Nonischemic Regions Research Articles

Cardiomyocytes of Chronically Ischemic Pig Hearts Express the MDR-1 Gene-encoded P-glycoprotein

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.

Correlation between endocardial voltage mapping and myocardial perfusion: implications for the assessment of myocardial ischemia

Recent clinical studies using a non-fluoroscopic three-dimensional (3D) left ventricular (LV) mapping system suggested reduced endocardial voltage amplitudes measured in zones sustaining myocardial ischemia or infarction. However, the direct relationship between myocardial perfusion and endomyocardial voltage amplitudes has not been fully elucidated. In a pig model of chronic myocardial ischemia (n = 20), LV endocardial unipolar voltage (UpV) mapping was performed using the Biosense 3D navigation system (Johnson and Johnson, Warren, New Jersey, USA) 4 weeks after ameroid constrictor placement around the left circumflex coronary artery. Echocardiography was used to assess regional changes in myocardial wall thickening (MT) and fluorescent microspheres (4 x 10/injection) were used to quantify rest regional myocardial blood flow (MBF) in ischemic (left circumflex) and remote non-ischemic (left anterior descending) regions. UpV measurements were reduced in ischemic compared to non-ischemic zones (9.9+/-3.3 compared with 13.3+/-3.3 mV, P = 0.03). This corresponded to changes in endocardial MBF and MT, which were both noted to be significantly reduced in the ischemic compared to the non-ischemic area (MBF, 0.50+/-0.16 compared with 0.74+/-0.15 ml/g per min, P = 0.001; MT, 26.1+/-12.0 compared with 37.4+/-10.1%, P=0.003). A positive linear correlation was found between UpV at rest and endomyocardial (but not epicardial) perfusion: UpV (mV) = 7.8+5.9xMBFendocardial (r = 0.32, P = 0.05). Chronic myocardial ischemia, resulting in reduced perfusion and function at rest (that is, hibernating myocardium), is characterized by a significant reduction ( approximately 25%) in endocardial UpV potentials, which correlates with reduced endomyocardial blood flow and tissue perfusion at rest.

Coexistence of Two Types of Ventricular Fibrillation During Acute Regional Ischemia in Rabbit Ventricle

We previously reported that a normal ventricle can demonstrate two types of ventricular fibrillation (VF), depending on the underlying electrophysiologic characteristics at the time of VF induction. We hypothesize that the two types of VF can coexist in acutely ischemic ventricles. Optical mapping studies were performed with di-4ANEPPS in 15 Langendorff-perfused rabbit hearts. Coronary artery branches were ligated to create regional ischemia in 10 hearts. Action potential duration measured to 50% repolarization (APD50) during ischemia showed an area with uniformly shortened APD50 (zone 1), an area with normal or lengthened APD50 (zone 3), and an area in between with an APD50 gradient (zone 2). Ischemia flattened APD restitution (APDR) slope and reduced conduction velocity in zone 1, creating a condition for type II VF. APDR steepened and the conduction velocity changed little in the nonischemic zone (zone 3), creating a condition for type I VF. During induced VF, the dominant frequency in zones 2 and 3 progressively increased after ischemia onset. The dominant frequency in zone 1 (ischemic zone) first decreased and then slightly increased but typically remained less than the dominant frequency in zone 3. The number of wavebreaks increased with time in all three zones (baseline: 4.3 +/- 1.5; 30 min: 11.7 +/- 5.6; 60 min: 15.6 +/- 11 per frame; P < 0.01). Two types of VF can coexist during acute regional ischemia. Both ischemic and nonischemic regions develop proarrhythmic changes during regional ischemia, thus contributing to increased ventricular vulnerability to VF and sudden death during acute coronary occlusion.

Cytoprotection by bcl-2 gene transfer against ischemic liver injuries together with repressed lipid peroxidation and increased ascorbic acid in livers and serum.

