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Introduction: IMPROVE-IT trial showed that ezetimibe plus statin treatment, as compared with statin alone, decreased cardiovascular events in patients with acute coronary syndrome (ACS). However, proir studies have failed to show a beneficial effect of ezetimibe on carotid plaque progression when added to statin treatment. Hypothesis: The addition of ezetimibe to statin therapy affects coronary plaque behavior in the non-culprit vessel. Methods: We conducted a prospective, randomized open-label parallel group study with blind endpoint evaluation conducted at 10 centers in Japan. A total of 128 statin naïve patients with ACS undergoing intravascular ultrasound (IVUS) guided percutaneous coronary intervention were randomized and nonculprit coronary lesions associated with mild-to-moderate stenosis in 103 patients had evaluable IVUS examinations at baseline and at 8 to 12 months follow-up. Conventional IVUS and integrated backscatter (IB)-IVUS measurements at 1-mm intervals were calculated. Patients were randomly assigned to receive either 2mg/day of pitavastatin plus 10mg/day ezetimibe or 2mg/day of pitavastatin. Primary endpoints were the percentage change in non-culprit coronary plaque volume and percent change in lipid plaque volume. Results: Mean low density lipoprotein cholesterol was reduced from 125mg/dl to 65mg/dl in the combination therapy group receiving statin plus ezetimibe (n=50) and 126mg/dl to 87 mg/dl in the statin alone group (n=53)(between group difference of 16.9%, P Conclusions: Among patients with acute coronary syndrome, combined therapy with ezetimibe and statin did not result in a significant change in coronary plaque regression or tissue component compared with statin alone.

Background— Fibrosis and progressive failure are prominent pathophysiological features of hearts after myocardial infarction (MI). We examined the effects of inhibiting transforming growth factor-β (TGF-β) signaling on post-MI cardiac fibrosis and ventricular remodeling and function. Methods and Results— MI was induced in mice by left coronary artery ligation. An adenovirus harboring soluble TGF-β type II receptor (Ad.CAG-sTβRII), a competitive inhibitor of TGF-β, was then injected into the hindlimb muscles on day 3 after MI (control, Ad.CAG-LacZ). Post-MI survival was significantly improved among sTβRII-treated mice (96% versus control at 71%), which also showed a significant attenuation of ventricular dilatation and improved function 4 weeks after MI. At the same time, histological analysis showed reduced fibrous tissue formation. Although MI size did not differ in the 2 groups, MI thickness was greater and circumference was smaller in the sTβRII-treated group; within the infarcted area, α-smooth muscle actin–positive cells were abundant, which might have contributed to infarct contraction. Apoptosis among myofibroblasts in granulation tissue during the subacute stage (10 days after MI) was less frequent in the sTβRII-treated group, and sTβRII directly inhibited Fas-induced apoptosis in cultured myofibroblasts. Finally, treatment of MI-bearing mice with sTβRII was ineffective if started during the chronic stage (4 weeks after MI). Conclusions— Postinfarction gene therapy aimed at suppressing TGF-β signaling mitigates cardiac remodeling by affecting cardiac fibrosis and infarct tissue dynamics (apoptosis inhibition and infarct contraction). This suggests that such therapy may represent a new approach to the treatment of post-MI heart failure, applicable during the subacute stage.

Background— We investigated whether the improvement of cardiac function and remodeling after myocardial infarction (MI) by granulocyte colony–stimulating factor (G-CSF) relates to acceleration of the healing process, in addition to myocardial regeneration. Methods and Results— In a 30-minute coronary occlusion and reperfusion rabbit model, saline (S) or 10 μg · kg −1 · d −1 of human recombinant G-CSF (G) was injected subcutaneously from 1 to 5 days after MI. Smaller left ventricular (LV) dimension, increased LV ejection fraction, and thicker infarct-LV wall were seen in G at 3 months after MI. At 2, 7, and 14 days and 3 months after MI, necrotic tissue areas were 14.2±1.5/13.4±1.1, 0.4±0.1/1.8±0.5*, 0/0, and 0/0 mm 2 · slice −1 · kg −1 , granulation areas 0/0, 4.0±0.7/8.5±1.0*, 3.9±0.8/5.7±0.7,* and 0/0 mm 2 · slice −1 · kg −1 , and scar areas 0/0, 0/0, 0/0, and 4.2±0.5/7.9±0.9* mm 2 · slice −1 · kg −1 in G and S, respectively (* P Conclusions— The acceleration of the healing process and myocardial regeneration may play an important role for the beneficial effect of post-MI G-CSF treatment.

