Min Of Low-flow Ischemia Research Articles

B-AB01-04 INTERACTION BETWEEN ISCHEMIA AND SYMPATHETIC ACTIVITY ON CARDIAC ELECTROPHYSIOLOGY AND ARRHYTHMIA IN THE INNERVATED LANGENDORFF-PERFUSED MOUSE HEART

Sympathetic activation of the heart plays a key role in the modulation of cardiac electrophysiology and arrhythmogenesis. It is generally believed that sympathetic activation is proarrhythmic in the setting of cardiac ischemia. However, few studies have looked at the dynamic effects of physiological sympathetic nerve activation on cardiac electrophysiological activities during ischemia. To investigate the effects of sympathetic activation in the innervated Langendorff-perfused mouse heart subjected to acute ischemia. The heart and posterior thoracic cavity from mice (C57Bl6, N=4) were dissected and perfused through the descending aorta for dual optical mapping of transmembrane potential (Vm) and intracellular Ca2+ to study the effects of sympathetic nerve stimulation (SNS). SNS, achieved through spinal cord stimulation at T1-T3, was performed before, during and immediately after 5 min of low-flow ischemia (10% of pre-ischemic flow rate). Early afterdepolarizations (EADs) and ventricular tachycardia (VT) occurred in all 4 animals within 5 min of ischemia and immediately upon reperfusion. Both SNS and ventricular pacing, during either ischemia or reperfusion, completely abolished EADs and VT induced by global low-flow ischemia and reperfusion (Fig). In the isolated innervated mouse heart, we showed that sympathetic stimulation protects against ischemia-induced cardiac arrhythmias potentially by over-driving ventricular ectopic activity.

Coronary microembolization during early reperfusion: infarct extension, but protection by ischaemic postconditioning

Reperfusion injury following acute myocardial infarction impacts not only on the myocardium but also on the coronary microcirculation, and microembolization from the culprit lesion contributes to microvascular obstruction. Prior experimental studies have not accounted for microembolization in ischaemia/reperfusion injury and not considered microembolization as a confounder and target of protection by ischaemic postconditioning. We therefore investigated the impact of microembolization during reperfusion on infarct size and cardioprotection by postconditioning. Anaesthetized, open-chest pigs were subjected to 90 min low-flow ischaemia. Immediate full reperfusion (n = 8) served as the control. Microembolization was induced by intracoronary infusion of 42 µm microspheres with the onset of reperfusion (n = 8). In a second step, postconditioning was induced by six cycles of 20s reperfusion/20s re-occlusion without (n = 8) and with superimposed microembolization (n = 8). Transmural blood flow and area at risk were determined by radioactive microspheres, infarct size by triphenyl tetrazolium chloride staining. Area at risk and transmural blood flow were not different between groups. Microembolization increased infarct size from 32 ± 3% of the area at risk to 47 ± 3% (P < 0.05). Embolizing particles were re-distributed away from the central infarcted area and accumulated in the infarct border, thus contributing to infarct extension. Postconditioning reduced infarct size without (21 ± 3%; P < 0.05 vs. immediate full reperfusion) and also with additional microembolization (26 ± 5%; P < 0.05 vs. immediate full reperfusion and microembolization); embolizing particles did not accumulate in the infarct border. Microembolization at reperfusion augments infarct size, but postconditioning in the presence of microembolization still reduces infarct size and attenuates infarct expansion.

Abstract 9145: Tyrosine 705 Phosphorylation of STAT3 is Causally Involved in Cardioprotection by Ischemic Postconditioning in Pigs

