, while 10 microM KB-R was needed for SD rats. In subsequent studies using aged F rats, there was better recovery of LV systolic function and high-energy phosphates with reduced creatine kinase release and the duration of reperfusion arrhythmias. 45Ca(2+) uptake via the reverse mode of Na(+)/Ca(2+) exchange was also inhibited with 1 nM of KB-R in young F rats, but not in young SD rats. Although the amount of NCX1 was not different among young SD, young F, and aged F rat hearts. These results demonstrated that KB-R could protect aged F rat hearts as well as young hearts of both strains against ischemia-reperfusion injury. Moreover, the sensitivity to KB-R is very different between these strains, suggesting serious caution when the agent is applied to human beings.

Calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart: influence of age

In the present study, we examined whether age-related reduction of ischemic preconditioning is related to calcitonin gene-related peptide (CGRP) release in the rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant decrease of cardiac function and a marked increase of creatine kinase (CK) release at 2, 6 and 20 months of age. Ischemic preconditioning and pretreatment with CGRP for 5 min significantly improved cardiac function and reduced CK release during reperfusion at 2 and 6 months of age but not at 20 months of age. The content of CGRP in the coronary effluent during ischemic preconditioning was significantly increased in the first cycle at 2, 6 months of age but not at 20 months of age. These results suggest that the protection afforded by ischemic preconditioning is decreased in aging hearts, and the age-related change may be related to reduction of the release and effect of CGRP in the rat heart.

Effects of Ketamine and Its Isomers on Ischemic Preconditioning.

Effects of Ketamine and Its Isomers on Ischemic Preconditioning.Molojavyi et al. and Mullenheim et al.(page 623) and (page 630)Ischemic preconditioning, in which brief periods of myocardial ischemia followed by reperfusion provide protection from subsequent ischemic injury, has been shown to occur in animals as well as humans. Current evidence suggests that opening of the adenosine triphosphate–sensitive potassium channel is a key mechanism. Based on this evidence and the finding of a concentration-dependent inhibitory effect of ketamine on adenosine triphosphate–sensitive potassium channel activity, two research teams from the same institution investigated the effects of ketamine and its stereoisomers on preconditioning in animal models.In the first study, Molojavyi et al. subjected 80 isolated rat hearts to 30 min of no-flow ischemia followed by 60 min of reperfusion. The hearts were assigned randomly to one of 10 groups: two groups of eight hearts received no preconditioning stimulus before undergoing the study protocol; in another group of eight hearts, preconditioning was elicited by two 5-min periods of ischemia before the 30-min ischemic period. In six treatment groups, ketamine, R (−)-ketamine, or S (+)-ketamine were administered at concentrations of 2 or 20 μg/ml before the preconditioning stimulus. A final group of eight hearts received 20 μg/ml R (−)-ketamine before ischemia. Left ventricular (LV) end-diastole pressure, LV developed pressure, and maximum and minimum dP/dt were obtained from digitized signals and later processed on a personal computer. LV end-diastole was determined as the point at which dP/dt started its rapid upstroke after crossing the zero line.Baseline LV developed pressure was similar in all groups of hearts. Those hearts in the control groups showed the poorest recovery of LV developed pressure, as well as a high creatine kinase release (evidence of cellular injury) after ischemia. Preconditioning improved recovery of LV developed pressure and reduced creatine kinase release. Administration of 2 μg/ml ketamine or 2 or 20 μg/ml S (+)-ketamine did not influence recovery of LV developed pressure. After 20 μg/ml ketamine or 2 or 20 μg/ml R (−) ketamine, the protective effects of preconditioning were abolished.In vivo , the pathophysiology of preconditioning and ischemia–reperfusion injury is more complex. Accordingly, Mullenheim et al. investigated the effects of ketamine and S (+)-ketamine on preconditioning in rabbit hearts in vivo . Forty-eight male New Zealand white rabbits were anesthetized with α-chloralose and instrumented for measurement of left ventricular pressure, cardiac output, and myocardial infarct size. Rabbits were assigned to one of five groups: a control group of 10 rabbits that received no pretreatment or conditioning before ischemia; a preconditioning group (n = 10) that underwent 30 min of coronary artery occlusion followed by 60 min of reperfusion; a group to which either 10 mg/kg ketamine (n = 9) or 10 mg/kg S (+)-ketamine (n = 8) was administered before preconditioning; and a final group of nine rabbits to which only 10 mg/kg−2ketamine was administered before ischemia. Two rabbits died during coronary artery occlusion, so the researchers were able to evaluate a total of 46 rabbits for analysis.All rabbits underwent 30 min of coronary occlusion, during which left ventricular pressure was reduced to 83 ± 14% of baseline values, and cardiac output was reduced to 84 ± 19% of baseline values. Functional recovery after 2 h of reperfusion did not differ significantly among groups. Infarct size (assessed by triphenyltetrazolium staining) was reduced from 45 ± 16% of the area at risk in controls to 24 ± 17% in the preconditioning group. Administration of ketamine had no effect on infarct size in animals without preconditioning but abolished the cardioprotective effects of preconditioning. S (+)-ketamine, however, had no effect, so the researchers conclude that the influence of ketamine on ischemic preconditioning is most likely enantiomer specific.

