The effect of taurine on the density of adrenergic nerve endings and the recovery of cardiac function after ischemia

Isolated guinea-pig hearts perfused at 37 degrees C with Krebs-Henseleit buffer through aorta. Mechanical function was evaluated by isovolumic pressure in a latex balloon inserted into left ventricle. The density of catecholamine-containing adrenergic nerve plexus was measured in both ventricles after 40 min of total ischemia and 30 min of reperfusion. Heart preparations were treated with 2% glyoxylic acid and the relative area occupied by the plexus was determined alanimetrically. Without taurine (control) the adrenergic plexus density was 4-(right ventricule) and 6-fold (left ventricule) lower compared with that in freshly isolated hearts. When taurine was added to the perfusion solution after ischemia, the plexus density increased about 2.7-fold; if it was added prior to ischemia, the density was nearly equal to the original value. In no experiment with taurine addition during reperfusion fibrillation did occur, and about 2-fold more rapid restoration of regular rhythm was observed comparing with the control and experiments when taurine was added prior to ischemia. Both in the latter and control experiments spontaneously disappearing fibrillation occurred. The restoration of pressure and contraction frequency were virtually the same in all experiments. These findings show that taurine is able to preserve the catecholamine stores in the myocardium.     

Dynamics of Ca(2+)/calmodulin-dependent protein kinase II following acute myocardial ischemia-translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.     

Ischemic preconditioning prevents I/R-induced alterations in SR calcium-calmodulin protein kinase II

Although Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) is known to modulate the function of cardiac sarcoplasmic reticulum (SR) under physiological conditions, the status of SR CaMK II in ischemic preconditioning (IP) of the heart is not known. IP was induced by subjecting the isolated perfused rat hearts to three cycles of brief ischemia-reperfusion (I/R; 5 min ischemia and 5 min reperfusion), whereas the control hearts were perfused for 30 min with oxygenated medium. Sustained I/R in control and IP groups was induced by 30 min of global ischemia followed by 30 min of reperfusion. The left ventricular developed pressure, rate of the left ventricular pressure, as well as SR Ca(2+)-uptake activity and SR Ca(2+)-pump ATPase activity were depressed in the control I/R hearts; these changes were prevented upon subjecting the hearts to IP. The beneficial effects of IP on the I/R-induced changes in contractile activity and SR Ca(2+) pump were lost upon treating the hearts with KN-93, a specific CaMK II inhibitor. IP also prevented the I/R-induced depression in Ca(2+)/calmodulin-dependent SR Ca(2+)-uptake activity and the I/R-induced decrease in the SR CaMK II activity; these effects of IP were blocked by KN-93. The results indicate that IP may prevent the I/R-induced alterations in SR Ca(2+) handling abilities by preserving the SR CaMK II activity, and it is suggested that CaMK II may play a role in mediating the beneficial effects of IP on heart function.     

The mechanism of large, excitation-dependent influx of 45Ca in the guinea-pig ventricular myocardium

In the previous papers (Lewartowski et al. 1982; Pytkowski et al. 1983) we found that excitation-dependent uptake of 45Ca (EDU) ranges in the vascularly perfused guinea-pig ventricular myocardium from 40-359 mumol/kg of wet weight per single steady-state beat or post-rest beat. The present paper describes an attempt to find whether slow calcium channel or Na/Ca exchange provides the route of this large 45Ca influx. We found that EDU during steady-state stimulation (60/min) was completely blocked by both D-600 (1 mg/l) and Ni (2 mmol/l) whereas EDU in post-rest beats was blocked only by Ni. Low Na+ perfusion (50 mmol/l) increased transiently EDU in steady-state beats. This surplus EDU was not blocked by D-600 nor by Ni. Noradrenaline infused at the rate sufficient to increase contractile force by 50% at least doubled EDU both in the steady-state and in post-rest beats. It is proposed that Na/Ca exchange does not participate significantly to EDU under physiological conditions. The changes in this uptake evoked by the applied interventions could be expected if its route was provided by the slow Ca channel.     