Background— Granulation tissue cells at the subacute stage of myocardial infarction (MI) are eliminated by apoptosis to finally make a scar at the chronic stage. We hypothesized that postinfarct inhibition of apoptosis might preserve myofibroblasts and endothelial cells in granulation and modulate chronic left ventricular (LV) remodeling and heart failure. Methods and Results— A pancaspase inhibitor, Boc-Asp-fmk (BAF, 10 μmol/kg per day), or vehicle (control) was given to rats with experimental large MI. The treatment was started on the third day after MI and continued until 4-week-old MI. Two weeks later, the apoptosis of granulation tissue cells was significantly reduced and conversely, the cell population was greater in BAF. Twelve weeks later, BAF showed significantly greater survival rates (84% versus 42%) with significantly smaller LV cavity, lower LV end-diastolic pressure and central venous pressure, and higher LV dP/dt, which indicated improvement of LV remodeling and dysfunction. A scar was established in old infarct of control subjects, but in BAF, the infarct wall was thicker because of greater old infarct area, which contained abundant myofibroblasts and vessels. Surprisingly, many of the α-smooth muscle actin–positive myofibroblast-like cells in BAF, making bundles and running parallel to the survived cardiomyocytes, were ultrastructurally mature smooth muscle cells with contractile phenotype. Cardiomyocyte apoptosis in the infarct area was equally rare in each group. Conclusions— The postinfarct treatment with BAF improved LV remodeling and dysfunction through inhibition of granulation tissue cell apoptosis. These findings imply a new therapeutic strategy against postinfarct heart failure.

Background — The purpose of the present study was to define whether integrated backscatter (IB) combined with conventional intravascular ultrasound (IVUS) makes tissue characterization of coronary arterial plaques possible. Methods and Results — IB-IVUS was performed in coronary arteries (total 18 segments) of 9 patients at autopsy, and the findings were compared with the histology. RF signals, which were digitized at 2 GHz in 8-bit resolution, were obtained with an IVUS system with a 40-MHz catheter. IB values of the RF signal from the region of interest (ROI) (100-μm depth, 1.4° per line) were calculated by use of a personal computer. IB values on the ROIs were divided into 5 categories, compared with each of the plaque histologies: category 1 (thrombus), −88 < IB ≤ −80; category 2 (intimal hyperplasia or lipid core), −73 < IB ≤ −63; category 3 (fibrous tissue), −63 < IB ≤ −55; category 4 (mixed lesions), −55 < IB ≤ −30; and category 5 (calcification), −30 < IB ≤ −23. On the basis of these categories, we analyzed 5120 ROIs per segment in each ring-like arterial specimen. Color-coded maps of plaques were constructed by use of these IB data and conventional IVUS data, which reflected the plaque histology of autopsied coronary arteries well. Then, the same method was undertaken in 24 segments with plaque from 12 patients in vivo with angina pectoris. Comparisons between coronary angioscopy and IB-IVUS revealed that the surface color of plaques in angioscopy reflected the thickness of the fibrous cap rather than the size of the lipid core. Conclusions — IB-IVUS represents a new and useful tool for evaluating the tissue structure of human coronary arterial plaques.

Background —It has been reported that tumor necrosis factor-α (TNF-α) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-α on viral myocarditis, however, have not been fully elucidated. Methods and Results —To investigate the role of TNF-α in the progression of viral myocarditis, we used TNF-α gene–deficient mice (TNF-α −/− ) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-α −/− mice after EMCV infection was significantly lower than that of TNF-α +/+ mice (0% versus 67% on day 14). Injection of recombinant human TNF-α (0.2 to 4.0 μg/mouse IV) improved the survival of TNF-α −/− mice in a dose-dependent manner, indicating that TNF-α is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-α −/− than in TNF-α +/+ mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-α −/− than in TNF-α +/+ mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-α −/− mice compared with TNF-α +/+ mice. Conclusions —These observations suggested that TNF-α is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF-α plays a protective role in the acute stage of viral myocarditis.

Background —The process of progression in coronary artery disease is unknown. Methods and Results —The subjects were 36 patients with 36 objective vessels with clinically significant progression of coronary artery disease (≥15% per year) in whom 4 serial coronary arteriograms (CAGs) were performed at intervals of ≈4 months in a 1-year period. The degree of progression of percent stenosis between each of 2 serial CAGs was classified as marked (M: ≥15%), slight (S: 5% to 14%), and no progression (N: P P Conclusions —Two types of stenosis progression provide a new insight into the mechanism of coronary artery disease.

Background — N -methyl-1-deoxynojirimycin (MOR-14), an α-glucosidase inhibitor, reduces the glycogenolytic rate by inhibiting the α-1,6-glucosidase of glycogen-debranching enzyme in the liver, in addition to possessing an antihyperglycemic action by blocking α-1,4-glucosidase in the intestine. Because the reduction of the glycogenolytic rate may be one of the mechanisms of myocardial protection in ischemic preconditioning, the compounds inhibiting myocardial α-1,6-glucosidase may be protective against ischemic damage. Thus, we investigated whether MOR-14 could inhibit α-1,6-glucosidase and reduce the infarct size in rabbit hearts without collateral circulation. Methods and Results —MOR-14 dose-dependently decreased the α-1,6-glucosidase activity in rabbit heart extract. A tracer study demonstrated the myocardial uptake of a considerable amount of MOR-14 sufficient to fully inhibit α-1,6-glucosidase. To assess the infarct size–reducing effect of MOR-14, 54 rabbits were subjected to 30-minute coronary occlusion followed by 48-hour reperfusion. Preischemic treatment with 25, 50, and 100 mg/kg of MOR-14 dose-dependently reduced the infarct size (to 26±4%, 19±3%, and 14±2% of the area at risk, respectively), compared with the saline control (45±5%) without altering the blood pressure or heart rate. Another 40 rabbits given 100 mg of MOR-14 or saline 10 minutes before ischemia were euthanized at 10 or 30 minutes of ischemia for biochemical analysis. MOR-14 decreased the α-1,6-glucosidase activity to ≈20% in vivo, reduced the glycogen breakdown, and attenuated the lactate accumulation at both 10 and 30 minutes of ischemia. Conclusions —Preischemic treatment with MOR-14 preserved glycogen, attenuated the accumulation of lactate, and reduced the myocardial infarct size by 69%. This cardioprotective effect was independent of changes of blood pressure and heart rate or regional blood flow. It may be associated with α-1,6-glucosidase inhibition, because MOR-14 markedly decreased the α-1,6-glucosidase activity in the heart.