In mice, the signal transducer and activator of transcription (STAT) 3 is causally involved in protection by ischemic postconditioning (PoCo) against myocardial infarction. However, signal transduction in rodents differs from that in larger mammals. We have now determined the impact of PoCo on STAT3 activation/phosphorylation in pig hearts, which more closely resemble human hearts in size, coronary anatomy, heart rate, and infarct development. Anesthetized pigs were subjected to 90 min low flow ischemia and 120 min reperfusion, initiated with either PoCo (six cycles of 20 s reperfusion/re-occlusion, n=8) or immediate full reperfusion (IFR, n=8). Eight additional pigs were treated with the JAK/STAT inhibitor AG490 (9 µg/kg/min i.c.; 10 min before ischemia until the end of reperfusion; AG-PoCo n=4, AG-IFR n=4). Myocardial biopsies were taken at baseline, 85 min ischemia and 10, 30, and 120 min reperfusion. STAT3 phosphorylation and total STAT3 protein were quantified by Western blot analysis. Area at risk (AAR) and regional myocardial blood flow (microspheres) during ischemia were not different between groups. PoCo reduced infarct size (TTC staining) from 38±2 to 26±3 % of the AAR (p&lt;0.05). AG490 abolished this infarct size reduction (AG-IFR: 36±4 vs. AG-PoCo: 36±1 % of AAR). STAT3 tyr 705 phosphorylation normalized to total STAT3 protein (Table 1) increased during ischemia/reperfusion; in pigs subjected to PoCo the STAT3 tyr 705 phosphorylation was even greater than with IFR (p&lt;0.05, ANOVA). STAT3 ser 727 phosphorylation was increased with PoCo and IFR to a similar extent. AG490 attenuated the increases in STAT3 phosphorylation in both groups to values below those with IFR and PoCo. Conclusion: In pigs, PoCo further increases STAT3 tyr 705 phosphorylation and protects the myocardium from infarction. AG490 attenuates the STAT3 tyr 705 activation at reperfusion and abolishes PoCo's protection. Thus, in pigs STAT3 is causally involved in cardioprotection by PoCo.

Upregulation of eNOS and unchanged energy metabolism in increased susceptibility of the aging type 2 diabetic GK rat heart to ischemic injury

We investigated the tolerance of the insulin-resistant diabetic heart to ischemic injury in the male Goto-Kakizaki (GK) rat, a model of type 2 diabetes. Changes in energy metabolism, nitric oxide (NO) pathway, and cardiac function were assessed in the presence of physiological substrates. Age-matched control Wistar (n = 19) and GK (n = 18) isolated rat hearts were perfused with 0.4 mM palmitate, 3% albumin, 11 mM glucose, 3 U/l insulin, 0.2 mM pyruvate, and 0.8 mM lactate for 24 min before switching to 1.2 mM palmitate (11 rats/group) during 32 min low-flow (0.5 ml·min(-1)·g wet wt(-1)) ischemia. Next, flow was restored with 0.4 mM palmitate buffer for 32 min. A subset of hearts from each group (n = 8 for control and n = 7 for GK groups) were freeze-clamped for determining baseline values after the initial perfusion of 24 min. ATP, phosphocreatine (PCr), and intracellular pH (pH(i)) were followed using (31)P magnetic resonance spectroscopy with simultaneous measurement of contractile function. The NO pathway was determined by nitric oxide synthase (NOS) isoform expression and total nitrate concentration (NOx) in hearts. We found that coronary flow was 26% lower (P < 0.05) during baseline conditions and 61% lower (P < 0.05) during reperfusion in GK vs. control rat hearts. Rate pressure product was lower during reperfusion in GK vs. control rat hearts (P < 0.05). ATP, PCr, and pH(i) during ischemia-reperfusion were similar in both groups. Endothelial NOS expression was increased in GK rat hearts during baseline conditions (P < 0.05). NOx was increased during baseline conditions (P < 0.05) and after reperfusion (P < 0.05) in GK rat hearts. We report increased susceptibility of type 2 diabetic GK rat heart to ischemic injury that is not associated with impaired energy metabolism. Reduced coronary flow, upregulation of eNOS expression, and increased total NOx levels confirm NO pathway modifications in this model, presumably related to increased oxidative stress. Modifications in the NO pathway may play a major role in ischemia-reperfusion injury of the type 2 diabetic GK rat heart.