The maximum gene exhibition was shown to be achieved at 48 h after transfection with human bcl-2 (hbcl-2) genes built in an SV40 early promoter-based plasmid vector and HVJ-liposome for cultured rat hepatocytes. The similar procedure of hbcl-2 transfection was therefore conducted for livers in rats via the portal vein, and after 48 h followed by post-ischemic reperfusion (I/R) operation for some hepatic lobes. The I/R-induced hepatic injuries were in situ observed as both cell morphological degeneration and cellular DNA strand cleavages around capillary vessels of the ischemic liver lobes as detected by HE stain and TUNEL assay, and were biochemically observed as release of two hepatic marker enzymes AST and ALT into serum. All the I/R-induced injuries examined were appreciably repressed for rats transfected with hbcl-2; hbcl-2 was expressed in hepatocytes around the capillaries of ischemic regions such as the median lobe and the left lobe, but scarcely around those of non-ischemic regions. Thus cytoprotection against I/R-induced injuries may be attributed to the I/R-promoted expression of transferred hbcl-2 genes. The possibility was examined firstly by methylphenylindole method, which showed that I/R-enhanced lipid peroxidation in the reference vector-transfected livers were markedly repressed in the hbcl-2-transfected livers. Contents of ascorbic acid (Asc) in serum and livers of hbcl-2-transfected rats were enriched, unexpectedly, versus those of non-transfected rats, and were as abundant as 1.90-fold and 1.95- to 2.60-fold versus those in the pre-ischemic state, respectively. After I/R, an immediate decline in serum Asc occurred in hbcl-2-transfectants, and was followed by prompt restoration up to the pre-ischemic Asc levels in contrast to the unaltered lower Asc levels in non-transfectants except a transient delayed increase. Hepatic Asc contents were also diminished appreciably at the initial stage after I/R in the ischemic lobes of hbcl-2-transfectants, which however retained more abundant Asc versus non-transfectants especially at the initial I/R stage when scavenging of the oxidative stress should be most necessary for cytoprotection. The results showed a close correlation between cytoprotection by exogenously transferred hbcl-2 and repressive effects on the lipid peroxidation associated with Asc consumption or redistribution.

Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats

Focal ischemia in the cerebral cortex results in acute and delayed cell death in the ischemic cortex and non-ischemic thalamus. We examined the hypothesis that neurons in ischemic and non-ischemic regions died from different mechanisms; specifically, we tested whether a mixed form of cell death containing both necrotic and apoptotic changes could be identified in individual cells.Focal barrel cortex ischemia in rats was induced by occlusion of small branches of the middle cerebral artery (MCA) corresponding to the barrel cortex, local blood flow was measured by quantitative autoradiography. Cell death was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin–eosin (H&E) staining, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 staining 1 to 10 days after the ischemia. Electron microscopy was used for ultrastructural examination. Cell death occurred in the ipsilateral cortex 24 h after ischemia, followed by selective neuronal death in the ventrobasal (VB) thalamus 3 days later. TUNEL positive neurons were found in these two regions, but with striking morphological differences, designated as type I and type II TUNEL positive cells. The type I TUNEL positive cells in the ischemic cortex underwent necrotic changes. The type II TUNEL positive cells in the thalamus and the cortex penumbra region represented a hybrid death, featured by concurrent apoptotic and necrotic alterations in individual cells, including marked caspase-3 activation, nuclear condensation/fragmentation, but with swollen cytoplasm, damaged organelles and deteriorated membranes. Cell death in the thalamus and the cortex penumbra were attenuated by delayed administration of the caspase inhibitor benzyloxycarbonyl-Val–Ala–Asp fluoromethylketone (Z-VAD-FMK). Our data suggest that TUNEL staining should be evaluated with morphological changes, the hybrid death but not typical apoptosis occurs in the penumbra region and non-ischemic thalamus after cerebral ischemia.

Protection against ventricular arrhythmias and cardiac death using adenosine and lidocaine during regional ischemia in the in vivo rat.