BACKGROUND Brain natriuretic peptide (BNP), as a cardiac hormone, is expressed together with atrial natriuretic peptide (ANP) in the ventricles in congestive heart failure. However, the ventricular expression of BNP in hypertrophic cardiomyopathy (HCM) with normal systolic function is still unclear. METHODS AND RESULTS The study population consisted of 39 HCM patients with asymmetric septal hypertrophy and 10 control subjects without any specific cardiac disease. Eleven cases of HCM were obstructive (HOCM), and the other 28 cases were nonobstructive (HNCM). All of these patients had a normal ejection fraction. Immunohistochemical analysis of endomyocardial biopsy specimens with specific monoclonal antibodies showed BNP immunoreactivity in the HOCM group (5/10, 50%) but not in the HNCM group (0/22) or in control subjects (0/5). In HOCM, left ventricular end-diastolic pressure was significantly higher in the BNP-positive patients than the BNP-negative patients. Histological changes such as myocardial fiber disarray, hypertrophy of myocytes, and fibrosis were greater in BNP-positive patients than BNP-negative patients in HCM. However, the expression had no significant relation with other clinical parameters. The elevation of the BNP plasma level versus control subjects was marked in both HOCM (85-fold) and HNCM (23-fold). By contrast, the elevation of the ANP plasma level versus control subjects was mild in HOCM (5.7-fold) and HNCM (4.2-fold). The ratio of BNP level to ANP level was higher in HOCM (4.16) than in HNCM (1.46) and control subjects (0.28), and it was higher than the ratio previously reported for severe congestive heart failure (1.72). CONCLUSIONS These findings suggest that BNP is expressed in the ventricular myocytes of HCM with normal systolic function. In HOCM, ventricular expression of BNP may be augmented in response to both obstruction and diastolic dysfunction.

BACKGROUND It has been reported that a brief period of coronary occlusion and reperfusion slows the rate of ATP depletion during subsequent sustained ischemia as well as limiting infarct size. However, it has not yet been determined whether ischemic preconditioning also has an effect on the functional and metabolic recovery of stunned myocardium. Our study was designed to address this problem. METHODS AND RESULTS Farm pigs were anesthetized with fluothane and randomly assigned to either a control group or a preconditioned group. The control group (n = 15) underwent 15 minutes of coronary occlusion followed by 120 minutes of reperfusion. The preconditioned group (n = 14) underwent two episodes of 5-minute occlusion and 5-minute reperfusion followed by 15 minutes of occlusion and 120 minutes of reperfusion. This protocol was designed to exclude the stunning effect of the preconditioning procedure itself as much as possible besides preconditioning the heart. A pair of ultrasonic crystals was implanted in the area at risk perfused by the left anterior descending coronary artery. 31P-nuclear magnetic resonance spectroscopy and sonomicrometry were performed alternately. Regional myocardial blood flow (RMBF) was determined with colored microspheres. At 15 minutes of sustained ischemia, phosphocreatine (Pcr), ATP, and intracellular pH were significantly better preserved in the preconditioned group (Pcr: control/preconditioned, 1 +/- 1%/14 +/- 1%; ATP:control/preconditioned, 66 +/- 2%/74 +/- 2%; pH:control/preconditioned, 6.32 +/- 0.07/6.52 +/- 0.05; P < .05). After reperfusion, ATP increased progressively and was almost normalized at 120 minutes of reperfusion in the preconditioned group (control/preconditioned, 73 +/- 4%/95 +/- 3%; P < .05). Overshoot of Pcr (which indicates that the energy generating system is operating better than energy utilizing system) persisted in preconditioned hearts but disappeared rapidly in controls (control/preconditioned, 104 +/- 3%/130 +/- 3% after 120 minutes of reperfusion). There was no significant difference in percent segment shortening (%SS), RMBF, and hemodynamics between the two groups throughout the experiment (%SS: control/preconditioned, 29.8 +/- 5.9%/28.8 +/- 6.3% of baseline after 120 minutes of reperfusion). CONCLUSIONS Preconditioning improves energy metabolism during reperfusion, although it does not attenuate myocardial stunning for at least 2 hours after reperfusion.