Effects of ketamine and its isomers on ischemic preconditioning in the isolated rat heart.

Ischemic preconditioning protects the heart against subsequent ischemia. Opening of the adenosine triphosphate-sensitive potassium (KATP) channel is a key mechanism of preconditioning. Ketamine blocks KATP channels of isolated cardiomyocytes. The authors investigated the effects of ketamine and its stereoisomers on preconditioning. Isolated rat hearts (n = 80) underwent 30 min of no-flow ischemia and 60 min of reperfusion. Two groups with eight hearts each underwent the protocol without intervention (control-1 and control-2), and, in eight hearts, preconditioning was elicited by two 5-min periods of ischemia before the 30 min ischemia. In the six treatment groups (each n = 8), ketamine, R(-)- or S(+)-ketamine were administered at concentrations of 2 or 20 microg/ml before preconditioning. Eight hearts received 20 microg/ml R(-)-ketamine before ischemia. Left ventricular (LV) developed pressure and creatine kinase (CK) release during reperfusion were determined as variables of ventricular function and cellular injury. Baseline LV developed pressure was similar in all groups: 104 +/- 28 mmHg (mean +/- SD). Controls showed a poor recovery of LV developed pressure (17 +/- 8% of baseline) and a high CK release (70 +/- 17 IU/g). Ischemic preconditioning improved recovery of LV developed pressure (46 +/- 14%) and reduced CK release (47 +/- 17 IU/g, both P < 0.05 vs. control-1). Ketamine (2 microg/ml) and 2 or 20 microg/ml S(+)-ketamine had no influence on recovery of LV developed pressure compared with preconditioning (47 +/- 18, 43 +/- 8, 49 +/- 36%) and CK release (39 +/- 8, 30 +/- 14, 41 +/- 25 IU/g). After administration of 20 microg/ml ketamine and 2 or 20 microg/ml R(-)-ketamine, the protective effects of preconditioning were abolished (LV developed pressure-recovery, 16 +/- 14, 22 +/- 21, 18 +/- 11%; CK release, 67 +/- 11, 80 +/- 21, 82 +/- 41 IU/g; each P < 0.05 vs. preconditioning). Preischemic treatment with R(-)-ketamine had no effect on CK release (74 +/- 8 vs. 69 +/- 9 IU/g in control-2, P = 0.6) and functional recovery (LV developed pressure 12 +/- 4 vs. 9 +/- 2 mmHg in control-2, P = 0.5). Ketamine can block the cardioprotective effects of ischemic preconditioning. This effect is caused by the R(-)-isomer.

Protective Effect of FR183998, a Na+H+ Exchange Inhibitor, Against Postischemic Injury After Normothermic and Prolonged Hypothermic Ischemia in Isolated Perfused Rat Hearts

Inhibition of Na+/H+ exchanger has been reported to protect hearts from ischemia and reperfusion injury. However, the effect of Na+/H+ exchange inhibition on hypothermic ischemic injury has not been extensively studied and the results are inconsistent. The purpose of this study was to investigate whether inhibition of Na+/H+ exchange with FR183998 (5-(2,5-dichlorothiphen-3-yl)-3-[(2-dimethylamino-ethyl)carbamoyl]benzoylguanidine dihydrochloride), a potent Na+/H+ exchange inhibitor, would show protective effects against postischemic cardiac dysfunction after hypothermic as well as normothermic ischemia and furthermore, after hypothermic cardioplegic arrest in isolated rat hearts. FR183998 (3.2 χ 10 -8 -3.2 χ 10 -7 M) improved post-ischemic recovery of left ventricular developed pressure and suppressed the increase of left ventricular end diastolic pressure in a dose-dependent manner, after not only 45 min of normothermic ischemia but also 6 h of hypothermic ischemia. Furthermore, FR183998 (10 -7 - 10 -6 M) significantly reduced creatine kinase release during reperfusion after 3 h of hypothermic ischemia with cardioplegia. These results indicate that FR183998 has a potent protective effect on postischemic cardiac dysfunction after normothermic and hypothermic ischemia, and also on reperfusion injury after hypothermic cardioplegic arrest, suggesting that its effect would be additive to cardioplegia.