Despite decades of research, there are few effective ways to treat ventricular fibrillation (VF), ventricular tachycardia (VT), or cardiac ischemia that show a significant survival benefit. Our aim was to investigate the combined therapeutic effect of two common antiarrhythmic compounds, adenosine and lidocaine (AL), on mortality, arrhythmia frequency and duration, and infarct size in the rat model of regional ischemia. Sprague-Dawley rats (n = 49) were anesthetized with pentobarbital sodium (60 mg.ml(-1).kg(-1) i.p.) and instrumented for regional coronary occlusion (30 min) and reperfusion (120 min). Heart rate, blood pressure, and a lead II electrocardiogram were recorded. Intravenous pretreatment began 5 min before ischemia and extended throughout ischemia, terminating at the start of reperfusion. After 120 min, hearts were removed for infarct size measurement. Mortality occurred in 58% of saline controls (n = 12), 50% of adenosine only (305 microg.kg(-1).min(-1), n = 8), 0% in lidocaine only (608 microg.kg(-1).min(-1), n = 8), and 0% in AL at any dose (152, 305, or 407 microg.kg(-1).min(-1) adenosine plus 608 microg.kg(-1).min(-1) lidocaine, n = 7, 8, and 6). VT occurred in 100% of saline controls (18 +/- 9 episodes), 50% of adenosine-only (11 +/- 7 episodes), 83% of lidocaine-only (23 +/- 11 episodes), 60% of low-dose AL (2 +/- 1 episodes, P < 0.05), 57% of mid-dose AL (2 +/- 1 episodes, P < 0.05), and 67% of high-dose AL rats (6 +/- 3 episodes). VF occurred in 75% of saline controls (4 +/- 3 episodes), 100% of adenosine-only-treated rats (3 +/- 2 episodes), and 33% lidocaine-only-treated rats (2 +/- 1 episodes) of the rats tested. There was no deaths and no VF in the low- and mid-dose AL-treated rats during ischemia, and only one high-dose AL-treated rat experienced VF (25.5 sec). Infarct size was lower in all AL-treated rats but only reached significance with the mid-dose treatment (saline controls 61 +/- 5% vs. 38 +/- 6%, P < 0.05). We conclude that a constant infusion of a solution containing AL virtually abolished severe arrhythmias and prevented cardiac death in an in vivo rat model of acute myocardial ischemia and reperfusion. AL combinational therapy may provide a primary prevention therapeutic window in ischemic and nonischemic regions of the heart.

ANP and BNP but not VEGF are regionally overexpressed in ischemic human myocardium

Angiogenic gene therapy in angina pectoris has been disappointing so far. Reasons might be that the administered genes already are overexpressed in ischemic myocardium, or that atrial and brain natriuretic peptides (ANP and BNP) are overexpressed, as they have anti-angiogenic effects. Five stable angina pectoris patients without heart failure were studied. Left ventricular biopsies were taken during coronary by-pass surgery from a region with stress-inducible ischemia and from a normal region. Both ANP and BNP but not vascular endothelial growth factor (VEGF) and VEGF-receptor 1 and 2 were overexpressed in ischemic regions compared to non-ischemic regions as measured by real-time PCR. The expression of 15 other angiogenic genes measured by oligonucleotide arrays was not consistently increased in ischemic regions. The overexpression of ANP and BNP suggests an anti-angiogenic effect in ischemic heart disease. The lack of overexpression of angiogenic genes supports the concept of therapeutic overexpression of these genes.

Alterations of β-adrenoceptor responsiveness in postischemic myocardium after 72 h of reperfusion