BACKGROUND Ischemic preconditioning slows ATP depletion and ultrastructural damage during the final episode of ischemia. To define the influence of creatine phosphate (CP) and intracellular pH (pHi) on this effect, CP and pHi were serially measured in porcine hearts without collateral circulation by using 31P-NMR spectroscopy and ultrastructural examination. METHODS AND RESULTS Farm pigs weighing 12-15 kg were anesthetized with Fluothane. The control group underwent a single occlusion (20 minutes or 60 minutes); the preconditioned group underwent four episodes of 5-minute occlusion and 5-minute reperfusion followed by a sustained occlusion (20 minutes or 60 minutes). After ischemic preconditioning, CP increased to 115 +/- 11% (p less than 0.05) of preischemic value and ATP decreased to 84 +/- 8% (p less than 0.05) of preischemic value, but pHi returned to preischemic value. At 5 and 10 minutes of sustained ischemia, CP was significantly preserved in the preconditioned group (control group, 19 +/- 3% versus preconditioned group, 29 +/- 4% at 5 minutes; control group, 5 +/- 3% versus preconditioned group, 11 +/- 3% at 10 minutes; p less than 0.05). At 15 and 20 minutes of sustained ischemia, ATP was significantly preserved in the preconditioned group (control group, 64 +/- 3% versus preconditioned group, 73 +/- 3% at 15 minutes; control group, 51 +/- 7% versus preconditioned group, 62 +/- 2% at 20 minutes; p less than 0.05). At 10, 15, 20, and 25 minutes of sustained ischemia, pHi was significantly higher in the preconditioned group (control group, 6.5 +/- 0.05 versus preconditioned group, 6.7 +/- 0.1 at 10 minutes; control group, 6.3 +/- 0.05 versus preconditioned group, 6.6 +/- 0.06 at 15 minutes; control group, 6.1 +/- 0.1 versus preconditioned group, 6.4 +/- 0.1 at 20 minutes; control group, 6.0 +/- 0.2 versus preconditioned group, 6.3 +/- 0.1 at 25 minutes; p less than 0.05). Ultrastructural changes were milder in the preconditioned group at 20 minutes of sustained ischemia. CONCLUSIONS In addition to ATP and ultrastructure, preconditioning preserved CP and pHi during sustained ischemia. These protective effects might be due to overshoot phenomenon of CP and/or reduced ATP consumption. The relatively longer period of preservation of pHi, which probably is the result of reduced ATP consumption, indicates its greater contribution to reducing infarct size than that of CP and ATP.

BACKGROUND Although first isolated from the porcine brain, brain natriuretic peptide (BNP) is also found in the heart, particularly in the atria. METHODS AND RESULTS We examined the immunocytochemical localization of BNP in relation to atrial natriuretic peptide (ANP) in five porcine atria using highly specific monoclonal antibodies. With the use of an indirect immunoperoxidase method, serial sections examined by light microscopy showed foci of multihormonal myocytes containing both ANP and BNP localized in the subendocardium. Monohormonal myocytes containing ANP only were observed in the subepicardium and part of the subendocardium. The staining intensity of ANP and BNP showed a transmural gradient from the subendocardium to the subepicardium. At the electron microscopic level, double immunocytochemistry using a two-face immunogold-staining method revealed two types of granules: Type 1 is a monohormonal granule containing ANP alone, and type 2 is a multihormonal granule containing both ANP and BNP. Although most atrial myocytes contained both, type 1 granules were frequently observed in the epicardial side, and type 2 granules were frequently observed in the endocardial side. We observed a few type 2 granules even in the light microscopically monohormonal myocytes. CONCLUSIONS These data suggest that the atrial myocyte population comprises single, multipotential cells able to synthesize both natriuretic peptides in varying proportions. The atrial transmural gradient of BNP and ANP may be related to responses to wall stress.

Background: Acarbose, an anti-diabetic drug, is an α– glucosidase inhibitor that can inhibit glucose absorption in the intestine. A recent large-scale clinical trial, STOP-NIDDM, showed that acarbose reduces the risk of myocardial infarction. We examined whether acarbose reduces myocardial infarct size and investigated its mechanisms. Methods and Results: Rabbits were fed with one of four diets in this study: normal chow, 30 mg acarbose/100 g chow, 8 g sucrose/100 g chow, or 30 mg acarbose plus 8 g sucrose/100 g chow for 7 days. Rabbits were assigned randomly to one of eight groups: control (n=10), acarbose (n=10), sucrose (n=10), acarbose+sucrose (n=10), acarbose+i.v. 5-hydroxydecanoate (5HD, a mitochondrial KATP channel blocker, 5 mg/kg) (n=10), 5HD (i.v. 5 mg/kg, n=10), acarbose+exendin(9 –39) (n=10, a Glucagon-Like Peptide-1(GLP-1) receptor blocker, 3 nmol/l) (45.4±1.2%) and exendin(9 –39)(n=10). Rabbits then underwent 30 min of coronary occlusion followed by 48 h reperfusion. Postprandial blood glucose levels were highest in the sucrose group and higher in the control groups, but decreased in the acarbose and sucrose+acarbose groups. The infarct size as a percentage of the left ventricular area at risk was reduced significantly in the acarbose (19.4±2.3%) and sucrose+acarbose groups (19.0±5.6%) as compared with the sucrose (47.4±4.2%) and control groups (42.8±5.4%). The infarct size-reducing effect of acarbose was abolished by 5HD (43.4±4.7%) and exendin(9 –39) (45.4±1.2%). The 5HD and exendin(9 –39) by itself did not affect the infarct size. Myocardial interstitial 2,5-DHBA levels, an indicator of hydroxyl radicals, increased during reperfusion after 30 min of ischemia but this increase was inhibited in the acarbose group. Conclusions: Acarbose reduces myocardial infarct size via stimulation of GLP-1 receptors, opening mitochondrial KATP channels, and preventing hydroxyl radical production.