Exercise training improves functional post-ischemic recovery in senescent heart

The incidence of ischemic cardiac diseases increases with age and elderly subjects are more vulnerable to myocardial infarction. Endurance exercise (e.g. treadmill training) provides cardioprotection against an ischemia and reperfusion (IR) event in adult heart but such a potential beneficial effect of regular exercise has never been evaluated during aging. Therefore, this study investigated the effects of moderate running training on post-ischemic recovery of contractile function and coronary perfusion in senescent myocardium. Isolated hearts of sedentary (24 mo-sedentary; n = 10) and trained senescent (24 mo-trained; n = 11; moderate running: 1 h/day, 5 days/week for 12 weeks) rats were submitted to 45 min low-flow ischemia (15% of initial coronary flow (CF)) followed by 30 min reperfusion. Active tension (AT) and CF were recorded at baseline and after 1, 3, 5, 10, 15, 20, 25 and 30 min of reperfusion. Left ventricular protein carbonylation, and both heat-shock-protein 70 (HSP70) and endothelial nitric oxide synthase (eNOS) contents were determined by Oxyblotting and Western blotting, respectively. Regular physical exercise improves impairment of functional post-ischemic recovery (AT and CF) of aged hearts during reperfusion and this cardioprotection is associated to limited protein oxidation and increased HSP70 and eNOS myocardial contents.

Thiazolidinedione drugs block cardiac KATP channels and may increase propensity for ischaemic ventricular fibrillation in pigs.

Opening of ATP-sensitive potassium (K(ATP)) channels during myocardial ischaemia shortens action potential duration and is believed to be an adaptive, energy-sparing response. Thiazolidinedione drugs block K(ATP) channels in non-cardiac cells in vitro. This study determined whether thiazolidinedione drugs block cardiac K(ATP) channels in vivo. Experiments in 68 anaesthetised pigs determined: (1) effects of inert vehicle, troglitazone (10 mg/kg i.v.) or rosiglitazone (0.1 or 1.0 mg/kg i.v.) on epicardial monophasic action potential (MAP) during 90 min low-flow ischaemia; (2) effects of troglitazone, rosiglitazone or pioglitazone (1 mg/kg i.v.) on response of MAP to intracoronary infusion of a K(ATP) channel opener, levcromakalim; and (3) effects of inert vehicle, rosiglitazone (1 mg/kg i.v.) or the sarcolemmal K(ATP) blocker HMR-1098 on time to onset of ventricular fibrillation following complete coronary occlusion. With vehicle, epicardial MAP shortened by 44+/-9 ms during ischaemia. This effect was attenuated to 12+/-8 ms with troglitazone and 6+/-6 ms with rosiglitazone (p<0.01 for both vs vehicle), suggesting K(ATP) blockade. Intracoronary levcromakalim shortened MAP by 38+/-10 ms, an effect attenuated to 12+/-8, 13+/-4 and 9+/-5 ms during co-treatment with troglitazone, rosiglitazone or pioglitazone (p<0.05 for each), confirming K(ATP) blockade. During coronary occlusion, median time to ventricular fibrillation was 29 min in pigs treated with vehicle and 6 min in pigs treated with rosiglitazone or HMR-1098 (p<0.05 for both vs vehicle), indicating that K(ATP) blockade promotes ischaemic ventricular fibrillation in this model. Thiazolidinedione drugs block cardiac K(ATP) channels at clinically relevant doses and promote onset of ventricular fibrillation during severe ischaemia.

Abstract 516: Pathologic Remodeling of Myocardial Cardiolipin in a Porcine Model of Diet-Induced Obesity and Insulin Resistance