To determine the effect of a completely developed reperfused myocardial infarction model on β-adrenoceptor responsiveness, we induced a 90-min regional ischemia followed by 72 h of reperfusion in dog hearts. Regional myocardial blood flow was determined after 60 min of ischemia using radioactive microspheres. β-adrenoceptor density was reduced in the ischemic endocardium (95±16 fmol/mg) and epicardium (160±13 fmol/mg) compared to the nonischemic region (304±21 fmol/mg). β-adrenoceptor density in the ischemic endocardium varied with the degree of collateral blood flow measured ( r 2=0.79, P<0.05); this relation was the opposite of that in the ischemic epicardium ( r 2=0.77, P<0.05). Higher levels of tissue catecholamines and G protein-coupled receptor kinase 2 (GRK2) were observed in the ischemic epicardium as compared to nonischemic tissue. Forskolin-induced adenylyl cyclase activities were depressed in both ischemic regions as compared to nonischemic region, correlating with a reduction in regional myocardial blood flow. Using forskolin stimulation as covariate, no difference in isoproterenol-induced adenylyl cyclase activity was identified in the different regions. It is concluded that cAMP production induced by β-adrenoceptor activation is dependent upon adenylyl cyclase enzyme activity rather than β-adrenoceptor density in the ischemic myocardium. However, the density of the β-adrenoceptor in the viable ischemic regions can be modified by the presence of GRK2 and tissue catecholamines, an index of regional sympathetic efferent postganglionic nerve terminal activity.

Effects of tachycardia on regional wall motion in acute ischemic canine heart.

Tachycardia accompanies the preload reduction. Our aim is to assess the effect of the heart rate change on wall motion in ischemic heart. In 8 dogs with occlusion of left anterior descending artery, we changed the heart rate (heart rate 90, 120, and 150 beats/minute) after using UL-FS49, a selective bradycardic agent, with atrial pacing. Preload was changed by inferior vena caval occlusion at a heart rate of 90 beats/minute. With either an increase in heart rate or an inferior vena caval occlusion, the end-diastolic length was decreased, but the end-diastolic length relationships between the non-ischemic and the ischemic region made different lines from those of the heart rate change and inferior vena caval occlusion. When increasing the heart rate, isovolumetric shortening was unchanged in the non-ischemic region with more expansion in the ischemic region. While inferior vena caval occlusion at a heart rate of 90 beats/minute, isovolumetric shortening was increased in the non-ischemic region, with more expansion in the ischemic region. Both in tachycardia and by the inferior vena caval occlusion, ejectional shortenings decreased in the non-ischemic and ischemic regions. Our results suggest that, in ischemic heart, tachycardia changes both in the end-diastolic length relationship between the non-ischemic and the ischemic region and at the isovolumetric contraction phase. The changes seem to be not only due to the inferior vena caval occlusion, but also due to tachycardia itself.

Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia

To test the hypothesis that presence of metalloproteases (MMPs), their inhibitors (TIMPs) and their substrate laminin-5 differs between the ischemic core and the surrounding tissue, we examined the impact of middle cerebral artery occlusion/reperfusion (MCA:O/R) on these molecules in different regions of the infarct. We also investigated the influence of hypothermia on the progression of the ischemic lesion and MMP activity. Brain sections from 64 Wistar rats subjected to MCA:O/R were examined by means of cytohistochemistry and zymography. The artery was occluded for 2 h followed by 3, 5, 8 and 12 h of reperfusion. Well characterized antibodies against laminin-5, MMPs and TIMP-2 were used. A total of 32 rats were treated with hypothermia. The presence of each antigen was related to the following regions of interest: ischemic core with BBB breakdown (I c), surrounding ischemic tissue without BBB breakdown (I r), and the contralateral non-ischemic region (N). Regions of interest were defined by MRI. The I c increased over time at the cost of the I r. BBB breakdown occurred early in the ischemic core and increased over time. Hypothermia reduced the BBB breakdown at all time points. A graded decreased presence of laminin-5 was observed with 16.5±3.7(N)>10±2.8(I r)>4±1.4(I c) immunopositive microvessels/mm 2 at 3 h of reperfusion. MMP-9 showed a reverse pattern with 0 (N)<4±0.8(I r)<10±1.5(I c) immunopositive microvessels/mm 2. Hypothermia decreased the MMP activity measured by zymography. Laminin-5 and MMP presence relate directly to the degree of postischemic injury. Hypothermia reduces the conversion from the I r to ischemic core and the degree of BBB as well as MMP abundance.