Chronic Ethanol-induced Myocardial Protection Requires Activation of Mitochondrial KATPChannels

Moderate alcohol consumption protects against coronary heart disease by unclear mechanisms. We tested whether chronic ethanol preconditioning requires activation of mitochondrial KATPchannels. Rats were fed 18% (v/v) ethanol in drinking water for 10 months. Blood alcohol levels at sacrifice were 3 mmol/l (0.015 gram percent). Isolated crystalloid-perfused hearts were subjected to global ischemia and reperfusion on a modified Langendorff apparatus. Prior alcohol exposure doubled the recovery of LVDP during reperfusion (45±5%v 20±3% of baseline for controls, n=6, P<0.01) and blunted the rise in LVEDP (3.5±0.5 v 5.5±0.4 times baseline for controls, n=6, P<0.01). Ethanol feeding also reduced creatine kinase release during reperfusion. Inhibition of mitochondrial KATPchannels with 5-hydroxydecanoate had no effect on baseline LVDP, LVEDP, or coronary flow but abolished the beneficial effects of alcohol on LV contractile recovery and myocyte necrosis. We conclude that mitochondrial KATPchannel activity is required for chronic ethanol-induced protection.

Chelerythrine increases Na-K-ATPase activity and limits ischemic injury in isolated rat hearts.

Myocardial ischemia results in an increase in intracellular sodium concentration ([Na]i), which may lead to cellular injury via cellular swelling and calcium overload. Because protein kinase C (PKC) has been shown to reduce Na-K-ATPase activity, we postulated that pharmacological inhibition of PKC would directly increase Na-K-ATPase activity, reduce [Na]i during ischemia, and provide protection from ischemic injury. Isolated rat hearts were subjected to 30 min of global ischemia with and without the specific PKC inhibitor chelerythrine. Intracellular pH, ATP, and [Na]i were assessed using 31P and 23Na NMR spectroscopy, whereas Na-K-ATPase and PKC activity were determined using biochemical assays. Na/H exchanger activity was determined using the ammonium prepulse technique under nonischemic conditions. Chelerythrine increased Na-K-ATPase activity (13.76 +/- 0.89 vs. 10.89 +/- 0.80 mg ADP. h(-1). mg protein(-1); P = 0.01), reduced PKC activity in both the membrane and cytosolic fractions (39% and 28% of control, respectively), and reduced creatine kinase release on reperfusion (48 +/- 5 IU/g dry wt vs. 689 +/- 63 IU/g dry wt; P = 0.008). The rise in [Na](i) during ischemia was significantly reduced in hearts treated with chelerythrine (peak [Na](i) chelerythrine: 21.5 +/- 1.2 mM; control: 31.9 +/- 1.2 mM; P < 0.0001), without an effect on either acidosis (nadir pH 6.16 +/- 0.05 for chelerythrine vs. 6.08 +/- 0.04 for control), the rate of ATP depletion or Na/H exchanger activity. These data support the hypothesis that pharmacological inhibition of PKC before ischemia induces cardioprotection by reducing intracellular sodium overload via an increase in Na-K-ATPase activity.

Cardioadaptation Induced by Cyclic Ischemic Preconditioning Is Mediated by Translational Regulation ofde NovoProtein Synthesis