Background: Cardiolipin is a dimeric phospholipid required for normal mitochondrial function. The majority of cardiolipin molecules contain four linoleic acid (18:2) residues. Pathologic decrease in (18:2) 4 cardiolipin content has been associated with cardiomyopathy. Patients with obesity and insulin resistance have poorer outcomes after myocardial infarction compared with lean subjects. In the present study, we determined if cardiolipin content and composition are altered in a new porcine model of diet-induced obesity and insulin resistance. Methods: Yucatan micropigs (n=16) were assigned to a control diet (4% w/w fat, 2% simple sugars, quantity fed 30 g/kg/day) or an intervention diet (25% fat from coconut oil, 17% simple sugars, 50 g/kg/day) for 7 months. Pigs were then anesthetized and subjected to 45 min low-flow myocardial ischemia and 120 min reperfusion (I/R, n=11). Regional myocardial external work and O 2 consumption were measured. After euthanasia, neutral lipid content in myocardial samples was assayed by solid phase extraction and gas chromatographic mass spectrometry, and cardiolipin species were quantified by electrospray ionization mass spectrometry. Results: Compared with controls, the intervention diet group recapitulated characteristics of the clinical “metabolic syndrome” with obesity (73 ± 4 vs 40±4 kg), insulin resistance by glucose tolerance testing, hypertension, and hyperlipidemia. There was abnormal substitution of saturated fatty acyl moieties in all myocardial lipid pools. Myocardial content of normal cardiolipin (18:2) 4 was reduced by an average of 13% (p=0.02), while several smaller pools of abnormal cardiolipin species were increased. For example, cardiolipin with one myristic acid residue substituted for a linoleic acid residue was increased by an average of 400% (p=0.001). After I/R, external work was approximately 20% lower (p=0.2) and MVO 2 was 50% lower (p=0.07) in the intervention diet group. Conclusions: Pigs fed a diet high in saturated fat and energy content for 7 months develop a phenotype resembling the clinical metabolic syndrome. This is accompanied by pathologic remodeling of myocardial cardiolipin, which in turn may contribute to decreased contractile and metabolic performance after I/R.

Myocardial Release of Nitric Oxide During Ischaemia and Reperfusion: Effects of l-Arginine and Hypercholesterolaemia

Nitric oxide (NO) may modulate myocardial ischaemia/reperfusion (I/R) injury, but effects of hypercholesterolaemia on myocardial NO release during I/R are unknown. A NO-specific carbon fibre electrode continuously measured coronary sinus [NO] during 60 min low-flow ischaemia (1 ml/min) and 60 min free reperfusion (I/R) in isolated rabbit hearts. Experimental groups (n=7 per group) were control, L-arginine supplement (200 microM), N-nitro-L-arginine methyl ester (L-NAME) treatment (8 microM) and hypercholesterolaemic. During early I, NO release decreased markedly in control (-1356+/-286 pmol/min/g) and L-arginine (-1972+/-172) groups, but less in L-NAME (-441+/-89) and hypercholesterolaemic (-602+/-164) groups (both p<0.01 vs. controls). No increase in NO release during I was seen in any group. After R, NO release increased above baseline in control (+2333+/-591 pmol/min/g) and L-arginine (+1048+/-278) groups and hypercholesterolaemic (+1100+/-478) (p<0.05 vs. pre-ischaemia each group). There was little increase in NO release in the L-NAME group (+436+/-247 pmol/min/g, p<0.05 vs. controls). In each group, myocardial NO release declined towards pre-ischaemic levels during 60 min R. Hearts treated with L-arginine had similar NO release but better functional recovery than controls (p<0.01). Treatment with L-NAME was also associated with better functional recovery than in controls or hypercholesterolaemic hearts. Myocardial NO release declines rapidly during ischaemia, but increases above baseline during early reperfusion. Improved function after L-arginine treatment appears to be independent of effects upon NO release. Hypercholesterolaemia is associated with reduced myocardial NO release, under both baseline conditions and during ischaemia and reperfusion.

Low carbohydrate diet reduces myocardial glycogen stores and increases myocardial injury following ischemia‐reperfusion

Low carbohydrate diets (LCDs) are popular for weight loss, but the safety of such diets is unknown. Myocardial glycogen may be an important energy source during ischemia, when circulating energy substrate and O2 supplies are reduced. We hypothesized that LCD lowers cardiac glycogen and increases cardiac injury during ischemia and reperfusion. Rats were fed LCD (60% kcal from fat/30% protein/10% carbohydrate) or a low fat diet (LFD; 16%/19%/65%) for 2 weeks (N=18 each diet). 6 from each group were sacrificed for biochemical analysis, 6 were subjected to 30 min of low flow ischemia (LFI; 0.3ml/min), and 6 to a 60 min reperfusion protocol following 30 min of LFI after hearts were isolated and perfused with a physiologic perfusion mixture. LCD led to significant increase of circulating free fatty acids, 3-hydroxybutyrate and decrease in insulin, and a decrease in myocardial and liver glycogen (Fig B). LCD resulted in impaired left ventricular performance during LFI, with worsened ischemic contracture and increased total lactate dehydrogenase (LDH) release (Fig. A). Cardiac glycogen was lower after LFI (0.42±0.04 and 0.44±0.02 mg/g tissue; P < 0.05 vs. pre-LFI). These results demonstrate that LCD lowers cardiac glycogen and increases myocardial injury following ischemia-reperfusion, and suggest that glycogen stores play a critical role in prevention of ischemic injury. Figure A and BOpen in figure viewerPowerPoint A: Total LDH release during LFI and reperfusion. B: Glycogen content in myocardium and liver (mg glucose/g wet tissue weight). ∗ P<0.05 vs. LFD.