Calcium content of peripheral and central mitochondria in the guinea pig myocardium: electron probe analysis

We quantitated subcellular elemental concentrations in stimulated and resting guinea pig myocardium to determine whether species-specific properties of guinea pigs or the subcellular localization of mitochondria accounted for reports of higher mitochondrial Ca in guinea pigs than in other species. Small papillary muscles or trabeculae isolated from guinea pig ventricles were stimulated to raise cytosolic [Ca(2+)](i) by two methods: (1). tetanizing by rapid pacing preparations in which Ca(2+) uptake by the sarcoplasmic reticulum was inhibited with cyclopiazonic acid or (2). freeze trapping paced muscles near-peak systole. Electron probe X-ray microanalysis showed no significant difference between the (low, approximately 0.4 mmol/kg dry weight) mitochondrial Ca content of stimulated guinea pig hearts, compared to mitochondria of other species, such as rat and hamsters, and the Ca contents of peripheral and central mitochondria were also not significantly different.     

β(2)-adrenoreceptor mediates the cardioprotection of ischemic preconditioning on myocardial contraction in rats subjected to ischemia/reperfusion injury

The aim of the present study is to investigate the role of beta(2)-adrenoreceptor (beta(2)-AR) in ischemic preconditioning (IP) in isolated rat heart model of ischemia/reperfusion (I/R). Sprague-Dawley rat hearts were quickly removed, mounted on Langendorff apparatus, and perfused with Krebs-Henseleit (KH) solution. After the initial stabilization period, the rats were randomly divided into 6 groups including control group (perfused for an additional 20 min), IP group (4 cycles of 5 min of ischemia followed by 5 min of reflow), isoproterenol (ISO) group (10 nmol/L ISO perfusion for 5 min followed by 5 min washout), IP + ICI118551 group (55 nmol/L ICI118551 perfusion for 5 min before and throughout IP), ISO + ICI118551 group (55 nmol/L ICI118551 perfusion for 5 min before and throughout ISO treatment), ICI118551 group (55 nmol/L ICI118551 perfusion for 20 min). After these treatments, all hearts were followed by 30 min of no-flow ischemia and 30 min of reperfusion. A computer-based electrophysiological recorder system was used to measure changes of the maximal rate of pressure increase in systole phase (+dp/dt(max)), maximal rate of pressure decrease in diastole phase (-dp/dt(max)), and difference of left ventricular pressure (DeltaLVP). Then cardiomyocytes from these hearts were isolated by 5 min of Ca(2+)-free buffer perfusion and 25 min of collagenase perfusion. The ventricles were chopped and filtered. The myocytes were resuspended in KB buffer. The contraction and the viability of cardiomyocytes were measured. Lactate dehydrogenase (LDH) concentration in coronary effluent was assayed with assay kit. The results showed that both IP and ISO significantly increased the values of +/-dp/dt(max), DeltaLVP, the contraction and viability of cardiomyocytes, shortened the time-to-peak contraction (TTP), and decreased the release of LDH in coronary effluent. ICI118551, a selective beta(2)-AR antagonist, blocked these effects. Either the time-to-50% relaxation (R(50)) or the time-to-100% relaxation (R(100)) had no significant differences between groups. Our results indicate that the cardioprotection of IP was mediated by beta(2)-AR in isolated rat hearts subjected to I/R injury.     

The effects of acetylcholine and catecholamines on post-rest phenomena in rabbit and guinea-pig atria and on accompanying shifts of 45Ca in guinea-pig atrial muscle

We studied the pattern of post-rest activation and shifts of 45Ca in the isolated mammalian atrial muscle. The first contraction evoked in the rabbit and guinea-pig atrial muscle after 10 min rest was several times stronger than the steady-state beats at a rate of 60/min. Contractile force (CF) declined to 20-50% of control during the next 1-3 beats and recovered to the pre-rest level upon subsequent stimulation. The post-rest beats were negligibly affected by noradrenaline (NA), isoproterenol (IS) acetylcholine (ACh), carbachol (CCh) and Ni, whereas the steady-state beats were readily affected by all these drugs or ions. Post-rest potentiation was completely inhibited by caffeine in a concentration of 10 mM. The guinea-pig atria, equilibrated for 60 min in 45Ca containing solution and stimulated at a rate of 60/min, contained 4.47 +/- 0.16 mmol 45Ca/kg wet weight (w.w.). Ten min of rest resulted in a drop of this content to 3.52 +/- 0.13 mmol/kg w.w. despite the continued presence of 45Ca in the superfusing solution. Three initial post-rest beats resulted in further drop of the content of 45Ca to 2.62 +/- 0.14 mmol/kg w.w. Continued post-rest stimulation resulted in a recovery of the pre-rest 45Ca content. This recovery was inhibited by ACh and CCh. Both drugs inhibited 45Ca loss during the initial 3 beats. Neither this loss nor recovery were affected by IS. It is concluded that calcium (Ca) fraction described in the previous papers [11, 15] as Ca2 in the guinea-pig ventricular muscle plays an important role in the force-frequency relations also in the atrial muscle. However, unlike in ventricular muscle only about half of it is released from the cells upon rest whereas the remaining Ca is taken up by the release compartment and used to activate the strong post-rest contraction. It is thereafter extruded from the cells which results in severe depletion of intracellular Ca stores. Fraction Ca2 is re-accumulated during the post-rest stimulation resulting in recovery of the contractile force.     