Repetitive episodes of brief ischemia induce myocardial adaptation to prolonged ischemia. To investigate whether this myocardial adaptive response involves gene transcription andde novoprotein synthesis, this study examined the effects of actinomycin D (ActD) and cycloheximide (Chx) on the cardioprotection induced by repeated ischemic preconditioning. Isolated, perfused working rat hearts underwent cyclic ischemia (CI, four 5-min ischemic intervals, 37°C) with and without pretreatment with Chx (1.0 mg/kg, ip; translation inhibition) or ActD (1.5 mg/kg, ip; transcription inhibition) 3 hr prior to heart isolation. All hearts were subjected to 20 min global ischemia (37°C) and 40 min reperfusion (I/R). Coronary effluent was assayed for creatine kinase (CK) activity. Myocardial tissue was homogenized and crude protein content determined. CI preconditioning improved postischemic recovery of cardiac output (CO; 48 ± 5.1% vs 73 ± 2.8% for control and CI, respectively,P< 0.05) and reduced CK release (61 ± 8.5 U/L vs 38 ± 4.2 U/L for control and CI, respectively,P< 0.05). The beneficial effects of CI preconditioning on myocardial function and cellular integrity were abolished by Chx while ActD had no effect. Myocardial protein content was increased in CI preconditioned myocardium relative to control hearts (5082 ± 89 μg/g vs. 4459 ± 260 μg/g, respectively,P< 0.05). Similarly, pretreatment with Chx but not ActD prevented the increase in myocardial protein content (Chx + CI, 4020 ± 254 μg/g; ActD + CI, 5049 ± 68 μg/g,P< 0.05 Chx + CI vs CI or ActD + CI). Myocardial dry/wet weight ratios were not different between groups (P> 0.05). We conclude that CI preconditioning induces protein synthesis-dependent myocardial protection against I/R injuries. CI-inducedde novoprotein synthesis in the myocardium appears to be regulated at the translational level rather than by gene transcription.

Glycogen utilization and ischemic injury in the isolated rat heart.

Fasting increases myocardial glycogen content and has been shown to limit injury and improve recovery following no-flow ischaemia in the isolated heart. However, the protective role of glycogen loading per se in fed animals has been questioned by data in preconditioned animals showing that reduced glycogenolysis may be protective prior to no-flow ischemia. Therefore, we hypothesized that fasting protects the globally ischemic heart by mechanisms separate from glycogen loading. Isolated hearts from rats fasted for 24 h were retrogradely perfused using glucose substrate and subjected to 20 min of global no-flow ischemia. Fed rats were identically perfused either under control conditions (glucose substrate) or with an intervention chosen to increase tissue glycogen (glucose plus insulin, [insulin]) prior to ischemia. Functional recovery and creatine kinase (CK) release were measured during reperfusion. Nuclear magnetic resonance spectroscopy was used to measure intracellular pH, phosphorylated glycolytic intermediates and high-energy phosphates, while the lactate and pyruvate contents of the hearts were measured prior to and at the end of ischemia. Heart from fasted animals had significantly increased glycogen content prior to ischemia (76.6 +/- 2 vs. 40.9 +/- 3 mumol glu/gdw in control hearts, P < 0.05) as did hearts exposed to insulin (88.6 +/- 10 mumol glu/gdw), but only hearts from fasted animals had greater glycogen utilization during ischemia. Hearts from fasted animals also had lower levels of lactate relative to pyruvate (L/P) under baseline conditions and, on reperfusion, reduced CK release (fasted: 183 +/- 48 versus control: 756 +/- 56 IU/gdw, P < 0.05). Conversely, insulin hearts had increased CK release (1831 +/- 190 IU/gdw, P < 0.001 vs control) and worse functional and metabolic recovery on reperfusion. Compared to the insulin hearts, hearts from fasted animals had both less acidosis and less rapid depletion of ATP during ischemia, as well as lower accumulation of glycolytic intermediates. Fasting protects the heart from ischemic injury and is associated with a lower L/P ratio and increased glycogen utilization during ischemia. In contrast, increasing glycogen content in hearts from fed animals using insulin limits glycogen utilization, increases ischemic injury, and impairs both functional and metabolic recovery under conditions of 20 min of global no-flow ischemia.

Protection against cellular damage in the perfused rat heart by lowered pH

In this comparative study, rat hearts were perfused at 37°C with three clearly defined protocols: the Ca 2+ paradox, the O 2 paradox and with 20 mM caffeine. Each protocol involved an initial priming (Ca 2+ o depletion or anoxia; stage 1) and subsequent full activation (Ca 2+ o repletion or reoxygenation; stage 2) of the damage system of the sarcolemma. Creatine kinase release in stage 2 was completely inhibited ( P<0.001) in all three protocols when pH was reduced to 6.5 throughout the experiments, or only during stage 1, or only during stage 2. The inhibitor of the Na +/H + antiporter, amiloride (1 mM), completely prevented creatine kinase release in the Ca 2+ paradox ( P<0.001) and markedly reduced damage in the caffeine protocol. Amiloride had no significant effect on creatine kinase release in the O 2 paradox. The possible role of Na + i was studied in the caffeine protocol: ouabain (5×10 −6 M) had little effect whereas substitution of choline for Na + in the perfusion medium reduced creatine kinase release by about 50%. It is suggested that the same damage system is activated in stage 1 in all three protocols and that a key event is the intracellular production of H + which are exported via Na + o/H i exchange. Prevention of H + efflux by lowered pH o, even during stage 2, protected against creatine kinase release. The possible role of Na + movements in the genesis of sarcolemma damage is discussed.