CARDIAC ISCHEMIA‐REPERFUSION DAMAGE IN PARTIAL HEXOKINASE (HK) II KNOCKOUT MICE

Overexpression of HKI and HKII in cultured cells protects against oxidant-induced cell death. It is unknown whether cardiac alterations of HKII affect ischemia-reperfusion injury in the whole heart. In the present study we examined ischemia-reperfusion injury in isolated hearts of partial HKII (+/−) knockout (HKKO) and wild-type mice (WT). Langendorff-perfused hearts (HKKO: n=7; WT: n=8) were subjected to 40 min low-flow (5% of baseline flow) ischemia and 120 min reperfusion. Necrosis was determined by lactate dehydrogenase (LDH) enzyme release during the reperfusion period. At baseline, no significant differences were observed between groups: end-diastolic pressure (EDP) is 7.5 ± 1.0 mm Hg, developed left ventricular pressure (DLVP) is 98.7 ± 4.6 mm Hg, heart rate is 369 ± 12 beats/min and flow is 15.1 ± 1.4 ml/min/gww. Following 120 min reperfusion, recovery of DLVP was 68.3 ± 8.3 % (WT) versus 55.6 ± 9.1% (HKKO) and EDP was 11.6 ± 3.7 mm Hg (WT) versus 29.0 ± 6.8 mm Hg (HKKO). Between groups contracture development during ischemia reached EDP >10 mm Hg at different time points: 34 min for WT and 12 min for HKKO. LDH release was 1.8 ± 0.35 versus 3.3 ± 0.75 μmol/gww/120 min reperfusion for WT versus HKKO, respectively. The data suggest for the first time that HKII may be an important mediator of ischemia-reperfusion damage in the intact heart.

Decrease in fatty acid oxidation increases tolerance of the ageing heart to ischaemic injury

Ageing is associated with reduced tolerance to myocardial infarction. Alterations in mitochondrial oxidative metabolism are important determinants in the contractile recovery of the adult heart after ischaemia. However, the profile of substrate metabolism during reperfusion in the ageing heart is unknown. The aim of this study was to investigate energy substrate preference and functional recovery following low flow ischaemia in the ageing heart. Hearts from 6 and 24 mo male Wistar rats were perfused in an isovolumic mode with buffer containing 3% albumin and a physiological mixture of substrates including 0.3mM [U-13C] palmitate, 1mM [3-13C] lactate and 5mM glucose. Cardiac work was increased by dobutamine infusion for 20 min (2.8±0.1 μg/min/g heart weight) followed by 30 min low flow ischaemia and 30 min reperfusion. Ischaemia caused similar reductions in left ventricular developed pressure and cardiac work in adult and senescent hearts. On reperfusion, 24 mo hearts demonstrated improved functional recovery relative to 6-mo hearts. This was associated with a significant decline in palmitate oxidation and increase in lactate oxidation. Thus, the ageing heart has increased tolerance to severe ischaemia, which may be attributed to the reduction in fatty acid oxidation. JS is funded by a PhD scholarship from the University of Hull and Academic Dept of Cardiology.

Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning

Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline. These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.