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.     

Ca(2+) loading and adrenergic stimulation reveal male/female differences in susceptibility to ischemia-reperfusion injury

To compare ischemia-reperfusion injury in males versus females under hypercontractile conditions, perfused hearts from 129J mice pretreated with 3 mmol/l Ca(2+) or 10(-8) mol/l isoproterenol +/- 10(-6) mol/l N(omega)-nitro-L-arginine methyl ester (L-NAME) were subjected to 20 min of ischemia and 40 min of reperfusion while (31)P NMR spectra were acquired. Basal contractility increased equivalently in female versus male hearts with isoproterenol- or Ca(2+) treatment. Injury was equivalent in untreated male versus female hearts but was greater in isoproterenol or Ca(2+)-treated male than female hearts, as indicated by lower postischemic contractile function, ATP, and PCr. Endothelial nitric oxide (NO) synthase (eNOS) expression was higher in female than male hearts, neuronal NOS (nNOS) did not differ, and inducible NOS (iNOS) was undetectable. Ischemic NO production was higher in female than male hearts, and L-NAME increased injury in female isoproterenol-treated hearts. In summary, isoproterenol or high Ca(2+) pretreatment increased ischemia-reperfusion injury in males more than females. eNOS expression and NO production were higher in female than male hearts, and L-NAME blocked female protection. Females were therefore protected from the detrimental effects of adrenergic stimulation and Ca(2+) loading via a NOS-mediated mechanism.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms

Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.     

Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Extracellular ATP is known to augment cardiac contractility by increasing intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes; however, the status of ATP-mediated Ca2+ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subjected to 10-30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca2+]i was measured in purified cardiomyocytes by fura-2 microfluorometry. Reperfusion for 30 min of 20-min ischemic hearts, unlike 10-min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca2+]i; no changes in basal [Ca2+]i were evident in 10- or 20-min I/R preparations. On the other hand, reperfusion of 30-min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca2+]i and a dramatic increase in basal [Ca2+]i. The positive inotropic effect of extracellular ATP was attenuated, and the maximal binding characteristics of 35S-labeled adenosine 5'-[gamma-thio]triphosphate with crude membranes from hearts undergoing I/R was decreased. ATP-induced increase in [Ca2+]i in cardiomyocytes was depressed by verapamil and Cibacron Blue in both control and I/R hearts; however, this response in I/R hearts, unlike control hearts, was not affected by ryanodine. I/R-induced alterations in cardiac function and ATP-induced increase in [Ca2+]i were attenuated by treatment with an antioxidant mixture and by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2. The results suggest an impairment of extracellular ATP-induced Ca2+ mobilization in I/R hearts, and this defect appears to be mediated through oxidative stress.     

Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia

To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.     

Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10-30 min of ischemia followed by 5-30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(-)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon beta-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.     

Adenosine induced direct negative inotropic effect is abolished during global ischemia: role of protein kinase C and prostacyclin

Adenosine acts as a cardioprotective agent by producing coronary vasodilation, decreasing heart rate and by antagonizing the cardiostimulatory effect of catecholamines; adenosine also exerts a direct negative inotropic effect. Myocardial ischemia is known to be associated with enhanced levels of adenosine, increased protein kinase C (PKC) activity and prostacyclin (PGI2) release. The present study was conducted to determine if myocardial ischemia alters the cardioprotective effect of adenosine by increasing PKC activity and PGI2 release in the isolated rat heart perfused at 10 ml/min with Krebs-Henseleit buffer (KHB; 95% O2+5% CO2). Adenosine (10 mmol/min) reduced myocardial contractility as indicated by a decrease in contractility (dp/dtmax), heart rate (HR) and coronary perfusion pressure (PP). In hearts subjected to 30 min of ischemia (without perfusion) and then reperfused with KHB, adenosine failed to decrease dp/dtmax, HR or PP. However, during infusion of PKC inhibitor H-7 (1-(5-Isoquinolinesulfonyl)-2-methylpiperazine hydrochloride) (H-7; 6 mmol/min), which commenced 10 min before ischemia and continued throughout reperfusion, adenosine produced a decrease in dp/dtmax, HR and PP, similar to that before ischemia. Infusion of the PKC activator phorbol 12,13-dibutyrate (PDBu; 2 nmol/min) but not an inactive analogue in non-ischemic hearts prevented the adenosine induced decrease in dp/dtmax. During infusion of H-7, PDBu failed to block the direct negative inotropic effect of adenosine in non-ischemic hearts. In addition, pretreatment with H-7 or indomethacin (cyclooxygenase inhibitor) significantly reduced the PGI2 release following ischemia. This data suggest that PKC and PGI2 regulate the direct negative inotropic effect of adenosine, which is abolished during ischemia.     