A growth hormone secretagogue prevents ischemic-induced mortality independently of the growth hormone pathway in dogs with chronic dilated cardiomyopathy.

To determine the functional role of growth hormone (GH) secretagogue in myocardium with ischemia, left ventricular (LV) pressure gauge, wall thickness crystals, coronary occluder, pacers, and catheters were implanted in 26 dogs. Beginning 1 week after ventricular pacing (240 beats/min) was initiated, dogs were treated (s.c.) with GH releasing peptide-6 (GHRP-6, n = 8, 0.2 mg/kg/day), GH (n = 7, 0.06 mg/kg/day), or vehicle (n = 11). Two weeks of pacing was associated with similar decreases in LV pressure, rate of change of LV pressure, systolic wall thickening (WT), and an increase in left atrial pressure in all groups. Coronary artery occlusion (CAO) resulted in a similar loss of WT in ischemic regions, which did not recover during reperfusion period in all groups. WT in nonischemic regions, however, was enhanced in the GHRP-6 group compared with the GH and vehicle groups, e.g., increase of WT after 1 h of reperfusion was greater (p <0.05) in the GHRP-6 (+53 +/- 8%) than in the GH (+14 +/- 12%) or (+14 +/- 6%). There were no differences in myocardial blood flow, hemodynamics, or arrhythmic beats among all groups during CAO and reperfusion periods. Strikingly, no dogs in the GHRP-6 group died during CAO, whereas the survival rates for GH and vehicle groups were 57 and 55%, respectively. Our data demonstrate, for the first time, that chronic therapy with a GH secretagogue prevents sudden death in dogs with dilated cardiomyopathy subjected to acute ischemia. This seems to be related to an enhanced nonischemic compensatory mechanism mediated by the GH secretagogue receptors rather than via the GH/insulin growth factor-1 pathway.

Clinical biochemical tissue monitoring during ischaemia and reperfusion in major vascular surgery.

Major vascular surgery with aortic cross-clamping is associated with temporary ischaemia of the lower limb due to lack of tissue blood flow. The present study was designed to determine if the short-term changes in cellular metabolism occurring during this situation can be detected by subcutaneous microdialysis. It was also hoped to ascertain if this new technique is useful in the continuous bedside monitoring of metabolism during aortic surgery. In a controlled clinical study 20 patients undergoing elective aortic surgery were monitored using microdialysis probes that were inserted in the subcutaneous tissue of the left lower limb and a non-ischaemic region (shoulder). Interstitial fluid was obtained and the concentrations of glucose and lactate during lower limb ischaemia and during reperfusion were measured and compared with concentrations observed in fluid obtained from the non-ischaemic control tissue. Circulatory occlusion caused an immediate and significant decrease in the glucose/lactate ratio from 3.1+/-1.3 to 0.48+/-0.5 (P<0.05) that returned to preocclusion values within 2 h of commencing reperfusion. We suggest that microdialysis may be used both to assess acute changes in tissue metabolism during ischaemic periods and also to act as an additional tool for the detection of peri-operative acute variations in limb blood flow.

Analytical model for predicting mechanotransduction effects in engineered cardiac tissue.

Mechanochemical and mechanoelectrical signaling is imperative for cardiac organogenesis and underlies pathophysiological events. New techniques for engineering cardiac tissue allow unprecedented means of modeling these phenomena in vitro. However, experimental design is often hampered by a lack of models that can be adapted to the ideal conditions these methods allow. To address these deficiencies, we developed a mathematical model to calculate the distribution of stress and strain in fibrous cardiac tissue. The fluid-fiber-collagen model characterizes the mechanical behavior of cardiac tissue and is solved analytically for the distributions of stress and strain along the myocardial fibers. An example application of the model is presented: modeling the distribution of strains in the vicinity of an ischemic region. The ischemic region is stretched during systole, as has been shown in previous one-dimensional models. Our model predicts a complex distribution of stretch in the border zone surrounding the ischemic region and in nonischemic regions surrounding the border zone. These strain patterns may predict patterns of mechanochemical coupling that results in localized fibrosis, altered gene expression, or the mechanoelectrical signaling events that potentiate cardiac arrhythmias.