Calcitonin gene-related peptide does not interact with sympathetic activity in myocardial ischemia

The aim of the present study was to assess whether calcitonin gene-related peptide (CGRP) modulates exocytotic norepinephrine release in ischemic myocardium. In isolated rat hearts subjected to 30 min of low flow ischemia, CGRP release increased 2.8-fold whereas stimulation-induced norepinephrine release decreased 4.1-fold. Pretreatment of rats with capsaicin almost completely depleted cardiac CGRP stores; however, suppression of norepinephrine release by 30 min of low flow ischemia was not affected. At normal flow, exogenous CGRP (5 μmol l −1) had no effect on norepinephrine release. These findings suggest that CGRP release from sensory neurons does not interact with the cardiac sympathetic system during myocardial ischemia.

Limited effects of post-ischemic NHE blockade on [Na+]i and pHi in rat hearts explain its lack of cardioprotection.

Na+/H+ exchanger (NHE) blockade fails as reperfusion therapy in patients with acute myocardial infarction. In experimental studies, the reports on the efficacy of NHE blockade only during reperfusion are inconsistent. Differences in the severity of ischemia and in drug delivery may explain these inconsistencies. Little is known about the primary goal of post-ischemic NHE blockade, i.e. reduction of Na+i overload. Isolated rat hearts were subjected either to 60 min of low flow (0.2 ml/min) ischemia or 25 min of zero flow ischemia. Hearts were reperfused with or without the selective NHE blocker cariporide added to the perfusate. [Na+]i and pHi were measured with simultaneous 23Na and 31P NMR spectroscopy. After 60 min of low flow ischemia [Na+]i had risen to 424 +/- 14% of baseline and pHi was 6.36 +/- 0.03. After low flow ischemia [Na+]i and pHi recovered similarly in treated and untreated hearts. Recovery of the rate pressure product (RPP) was poorly in both groups. After 25 min of zero flow ischemia [Na+]i had risen to 279 +/- 7% of baseline and pHi was 6.12 +/- 0.02. NHE blockade after zero flow ischemia caused [Na+]i to decrease during the first 30 s of reperfusion, followed by a partial and transient rise during the second 30 s. Untreated hearts showed a very small rise in [Na+]i during the first minute. pHi recovered 30 s slower in cariporide treated hearts than in untreated hearts (p<0.05). No effect of cariporide on RPP could be detected since RPP recovered fully in untreated hearts. The end diastolic pressure, however, was increased during reperfusion to a similar extent in both groups. The lack of cardioprotection under these specific conditions of zero flow and low flow ischemia can be explained by the fact that NHE blockade only resulted in a small and transient effect on [Na+]i and pHi.

Human thrombocytes are able to induce a myocardial dysfunction in the ischemic and reperfused guinea pig heart mediated by free radicals-role of the GPIIb/IIIa-blocker tirofiban

In recent studies, we could demonstrate a myocardial dysfunction induced by homologous platelets in ischemic and reperfused guinea pig hearts. Aim of the current study was to find out whether or not this is a phenomenon specific for platelets isolated from guinea pigs and to further examine the mechanisms of a possible cardiodepressive effect of human platelets. Isolated guinea pig hearts were exposed to a 30 min low-flow ischemia (1 ml/min) and reperfused. Human thrombocytes were administered as bolus (20.000 thrombocytes/μl perfusion buffer) in the 15 th min of ischemia or in the 1 st or 5 th min of reperfusion in the presence of thrombin. Recovery of external heart work (REHW) and intracoronary platelet retention (RET) were quantified in percent. In additional experiments, the GPIIb/IIIa-blocker tirofiban (10 μg/ml perfusion buffer) or the radical scavenger superoxide dismutase (SOD-10 U/ml perfusion buffer) were added. Platelet application in the absence of tirofiban, either during ischemia (REHW 75.4 ± 4%, RET 22.2 ± 2%) or the 1st min (REHW 71.6 ± 1%, RET 31.2 ± 2%) or the 5th min of reperfusion (REHW 63.2 ± 4%, RET 40.5 ± 1%) led to a significant reduction of REHW and a significant increase of RET. The coapplication of tirofiban, on the other hand, prevented RET at all three times of platelet application (1.1 ± 1.7%, 0% or 2.1 ± 1.2%, respectively). An improvement of REHW, however, could only be noticed during ischemia (89 ± 2%), whereas coapplication of tirofiban in early (72.9 ± 3%) or in late reperfusion (74.6 ± 2%) did not lead to a significant increase of REHW. Coapplication of SOD, on the other hand, significantly improved REHW in early (88.1 ± 1) or late (95.9 ± 1) reperfusion but not during ischemia (83.5 ± 2). Corresponding to REHW, RET was changed significantly by coapplication of SOD during early (1 ± 2%) or late (0%) reperfusion but not during ischemia (21.1 ± 4%). We conclude that human thrombocytes are able to induce a myocardial dysfunction in ischemic and reperfused guinea pig hearts mediated by reactive oxygen species and independent of intracoronary platelet adhesion.