Differences in ischemia-reperfusion-induced endothelial changes in hearts perfused at constant flow and constant pressure

Isolated hearts subjected to ischemia-reperfusion (I/R) exhibit depressed cardiac performance and alterations in subcellular function. Since hearts perfused at constant flow (CF) and constant pressure (CP) show differences in their contractile response to I/R, this study was undertaken to examine mechanisms responsible for these I/R-induced alterations in CF-perfused and CP-perfused hearts. Rat hearts, perfused at CF (10 ml/min) or CP (80 mmHg), were subjected to I/R (30 min global ischemia followed by 60 min reperfusion), and changes in cardiac function as well as sarcolemmal (SL) Na(+)-K(+)-ATPase activity, sarcoplasmic reticulum (SR) Ca(2+) uptake, and endothelial function were monitored. The I/R-induced depressions in cardiac function, SL Na(+)-K(+)-ATPase, and SR Ca(2+)-uptake activities were greater in hearts perfused at CF than in hearts perfused at CP. In hearts perfused at CF, I/R-induced increase in calpain activity and decrease in nitric oxide (NO) synthase (endothelial NO synthase) protein content in the heart as well as decrease in NO concentration of the perfusate were greater than in hearts perfused at CP. These changes in contractile activity and biochemical parameters due to I/R in hearts perfused at CF were attenuated by treatment with l-arginine, a substrate for NO synthase, while those in hearts perfused at CP were augmented by treatment with N(G)-nitro-l-arginine methyl ester, an inhibitor of NO synthase. The results indicate that the I/R-induced differences in contractile responses and alterations in subcellular organelles between hearts perfused at CF and CP may partly be attributed to greater endothelial dysfunction in CF-perfused hearts than that in CP-perfused hearts.     

Intermittent hypoxia protects the rat heart against ischemia/reperfusion injury by activating protein kinase C

The aim of this study was to investigate whether and how protein kinase C (PKC) was involved in the protection afforded by intermittent hypoxia (IH) and the subcellular distribution of different PKC isozymes in rat left ventricle. Post-ischemic recovery of left ventricular developed pressure and +/-dP/dtmax in IH hearts were higher than those of normoxic hearts. Chelerythrine (CHE, 5 microM), a PKC antagonist, significantly inhibited the protective effects of IH, but had no influence on normoxic hearts. CHE significantly reduced the effect of IH on the time to maximal contracture (Tmc), but had no significant effect on the amplitude of maximal contracture (Amc) in IH group. In isolated normoxic cardiomyocytes, [Ca(2+)](i), measured as arbitrary units of fluorescence ratio (340 nm/380 nm) of fura-2, gradually increased during 20 min simulated ischemia and kept at high level during 30 min reperfusion. However, [Ca(2+)](i) kept at normal level during simulated ischemia and reperfusion in isolated IH cardiomyocytes. In normoxic myocytes, [Na(+)](i), indicated as actual concentration undergone calibration, gradually increased during 20 min simulated ischemia and quickly declined to almost the same level as that of pre-ischemia during 30 min simulated reperfusion. However, in IH myocytes, [Na(+)](i) increased to a level lower than the corresponding of normoxic myocytes during simulated ischemia and gradually reduced to the similar level as that of normoxic myocytes after simulated reperfusion. 5 microM CHE greatly increased the levels of [Ca(2+)](i) and [Na(+)](i) during ischemia and reperfusion in normoxic and IH myocytes. In addition, we demonstrated that IH up-regulated the baseline protein expression of particulate fraction of PKC-alpha, epsilon, delta isozymes. There is no significant difference of protein expression of PKC-alpha, epsilon, delta isozymes in cytosolic fraction between IH and normoxic group. The above results suggested that PKC contributed to the cardioprotection afforded by IH against ischemia/reperfusion (I/R) injury; the basal up-regulation of the particulate fraction of PKC-alpha, epsilon, delta isozymes in IH rat hearts and the contribution of PKC to the elimination of calcium and sodium overload might underlie the mechanisms of cardioprotection by IH.     