Therapeutic angiogenesis: a complex problem requiring a sophisticated approach.

Blood and vascular disorders underlie a plethora of pathologic conditions and are the single most frequent cause of human disease. Ischemia, involving restricted blood flow to tissues is the most common consequence of vessel dysfunction resulting in the disruption of oxygen and nutrient delivery and the accumulation of waste metabolites. Cells cannot survive extended severe ischemia but may be able to adapt to a moderate condition where diffusion to and from bordering nonischemic regions sustains vital functions. Under this condition, the secondary functions of effected cells are likely to be impaired, and a new metabolic equilibrium is established, determined by the level of cross-diffusion and degree of hypoxia. In tissues with a normally high metabolic turnover such as skeletal and cardiac muscle, even mild ischemia causes hypoxia, acidosis, and depressed function (contractility) and eventually threatens myocyte viability and organ function. Ischemic cardiac muscle is additionally vulnerable because reperfusion is essential for survival but reperfusion itself poses additional stress principally from increased production of free radicals during reoxygenation. The latter effect is called reperfusion injury and can cause as much damage as the ischemia. The treatment possibilities for ischemia-related vascular disease are limited. Lipid/cholesterol-lowering agents, diet and antiplatelet adherence (aspirin) therapy may help slow the progression of vessel disease in some instances; but surgical reconstruction may be the only option in advanced stages, and even this is not always an option. An alternative and rather obvious strategy to treat ischemia is to activate endogenous angiogenic or arteriogenic pathways to stimulate revascularization of the tissue. The feasibility of such a strategy has now been established through the results of studies over the past decade, and a new discipline called therapeutic angiogenesis has emerged. This review focuses on the application of therapeutic angiogenesis for treating ischemic muscle disease and includes a critical evaluation of the parameters and limitations of current procedures. The development of this technology has benefited from its application to both peripheral and coronary artery disease and results from both are reviewed here.

Dissociation of Stress-activated Protein Kinase (p38-MAPK and JNKs) Phosphorylation from the Protective Effect of Preconditioning in vivo

E. K. Iliodromitis, C. Gaitanaki, A. Lazou, E. Bofilis, G. K. Karavolias, I. Beis and D. Th. Kremastinos. Dissociation of Stress-activated Protein Kinase (p38-MAPK and JNKs) Phosphorylation from the Protective Effect of Preconditioning in vivo. Journal of Molecular and Cellular Cardiology (2002) 34, 1019–1028. The aim of the present study was to examine and compare the role of the stress-activated protein kinases in ischemic and stretch-induced preconditioning. A model of anesthetized rabbits was used, and the preconditioning protocol included one or three cycles of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload without or with the addition of the stretch blocker gadolinium. Infarct size was determined after 2h reperfusion and p38 MAPK and JNKs phosphorylation was determined after 20min of prolonged ischemia. Preconditioning stimuli were equally effective in reducing the infarct size (14.2±3.4%, 12.9±3.0%, 15.9±3.3%, P<0.01 vs control). The addition of the stretch channel blocker gadolinium abrogated the effect of stretch preconditioning only, without any effect on ischemic preconditioning. Comparing p38-MAPK and p46/p54 JNKs phosphorylation in the ischemic and non-ischemic regions of the heart at the time of sustained ischemia, activation was observed in the ischemic or mechanically preconditioned groups compared with the control. The addition of gadolinium abolished this activation. The above results indicate that the phosphorylation of p38-MAPK and p46/p54 JNKs is increased in preconditioning but this effect can be dissociated from the protective effect of ischemic preconditioning. Activation of the stress-activated protein kinases may be related to the increased contracture, a characteristic of ischemic preconditioning.

Mechanisms of coronary vessels' dilation in non-ischemic region during myocardial ischemia and reperfusion

Mechanisms of coronary vessels' dilation in non-ischemic region during myocardial ischemia and reperfusion

Increased cardiomyocyte apoptosis following ischemia and reperfusion in diet-induced hypercholesterolemia: relation to Bcl-2 and Bax proteins and caspase-3 activity.