P38 MAP kinase is a mediator of ischemic preconditioning in pigs.

The role of p38MAPK in ischemic preconditioning (IP) is still equivocal, insofar as the p38MAPK-inhibitor SB203580 abolished IP in rats, rabbits and dogs, but not in pigs. Blockade of p38MAPK prior to the sustained ischemia also generated contradictory findings, insofar as p38MAPK acted as trigger in dogs but as mediator in rats. We have now tested whether the two structurally unrelated p38MAPK-inhibitors, BIX-645 and SB203580, abolished infarct size (IS) reduction by IP in pigs and whether their effects depended on the time of administration. Sixty-five enflurane-anesthetized pigs underwent 90 min low-flow ischemia and 120 min reperfusion without or with one preceding cycle of 10 min preconditioning ischemia and 15 min reperfusion. Pigs received BIX-645 (1 mg/kg, i.v.) or SB203580 (10 microM, i.c.) prior to either IP or the sustained ischemia. IS (% TTC-staining) was reduced by IP [4.8+/-3.1(S.E.M.), P<0.05] compared to placebo (25.8+/-5.5). BIX-645 or SB203580 per se had no effect on IS (23.5+/-5.2 and 21.8+/-4.4, respectively). IS reduction by IP was abolished by BIX-645 (26.2+/-6.4 or 25.5+/-4.7) and SB203580 (19.9+/-4.3 or 16.7+/-4.7), given either prior to IP or the sustained ischemia, respectively. The supernatant of homogenized myocardial biopsies taken during the sustained ischemia from preconditioned pigs receiving either BIX-645 or SB203580 inhibited the anisomycin-stimulated ATF-2 phosphorylation in cultured Rat1 fibroblasts. This in vitro inhibition of ATF-2 phosphorylation correlated to the actual IS. The attenuation of the IS-reducing effect of IP depends on the effectiveness of blockade of p38MAPK activity. p38MAPK is a mediator of IP in pigs.

Effect of Treatment on Ventricular Function and Troponin I Proteolysis in Reperfused Myocardium

Effects of ischemia time and treatment interventions upon troponin I (TnI) proteolysis and function of reperfused myocardium were examined in isolated, perfused rabbit hearts. Hearts were randomized to 90 min aerobic perfusion, 15 min low-flow (1 ml/min) ischemia (I) and 60 min reperfusion (R) or 60 min low-flow I and 60 min R. Hearts subject to 60 min I and 60 min R received either no treatment, l -arginine treatment, or treatment with oxygen free radical (OFR) scavengers (mercapto-proponyl-glycine, catalase and superoxide dismutase). Hearts from cholesterol-fed rabbits were also studied after 60 min I and R. Isovolumic LV pressure and heart rate were recorded throughout and Western analysis of ventricular myocardium, using 3 specific antibodies, detected intact TnI (29 kDa) and TnI fragment (25 kDa). Hearts subject to 15 min I had minimal irreversible injury (TTC negative region=0.6±0.4% LV) but hearts subject to 60 min I had more extensive injury (TTC negative=40.7±5.8% LV). Recovery of rate–pressure product after 15 min I and 60 min R (56±9% of baseline) was better than after 60 min I and 60 min R (23±9%, P<0.01). Bothl -arginine and OFR scavengers were associated with better recovery of function after 60 min I, (66±7% and 72±3% of baseline respectively, P<0.01 v no treatment) but cholesterol hearts had poor recovery after 60 min I (37±8%). The 25 kDa TnI (% total TnI immunoreactivity) was 8.7±0.9% in controls, 10.0±1.6% after 15 min I and 60 min R, and 17.4±2.4% after 60 min I and 60 min R (P<0.01v controls and 15 min I). The proportion of 25 kDa TnI was increased in all hearts after 60 min I and did not change with treatment (l -arginine 16.8±1.8%, OFR scavengers 16.0±3.2%, cholesterol 14.0±1.9%). There was no relation between proportion of 25 kDa TnI and recovery of function. Samples from freshly excised rabbit hearts and human right atria also had 25 kDa TnI (relative intensities 8.5±2.3% and 5.1±2.6% respectively). Although TnI fragmentation increases after prolonged ischemia and reperfusion, the functional recovery of stunned myocardium is independent of degree of TnI fragmentation.