Drug-induced heat-shock preconditioning improves postischemic ventricular recovery after cardiopulmonary bypass.

Heat-stress preconditioning of mammalian heart has been found to confer protection against ischemic reperfusion injury. Heat shock is generally provided by warming the animal by mechanical means, which is often impractical in a clinical setting. Amphetamine, a sympathomimetic drug, can elevate the body temperature as a result of enhanced endogenous lipolysis. In this study, we examined the effects of heat shock induced by amphetamine on postischemic myocardial recovery in a setting of coronary revascularization for acute myocardial infarction. Adult Yorkshire swine were injected with amphetamine (3 mg/kg IM) (n = 12), and body temperature was continuously monitored. For control studies, the pigs were injected with saline (n = 12). Five swine in each group were killed after 3 hours to obtain biopsies of vital organs to measure heat-shock protein (HSP) mRNAs. After 40 hours, the remaining 7 pigs in each group were placed on cardiopulmonary bypass, and the isolated, in situ heart preparations were subjected to 1 hour of occlusion of the left anterior descending coronary artery followed by 1 hour of global hypothermic cardioplegic arrest and 1 hour of reperfusion. Postischemic myocardial performance was monitored by measuring left ventricular (LV) pressure, its dP/dt, myocardial segment shortening, and coronary blood flow. Cellular injury was examined by measurement of creatine kinase release. The antioxidant enzymes superoxide dismutase and catalase were also assayed. Amphetamine treatment was associated with the induction of mRNAs for HSP 27, HSP 70, and HSP 89 in all the vital organs, including heart, lung, liver, kidney, and brain. Amphetamine also enhanced superoxide dismutase and catalase activities in the heart. Significantly greater recovery of LV contractile functions was noticed, as demonstrated by improved recovery of LV developed pressure (61% versus 52%), LV dP/dtmax (52% versus 44%), and segment shortening (46.2% versus 10%) and reduced creatine kinase release in the amphetamine group. The results demonstrate that amphetamine can induce whole-body heat shock that can precondition the heart, enhancing cellular tolerance to ischemia-reperfusion injury. Amphetamine is a sympathomimetic drug that may be used for preconditioning.

Dual Activation of the Damage System that Causes the Release of Cytosolic Proteins in the Perfused Rat Heart

Caffeine (10 m<i>M</i>) or 2, 4-dinitrophenol (DNP; 1 m<i>M</i>) caused release of creatine kinase (CK) in the perfused rat heart but only when previously activated by Ca²⁺o depletion. Simultaneous Ca²⁺&#8320; depletion and caffeine or DNP perfusion markedly reduced CK release. Activation by Ca²⁺&#8320; depletion was inhibited at 28°C. Both the Ca²⁺ paradox and caffeine-induced release of CK were inhibited by 1 <i>mM</i> amiloride. Perfusion of anoxic standard medium lacking glucose had no overt effects, but CK release began immediately on subsequent Ca²⁺ depletion. A small, but steady release of CK was observed after glucose-free, anoxic perfusion for 75 min. CK release produced by reoxygenation after anoxia (the O₂ paradox) is augmented if anoxic perfusion is lengthened from 20 to 45 min. The Ca²⁺ paradox is markedly inhibited by 2 m<i>M</i> iodoacetate (IAA), even when this is introduced after Ca²⁺o depletion. Since caffeine or DNP caused ultrastructural damage in the presence of extracellular Ca²⁺ (when no CK was released) it is concluded that the pathways of myofilament degradation and CK release can be independently activated. It is concluded that CK release is synergistically triggered by removal of extracellular Ca²⁺ and a rise in [Ca²⁺]i. It is suggested that Ca²⁺o depletion causes membrane perturbation that activates a trans-sarcolemma complex that is critically dependent on its mobility within the bilayer, is intimately linked to a Na⁺/H⁺ antiporter and is inhibited by IAA.