Background: It is well known that coronary plaques that show backward attenuation of ultrasound signal (AT) often cause distal flow disturbance during percutaneous coronary intervention (PCI). However, tissue characteristics of such plaques have not been precisely investigated. We previously developed an Integrated Backscatter Intravascular Ultrasound (IB-IVUS) for tissue characterization of coronary plaques. The aim of this study is to clarify the relationship among tissue components of coronary plaques, AT and distal embolization after stent deployment by use of IB-IVUS. Methods: In the pathological study, images were acquired from 221 segments of 18 coronary arteries from 13 cadavers obtained at autopsy. Twenty-three segments that showed AT (10%) were enrolled in this study. A total of 47 regions of interest (ROI) on histological images were compared with IB-IVUS color coded maps. Micro calcifications and cholesterol clefts were evaluated by von Kossa, Masson Trichrome and Hematoxylin-Eosin staining. Percentage of cholesterol clefts area (area of cholesterol cleft/area of ROI) and micro calcification area were calculated by a personal computer. In the clinical study, fourteen lesions which underwent elective stent deployment were enrolled. We evaluated the degrees of distal embolization by measuring serum troponin I (TnI) level before and after PCI and compared with IB-IVUS parameters. We defined the area of AT as the area where IB value is -6dB less than lipid pool. Results: In the pathological study, the attenuation of IB values of cholesterol cleft and micro calcification were3.58 ± 1.56 dB/0.1 mm and 3.51 ± 1.74 dB/0.1 mm, respectively. As the % cholesterol cleft area and % micro calcification area increased, the attenuation of IB values increased (r = 0.59 and 0.62, P < 0.001 and 0.001, respectively). Elevation of serum TnI have strong correlation to AT volume at the target lesions compared to the % lipid volume (lipid volume/plaque volume) (r = 0.92 and 0.56, P < 0.001 and 0.001, respectively). Conclusion: This is the first demonstration of the relationship among the attenuation of IB values, tissue component and distal embolizaion after PCI. This study provides new clinical insights to prevent and predict distal flow disturbance during PCI.

Background: We developed an Integrated Backscatter Intravascular Ultrasound (IB-IVUS) for tissue characterization of coronary plaques. The relationship between the coronary artery distensibility and tissue components has not been investigated. Methods: Fourty-seven plaques with moderate stenosis (30 to 50%) in 36 patients were evaluated by three-dimensional (3D) IB-IVUS at every one millimeter. Plaque distensibility, lumen distensibility, vessel area, lumen area (LA) and plaque area (PA) were measured. Plaque distensibility was defined as follows: [In (systolic BP/diastolic BP)]/[(diastolic PA - systolic PA)/diastolic PA]. Lumen distensibility was defined as follows: [In (systolic BP/diastolic BP)]/[(diastolic LA -systolic LA)/diastolic LA]. % lipid area (lipid area/plaque area) were automatically calculated by IB-IVUS. Results: As the % lipid area increased, the lumen distensibility also increased (r = 0.32, p = 0.040). There was a tendency that the plaque distensibility increased as the % lipid area increased (r = 0.31, p = 0.056). 3D plaque distensibility map showed the difference in the plaque distensibility of each segment well. Conclusion: This is the first clinical demonstration of the relationship between the % lipid area and plaque distensibility of coronary arteries. The plaque area with greater % lipid area were more likely to increase from systolic to diastolic phase. 3D IB-IVUS color-coded mapping is useful for the evaluation of tissue characteristics and dynamics of coronary plaques.