It has been reported that apoptosis is a significant contributor to myocardial cell death as a result of reperfusion injury. However, whether the extent of cardiomyocyte apoptosis following ischemia and reperfusion varies in different pathophysiological backgrounds is still uncertain. In this study, we examined whether hypercholesterolemia increases the extent of myocardial reperfusion injury by aggravating cardiomyocyte apoptosis and the effects of hypercholesterolemia on the expression of Bcl-2 and Bax proteins and the activation of caspase-3. Twenty-eight male New Zealand white rabbits were fed standard chow (control, n = 14) or chow supplemented with 10% cholesterol (hypercholesterolemic, n = 14) for 8 wk. Anesthetized rabbits were then subjected to 30 min of left circumflex artery occlusion followed by 4 h of reperfusion. Apoptosis was identified as "DNA ladders" by gel electrophoresis and confirmed histologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The infarct size (% of risk region) was significantly greater in hypercholesterolemic rabbits than in controls (39 +/- 6 vs. 23 +/- 2%, P = 0.02). Very few TUNEL-positive cardiomyocytes could be identified in the nonischemic regions in both groups, consistent with an absence of DNA laddering. In contrast, TUNEL-positive cardiomyocytes were significantly displayed in the ischemic, nonnecrotic myocardium, and DNA ladder occurred in all animals. The percentage of TUNEL-positive cardiomyocytes in the ischemic nonnecrotic myocardium was significantly higher in hypercholesterolemic rabbits compared with controls (40 +/- 5 vs. 17 +/- 11%, P < 0.001). Western blot analysis showed that, in the nonischemic myocardium, hypercholesterolemic rabbits exhibited an approximately 50% increase in the expression of Bcl-2 (P < 0.05), but not Bax, than control rabbits. However, compared with controls, hypercholesterolemic rabbits exhibited a more pronounced decrease in the expression of Bcl-2 (42 +/- 4 vs. 26 +/- 2%, P < 0.01) and a similar extent of increase in the expression of Bax in the ischemic myocardium. Furthermore, hypercholesterolemic rabbits were associated with a markedly increased activation of caspase-3 within the ischemic myocardium compared to control rabbits. This study demonstrates that although hypercholesterolemia is associated with an increased myocardial Bcl-2/Bax ratio at baseline, it still significantly exacerbates cardiac reperfusion injury, not only by increasing the infarct size but also by increasing the extent of cardiomyocyte apoptosis.

Role of augmented expression of intermediate-conductance Ca2+-activated K+ channels in postischaemic heart.

1. The rat intermediate conductance calcium-activated potassium channel (ImK) was cloned from a cDNA library of vascular smooth muscle cells (VSM) in rat pulmonary artery. The ImK distributes in a variety of tissue, including VSM, endothelial cells, leucocytes and fibroblasts. The ImK has a tyrosine phosphorylation consensus site in the proximal portion of the C-terminus and motifs exist for the DNA-binding protein AP-1 in the promoter, suggesting this channel is upregulated and active in cell cycle functions. The aim of the present study was to examine the role of ImK in postischaemic cardiovascular remodelling in relation to the angiotensin AT1 receptor-mediated AP-1 signalling pathway. 2. Rats underwent left coronary artery ligation for periods between 1 day and 3 weeks. The temporal profile of expression of ImK mRNA was analysed by RNase protection assay. To test the effect of AT1 receptor blockade, candesartan (3 mg/kg per day) was administered via an osmotic mini-pump implanted in the intraperitoneal space 3 days prior to coronary occlusion. 3. ImK expression in postischaemic hearts showed a significant increase with two distinct peaks; the first peak at day 3 (2.7-fold compared with control levels; P < 0.001) and the second after 2 weeks (1.5-fold; P < 0.01). Reperfusion following 30 min of ischaemia markedly accelerated and augmented the first peak at days 1-3 (4.8-fold), but completely abolished the second peak after 1-2 weeks (0.8-fold). In situ hybridization of ImK mRNA and immunostaining of ImK protein with specific antibody revealed that this was not only the result of the increase in ImK expression in vascular cells, but also related to infiltration of mononuclear leucocytes and fibroblasts into the ischaemic region. Candesartan inhibited cardiac hypertrophy and perivascular fibrosis of coronary arterioles in the non-ischaemic region. Candesartan also abrogated both peaks in ImK expression. 4. These findings indicate that both the inflammatory reaction and the postischaemic cardiovascular remodelling promote increased expression of ImK in postischaemic hearts via the AT1 receptor-mediated AP-1 signalling pathway.