Thiazolidinedione treatment normalizes insulin resistance and ischemic injury in the zucker Fatty rat heart.

Obesity is associated with risk factors for cardiovascular disease, including insulin resistance, and can lead to cardiac hypertrophy and congestive heart failure. Here, we used the insulin-sensitizing agent rosiglitazone to investigate the cellular mechanisms linking insulin resistance in the obese Zucker rat heart with increased susceptibility to ischemic injury. Rats were treated for 7 or 14 days with 3 mg/kg per os rosiglitazone. Hearts were isolated and perfused before and during insulin stimulation or during 32 min low-flow ischemia at 0.3 ml small middle dot min(-1) small middle dot grams wet wt(-1) and reperfusion. D[2-(3)H]glucose was used as a tracer of glucose uptake, and phosphorus-31 nuclear magnetic resonance spectroscopy was used to follow energetics during ischemia. At 12 months of age, obese rat hearts were insulin resistant with decreased GLUT4 protein expression. During ischemia, glucose uptake was lower and depletion of ATP was greater in obese rat hearts, thereby significantly impairing recovery of contractile function during reperfusion. Rosiglitazone treatment normalized the insulin resistance and restored GLUT4 protein levels in obese rat hearts. Glucose uptake during ischemia was also normalized by rosiglitazone treatment, thereby preventing the greater loss of ATP and restoring recovery of contractile function to that of lean rat hearts. We conclude that rosiglitazone treatment, by normalizing glucose uptake, protected obese rat hearts from ischemic injury.

Preconditioning of rat hearts by adenosine A 1 or A 3 receptor activation

Our study in rat hearts examined whether activation of adenosine A 1 or A 3 receptors improved functional recovery and reduced apoptosis resulting from low-flow ischemia. Prior to 30 min low-flow ischemia (0.6 ml/min; 6% of baseline flow), Langendorff rat hearts were preconditioned with two 5-min cycles of (a) ischemia (PC; n=7), (b) infusion of 250 nM adenosine A 1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA; n=6), or (c) infusion of 50 nM adenosine A 3 receptor agonist N 6-(3-iodobenzyl)-adenosine-5′- N-methyl-uronamide (IB-MECA; n=8). Recovery of function was improved in PC (71±3%), CCPA (68±6%) and IB-MECA (68±4%) groups compared to control hearts (46±5%; P<0.05). Cumulative release of total purines during ischemia–reperfusion was approx. 50% lower in PC, CCPA and IB-MECA groups compared to controls ( P<0.05) and was significantly correlated to the percentage functional recovery ( R 2=0.55; P<0.05). The number of cytosolic histone-associated-DNA fragments, a hallmark of apoptosis and measured by Enzyme Linked ImmunoSorbent Assay (ELISA), was small and not different between groups after 30 min reperfusion. However, CCPA (0.6±0.1 absorbance units) and MECA (0.7±0.1 units; P<0.05 vs. PC) decreased apoptosis after 150 min reperfusion compared to PC (1.4±0.3 units) and control (1.2±0.1 units) hearts. This study shows that adenosine triggers protection of function in preconditioned rat hearts via both the adenosine A 1 and A 3 receptor. In clinical practice, pharmacological stimulation of adenosine A 3 receptors may be advantageous over adenosine A 1 receptor activation due to a lack of contractile side-effects. In contrast to ischemic preconditioning, pharmacological stimulation of adenosine A 1 or A 3 receptors reduced apoptosis. Furthermore, total purine release may serve as a marker of the degree of functional protection.