Background— Doxorubicin is a highly effective antineoplastic drug, but its clinical use is limited by its adverse side effects on the heart. We investigated possible protective effects of erythropoietin against doxorubicin-induced cardiomyopathy. Methods and Results— Cardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15 mg/kg). In some cases, human recombinant erythropoietin (5000 U/kg) was started simultaneously. Two weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by erythropoietin treatment. Erythropoietin also protected hearts against doxorubicin-induced cardiomyocyte atrophy and degeneration, myocardial fibrosis, inflammatory cell infiltration, and downregulation of expression of GATA-4 and 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin. Cyclooxygenase-2 expression was upregulated in doxorubicin-treated hearts, and that, too, was attenuated by erythropoietin. No doxorubicin-induced apoptotic effects were seen, nor were any changes seen in the expression of tumor necrosis factor-α or transforming growth factor-β1. Antiatrophic and GATA-4 restoring effects of erythropoietin were demonstrated in the in vitro experiments with cultured cardiomyocytes exposed to doxorubicin, which indicated the direct cardioprotective effects of erythropoietin beyond erythropoiesis. Cardiac erythropoietin receptor expression was downregulated in doxorubicin-induced cardiomyopathy but was restored by erythropoietin. Among the downstream mediators of erythropoietin receptor signaling, activation of extracellular signal-regulated kinase was reduced by doxorubicin but restored by erythropoietin. By contrast, erythropoietin was ineffective when administered after cardiac dysfunction was established in the chronic stage. Conclusions— The present study indicates a protective effect of erythropoietin against doxorubicin-induced cardiomyopathy.

Background— Hepatocyte growth factor (HGF) is implicated in tissue regeneration, angiogenesis, and antiapoptosis. However, its chronic effects are undetermined on postinfarction left ventricular (LV) remodeling and heart failure. Methods and Results— In mice, on day 3 after myocardial infarction (MI), adenovirus encoding human HGF (Ad.CAG-HGF) was injected into the hindlimb muscles (n=13). As a control (n=15), LacZ gene was used. A persistent increase in plasma human HGF was confirmed in the treated mice: 1.0±0.2 ng/mL 4 weeks later. At 4 weeks after MI, the HGF-treated mice showed improved LV remodeling and dysfunction compared with controls, as indicated by the smaller LV cavity and heart/body weight ratio, greater % fractional shortening and LV ±dP/dt, and lower LV end-diastolic pressure. The cardiomyocytes near MI, including the papillary muscles and trabeculae, were greatly hypertrophied in the treated mice. The old infarct size was similar between the groups, but the infarct wall was thicker in the treated mice, where the density of noncardiomyocyte cells, including vessels, was greater. Fibrosis of the ventricular wall was significantly reduced in them. Examination of 10-day-old MI revealed no proliferation or apoptosis but showed augmented expression of c-Met/HGF receptor in cardiomyocytes near MI, whereas a greater proliferating activity and smaller apoptotic rate of granulation tissue cells in the HGF-treated hearts was observed compared with controls. Conclusions— Postinfarction HGF gene therapy improved LV remodeling and dysfunction through hypertrophy of cardiomyocytes, infarct wall thickening, preservation of vessels, and antifibrosis. These findings imply a novel therapeutic approach against postinfarction heart failure.

Background — Sensitivity to apoptotic signals, the clearance rate of apoptosis, and the apoptotic ultrastructure have not been studied in cells of the in vivo adult heart. Methods and Results — To minimize the systemic influence, soluble Fas ligand was injected directly into in vivo rat hearts and livers (as the control) at concentrations of 0, 0.5, 2, and 5 μg/mL (groups C, F0.5, F2, and F5). Apoptotic cardiomyocytes and apoptotic noncardiomyocytes of the heart were identified with similar incidences only in F5. Their incidences peaked at 12 hours after injection (2.0±0.09% in cardiomyocytes) and diminished markedly 24 hours later. Caspase-3 was activated only in F5. Boc-Asp-fmk, a pancaspase inhibitor, inhibited apoptosis, suggesting that the apoptosis sensitivity was regulated upstream of caspase-3. Apoptotic noncardiomyocytes showed typical ultrastructure. In addition to the typical ultrastructure, such as cellular shrinkage, chromatin condensation, and apoptotic bodies, however, apoptotic cardiomyocytes showed unique features: doughnut-like, but not half-moon– or crescent-like, chromatin condensation; frequent plasma membrane rupture even during the early stage; condensed mitochondria with wrinkled cristae inside; the appearance of cytoplasmic lipid-like droplets; and myofibrillar derangement. In the livers, typical apoptosis was induced in hepatocytes and nonhepatocytes of the liver even in the F0.5 group, which were cleared 24 hours later. Conclusions — Compared with liver cells, cardiomyocytes as well as noncardiomyocytes of the heart are more resistant against the apoptotic signal, but the clearance is similarly rapid (within 24 hours). The ultrastructure of apoptotic cardiomyocytes is unique. These findings provide new insights into the dynamics of cell death in the heart.