Biochemical pathways of cell damage during the oxygen paradox of the rat hear

1. 1. The standard O 2-paradox has been studied in the Langendorff-perfused rat heart. 2. 2. Perfusion of glucose-free saline under anoxia did not cause release of creatine kinase (CK) although, it is suggested, there was a progressive rise in [Ca 2+] i. 3. 3. Ca 2+ -depletion after anoxia caused CK release. 4. 4. Prolonged anoxic perfusion (55 min) produced a markedly reduced release of CK on Ca 2+-depletion because, it is suggested, of the reduction in substrates for the release mechanism. 5. 5. No protection against the O 2-paradox was found with oxygen radical scavengers and inhibitors. 6. 6. Lowering [Ca 2+] oduring reoxygenation to 0.1 mM did not reduce CK release. 7. 7. Neither 1 mM amiloride (Na +/H +antiporter inhibitor) nor 2 × 10 −6 M l-(5-isoquinolinesulphonyl) piperazine (protein kinase C inhibitor) reduced CK release, unlike their effects in the Ca 2+-paradox. 8. 8. An hypothesis for events in the O 2-paradox in presented: anoxia causes a loss of Ca 2+-homeostasis and a rise in [Ca 2+] i thereby activating a transmembrane NAD(P) oxido-reductase/diaphorase (stage 1); the return of O 2 synergistically activates this molecular complex and causes CK release (stage 2).

Attenuation of myocardial reperfusion injury by sulfhydryl-containing angiotensin converting enzyme inhibitors.

Recent studies have suggested the beneficial effects of angiotensin converting enzyme (ACE) inhibitors against myocardial ischemic-reperfusion injury. This study was designed to compare the cardioprotective effects of two sulfhydryl ACE inhibitors, captopril and zofenopril, with those of a nonsulfhydryl ACE inhibitor, fosinopril. The efficacy of these ACE inhibitors to scavenge oxygen radicals in vitro were also examined. Isolated rat hearts perfused by the Langendorff technique were preperfused in the presence or absence of ACE inhibitors (50 microns for 15 minutes, and the hearts were then subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Zofenopril and captopril, but not fosinopril, improved postischemic left ventricular functions and reduced myocardial cellular injury, as evidenced by improved recovery of the first derivative of left ventricular pressure development and reduced creatine kinase release compared with control (p less than .05). Coronary flow was significantly increased by captopril and zofenopril only. The same two drugs also inhibited the enhanced lipid peroxidation during reperfusion. Although significant differences were not noticed in the postischemic myocardial membrane phospholipid composition, captopril and zofenopril reduced nonesterified fatty acid contents, including palmitic, linoleic, oleic, and arachidonic acids. In vitro studies demonstrated that captopril and zofenopril were able to scavenge hydroxyl radicals. These results indicate that among three ACE inhibitors, two sulfhydryl-containing drugs, captopril and zofenopril, possess cardioprotective as well as free-radical scavenging abilities. Attenuation of phospholipid degradation and lipid peroxidation may be contributory to the protective effects observed in this study.

Salicylate Reduces Ventricular Dysfunction and Arrhythmias During Reperfusion in Isolated Rat Hearts

Recent studies have shown the ability of salicylic acid (SA) to trap hydroxyl radicals (OH.) generated during reperfusion in ischemic myocardium. Since OH. is implicated in the pathogenesis of reperfusion injury, we examined the effect of SA on reperfusion-induced arrhythmias and postischemic ventricular dysfunction. Isolated rat hearts perfused by the Langendorff technique were preperfused with Krebs-Henseleit buffer containing SA for 10 min. Hearts were then made ischemic for 30 min, followed by 30 min of reperfusion. In a separate group, SA was administered only at the onset of reperfusion. The left ventricular contractile functions, left ventricular developed pressure (LVDP) and its first derivative (LV dP/dt), coronary flow (CF), and creatine kinase (CK) release were determined before and after ischemia. Epicardial electrocardiogram (ECG) was also recorded to analyze the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF). SA improved LVDP, LV dp/dt, and CF recovery and reduced CK release compared to the control group. The incidence of VT and VF during reperfusion was also significantly reduced by SA. Analysis of tissue thiobarbituric acid-reactive products indicates that SA decreased oxidative stress during reperfusion. In conclusion, these results suggest that SA reduces myocardial reperfusion injury and attenuates ventricular arrhythmias by trapping OH. radicals upon reperfusion in isolated rat hearts.