A comparison of no-flow and low-flow ischemia in the rat heart: an energetic study

Heat production under no-flow ischemia (ISCH) and under hypoperfusion (HYP) conditions was measured in single isovolumetric contractions of perfused rat ventricles at 25 degrees C. Resting heat production (Hr) and resting pressure decreased when the perfusion rate was reduced from 6 to 1.5 mL min(-1) or lower flows (HYP) and by ISCH. Maximal developed pressure (P) decreased to 29% and 20% of control by HYP at 0.8 mL min(-1) and ISCH, respectively. The tension-independent heat (TIH) fraction attributed to Ca2+-binding, measured during single contractions, decreased under HYP with an increase in the ratio between the maximum relaxation rate and P (-P/P ratio). The TIH fractions (attributed to Ca2+ binding and Ca2+ removal processes) decreased under ISCH. The long duration TIH fraction associated with Ca2+-dependent mitochondrial activity disappeared at flow rates of 1.5 mL min(-1) or lower. The ratio between the tension-dependent energy release and P was decreased by ISCH but not by HYP, indicating that under ISCH there was an improvement in contractile economy, but this was not modified by HYP. Overall, the results indicate that no-flow and low-flow ischemias are energetically different models. While the contractile failure under HYP seems to be related to a decrease in myofilament Ca2+ sensitivity, under ISCH it appears to be related to decreased cytosolic Ca2+ availability combined with a more noticeable effect on a fraction of energy that has been attributed to mitochondrial activity. Furthermore, mechanical and energetic responses of both models (i.e., ISCH and HYP) found in the present work were not the same as those previously observed in severe hypoxia so that all these models should not be used indistinctly.     

Factors affecting the post-ischemic recuperation of isolated perfused rat heart

A number of cardioplegic solutions have been described for the reduction of cellular damage during ischemic cardiac arrest. Using an isolated working rat heart model, we have attempted to precise some of the factors affecting the post-ischemic recovery of myocardial tissue after a 30-min period of total ischemia at 37 degrees C. The results indicate that procaine (1 mM) is able to afford some protective against normothermic ischemia while this protective effect remains consistently lower than that of the St. Thomas' Hospital solution (procaine + high K+ + high Mg2+; JYNGE et al., 1977). On the other hand, hearts from rats of the Wistar strain consistently exhibit a significantly better degree of recovery than do hearts from rats of the Shermann strain. When hearts were perfused at different levels of preload (1 or 2 kPa) and afterload (8 or 10 kPa), post-ischemic recovery was better in hearts with lower levels of cardiac work. Glucose, insulin and DL-propranolol which have been shown to exert a protective effect in isolated rat hearts with regional ischemia failed to protect the heart in the present experimental conditions. No clear correlation does exist between the post-ischemic recovery and the enzymatic assessment of myocardial cell damage.     

Rat cardiac contractile dysfunction induced by Ca2+ overload: possible link to the proteolysis of alpha-fodrin

The aim of the present study was to examine the mechanisms of Ca2+ overload-induced contractile dysfunction in rat hearts independent of ischemia and acidosis. Experiments were performed on 30 excised cross-circulated rat heart preparations. After hearts were exposed to high Ca2+, there was a contractile failure associated with a parallel downward shift of the linear relation between myocardial O(2) consumption per beat and systolic pressure-volume area (index of a total mechanical energy per beat) in left ventricles from all seven hearts that underwent the protocol. This result suggested a decrease in O(2) consumption for total Ca2+ handling in excitation-contraction coupling. In the hearts that underwent the high Ca2+ protocol and had contractile failure, we found marked proteolysis of a cytoskeleton protein, alpha-fodrin, whereas other proteins were unaffected. A calpain inhibitor suppressed the contractile failure by high Ca2+, the decrease in O(2) consumption for total Ca2+ handling, and membrane alpha-fodrin degradation. We conclude that the exposure to high Ca2+ may induce contractile dysfunction possibly by suppressing total Ca2+ handling in excitation-contraction coupling and degradation of membrane alpha-fodrin via activation of calpain.