Background — Collateral fractional flow reserve (FFR coll ) is an index to quantify collateral blood flow, derived from coronary pressure measurements. Although well defined theoretically, its direct validation by myocardial perfusion imaging has not been established so far. Validating this index by myocardial perfusion imaging is the main aim of this study. Methods and Results — Twenty-four consecutive patients with stable angina and single left anterior descending artery stenosis underwent simultaneous measurement of aortic pressure (P a ), coronary wedge pressure (P w ), and central venous pressure (P v ) during balloon inflation. FFR coll was calculated and compared with the extent and severity of the defect during coronary occlusion using 99m Tc-sestamibi imaging at balloon inflation of the respective coronary artery. Although the pressure-derived collateral indexes (P w , P w /P a , and FFR coll ) ranged widely, they were closely correlated with extent and severity scores of the nuclear occlusion images and superior to the ECG for that purpose. Of all parameters, FFR coll correlated best with the severity score at imaging ( r =−0.88), followed by the P w /P a ratio ( r =−0.74) or P w alone ( r =−0.69). Conclusions — FFR coll , calculated from coronary pressure during balloon occlusion, is highly correlated with the extent and severity of the defect at myocardial perfusion of the territory of the occluded artery and can be used for quantitative assessment of collateral blood flow in conscious humans.

Background —It has been thought that the thrombi and bleeding in plaques that occur after plaque rupture or endothelial damage from vessels with mild stenosis suddenly occlude the lumen and cause acute myocardial infarction (AMI). However, our hypothesis is that thrombi and bleeding may not suddenly occlude the lumen. Methods and Results —The study group consisted of 20 patients who had coronary angiograms performed within 1 week (3±3 days) before AMI and 20 control patients who had coronary angiograms performed 6 to 18 months (282±49 days) before AMI. The features of infarct-related coronary segments (IRCS) at 3 days before AMI were the presence of a significant stenosis of >50% (95% in incidence and 71±12% diameter stenosis) and Ambrose’s type II eccentric lesions (plus multiple irregularities), an indicator of plaque rupture and/or thrombi (60% [70%]), and the features at 1 year before AMI were mild stenosis of Conclusions —The appearance of marked progression and Ambrose’s type II eccentric lesion on coronary angiograms 3 days before AMI suggests the presence of a considerable time from the onset of plaque rupture and/or thrombi until the onset of AMI. These features may be predictors of AMI. The concept provides new insight into the mechanism and prevention of human AMIs.

Background —The presence of apoptotic myocytes has been reported in human hearts with dilated cardiomyopathy (DCM) on the basis of a positive finding of DNA in situ nick end-labeling (TUNEL). However, ultrastructural evidence of myocyte apoptosis has not been obtained. Methods and Results —A total of 80 endomyocardial biopsies were obtained from right and left ventricles of 20 patients with DCM and 20 normal control subjects. TUNEL-positive myocytes were found by light microscope in 15% of DCM specimens (controls, 0%, P Taq polymerase–based DNA in situ ligation assay, which detects double-stranded DNA fragments more specifically than TUNEL, did not detect a positive reaction in any case. In mirror sections, all of the TUNEL-positive myocytes in DCM simultaneously expressed proliferating cell nuclear antigen, which is required for both DNA replication and repair, but Ki-67, a replication-associated antigen, was completely negative in all cases, which appeared to rule out cell proliferation activity. Conclusions —Most of the TUNEL-positive myocytes in hearts with DCM are not apoptotic but rather living cells with increasing activity of DNA repair.

Background —Modes of cell death have been defined morphologically as apoptosis and oncosis. Infarcted myocytes have been reported to show apoptosis, as revealed by DNA fragmentation by DNA ladder and by in situ terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) at the light microscopic level. We investigated whether TUNEL-positive infarcted myocytes have apoptotic or oncotic ultrastructures by using electron microscopic TUNEL, which can simultaneously observe the ultrastructure and DNA fragmentation of the same myocytes. Methods and Results —Thirty rabbits were divided into 5 groups (n=6 each) that were subjected to a sham operation or to 30-minute ischemia followed by 0-minute, 30-minute, 2-hour, or 4-hour reperfusion of a coronary artery. In the 2- and 4-hour reperfusion groups only, DNA electrophoresis showed a ladder pattern, and the light microscopic TUNEL finding was positive in the nuclei of myocytes localized in the infarcted area (6±2% and 11±3%, respectively). Electron microscopic TUNEL showed that nuclei with a significant accumulation of immunogold particles (indicating an electronic microscopic TUNEL–positive result) were observed only in the infarcted myocytes with irreversibly oncotic ultrastructures that were found in the hearts of the 2- and 4-hour reperfusion groups (41±3% and 83±4%, respectively). Irreversibly oncotic myocytes (indicated by swelling, inhomogeneously clumped chromatin in nuclei, dense bodies in mitochondria, and/or ruptured plasma membranes) were also seen in the 0- and 30-minute reperfusion groups, which did not exhibit TUNEL-positive myocytes. There was no evidence of apoptotic ultrastructures in the myocytes. Conclusions —DNA fragmentation occurs in the myocytes that had already shown irreversibly oncotic, but not apoptotic, ultrastructures with ischemia and/or reperfusion. Therefore, DNA fragmentation itself does not always mean apoptosis, and so-called apoptotic infarcted myocytes may belong to a category of cell death other than apoptosis.