Enhanced iNOS function in myocytes one day after brief ischemic episode.

There is increasing evidence that nitric oxide (NO) produced by inducible NO synthase (iNOS) plays a key role in cadioprotection during the "second window of protection" (SWOP). The goals of this study were to determine 1) whether a transient ischemic episode [10-min coronary artery occlusion (CAO), followed by full reperfusion] enhances NOS function in cardiac myocytes, 2) which specific NOS isoform is responsible for the enhanced NOS function in myocytes, and 3) to localize iNOS expression during SWOP. To address these questions, 10 dogs were instrumented to measure aortic and left ventricular pressures and wall thickness. At 1-2 wk after recovery, myocardial ischemia was induced regionally by a 10-min left circumflex CAO. After 24-h reperfusion, cardiac myocytes were isolated from the previously ischemic and nonischemic regions (n = 6). Myocyte contractile function was assessed using a video motion detector at 1 Hz (35 +/- 2 degrees C). At baseline, myocyte contractile function (% contraction) was similar in the two regions (ischemic 7.8 +/- 0.5% vs. nonischemic 7.8 +/- 0.2%). L-Arginine (1 mM) significantly reduced (P < 0.05) myocyte contraction in the ischemic (-34 +/- 3%, P < 0.05) but not (-7 +/- 4%) nonischemic regions; these responses were abolished by N(G)-nitro-L-arginine (1 mM), a nonspecific NOS inhibitor, as well as 2-amino-5,6-dihydro-6-methy-4H-1,3,thiazine (1 mM), a specific iNOS inhibitor. Immunohistochemistry also revealed enhanced iNOS expression in the myocardium and in particular the interstitial spaces in the ischemic zone. These results indicate that a brief ischemic episode upregulates iNOS function in myocytes as well as in the interstitial space between blood vessels and myocytes, strategically where it can regulate both vascular and myocyte function during the SWOP.

Post-endsystolic active shortening in the non-ischemic region impairs left ventricular pressure fall in acute ischemic heart.

To investigate the relation between the impairment of isovolumic relaxation and the regional wall motion in acute ischemia, the left ventricular pressure fall and regional myocardial motion were examined in the relaxation phase in dogs during both acute coronary artery occlusion (n = 12) and a regional coronary flow reduction (n = 6). Fifteen to 40 seconds after complete coronary artery occlusion or in the stable state after a regional coronary flow reduction by 70 to 90% of the control state, a shortening of the non-ischemic region at the early isovolumic relaxation phase (the post-endsystolic shortening) appeared, combined with lengthening of the ischemic region. In these situations, the logarithmic plots of the left ventricular pressure fall was composed of two components (time constant of early part [Ta] and at latter part [Tb]). Ta was greater than Tb (64.3 +/- 13.8 milliseconds vs. 36.6 +/- 10.4 milliseconds at 15 seconds after coronary occlusion, p < 0.01; 67.6 +/- 22.9 milliseconds vs. 45.1 +/- 17.5 milliseconds at flow reduction, p < 0.01) and the time constant at control (p < 0.01). These findings suggested that post-endsystolic shortening in the non-ischemic region played a role in a the non-uniformity of the left ventricular contraction and contributed to the impairment of the left ventricular pressure fall in acute regional ischemia, especially in early isovolumic relaxation.