Background The effects of angiotensin II (Ang II) on ATP-sensitive K + channels (K ATP ) were investigated in ventricular myocytes enzymatically isolated from adult guinea pig heart. Methods and Results In the whole-cell and cell-attached configurations (including open-cell-attached mode) of the patch-clamp technique, K ATP currents ( I KATP ) were activated through metabolic poisoning by the use of inhibitors of both glycolytic and oxidative ATP productions at 37°C. In the whole-cell mode, I KATP were reversibly suppressed by increasing extracellular glucose and Ang II (1 nmol/L). In the cell-attached mode, Ang II concentration-dependently inhibited single K ATP activities with an IC 50 value of 3.2±0.5 pmol/L (Hill coefficient=1.3±0.3). CV11974 (100 nmol/L), an angiotensin 1 (AT 1 ) receptor-selective antagonist, blocked the inhibitory action of Ang II. Preincubation of myocytes with pertussis toxin (5 μg/mL for >120 min at 37°C) virtually prevented subsequent Ang II action. The inhibitory effect of Ang II was also abolished in the open-cell–attached mode (achieved by a prior perfusion of streptolysin- O , 0.08 U/mL). In this mode, through tiny membrane holes, the intracellular ATP concentration can be controlled by bathing extracellular solutions containing a known ATP concentration. Conclusions The inhibitory actions of Ang II on K ATP appear to be mediated by an increase in the subsarcolemmal ATP concentration that results from the inhibition of adenylate cyclase activities via AT 1 receptors/PTX-sensitive G proteins.

Background In general, myocyte death in myocardial infarctions (MIs) is attributed to necrosis, but recently the involvement of apoptosis has been suggested. The ratio of bcl-2 protein, an inhibitor of apoptosis, to Bax protein, an inducer of apoptosis, determines survival or death after an apoptotic stimulus. We speculated that bcl-2 or Bax expression is induced by ischemia and that it may be related to myocyte death in human hearts. Methods and Results We studied immunohistochemically 37 autopsied human hearts (acute MI, n=15; old MI, n=12; normal hearts as a control, n=10) with the use of bcl-2 and Bax antibodies. There were no myocytes with positive bcl-2 immunoreactivity in the controls or hearts with old MI. However, myocytes with positive bcl-2 immunoreactivity were seen in 9 of 15 hearts (60%) with acute MI, in that it was localized only in salvaged areas surrounding the infarcted tissues. Myocytes with slightly positive Bax immunoreactivity were observed in the control hearts. In the salvaged myocytes surrounding the infarcted tissues, Bax was overexpressed in 2 of 15 hearts (13%) with acute MI but in 10 of 12 hearts (83%) with old MI. Conclusions bcl-2 protein is induced in salvaged myocytes at the acute stage of infarction, but Bax protein is overexpressed at the old stage. The expression of bcl-2 and the overexpression of Bax may play an important pathophysiological role in the protection or acceleration of the apoptosis of human myocytes after ischemia and/or reperfusion.

To investigate the ventricular expression of atrial natriuretic peptide (ANP) in human hypertrophic hearts, we conducted an immunohistochemical study of 130 endomyocardial biopsy specimens obtained from the right side of the ventricular septum (RVB), left ventricular free wall (LVB), or both from a total of 80 patients: 44 patients with hypertrophic cardiomyopathy (HCM), 14 with apical hypertrophic cardiomyopathy (APH), 13 with hypertensive hearts (HHD), and nine without hypertrophy (controls). No patients had apparent congestive heart failure. ANP was not seen in ventricular myocytes in controls but was identified in biopsy specimens of hypertrophic hearts, and its distribution was characteristic in each hypertrophic group: 15 RVB (37%) and two LVB (7%) of the HCM group, one RVB (7%) and two LVB (18%) of the APH group, and zero RVB (0%) and five LVB (46%) of the HHD group. Clinical data (including echocardiographic, hemodynamic, and angiographic data) were not directly related to ventricular ANP expression in HCM, APH, or HHD with one exception. In HHD patients, LVB specimens with ANP showed greater ventricular wall thickness than LVB specimens without ANP. According to histological data, however, the ANP-present RVB specimens of HCM or ANP-present LVB specimens of HHD had greater myocyte size than did the ANP-absent specimens. In addition, in HCM patients, the ANP-present RVB specimens showed more severe fibrosis and myofiber disarray than did the ANP-absent specimens. We conclude that a failing state and hemodynamic overload are not likely to be indispensable for ANP expression in human hypertrophic ventricles and that ventricular ANP expression occurs as a response to disease-specific changes: hemodynamic overload in HHD and histological changes such as myocardial fiber disarray, hypertrophy of myocytes, and fibrosis in HCM, which may reflect the characteristic distribution of intraventricular ANP.

To investigate the mechanism of expression of atrial natriuretic polypeptide (ANP) in human ventricles, we conducted an immunohistochemical study of ANP in biventricular endomyocardial biopsy specimens obtained from a total of 49 patients with cardiac dilatation due to dilated cardiomyopathy (21 patients), postmyocarditis (18 patients), or volume overload (five patients) and subjects with no dilatation as controls (five patients). Four-micron thick sections were stained by an indirect immunoperoxidase method using monoclonal antibody to alpha-human ANP as the primary antibody. The frequency of ANP-present myocytes was calculated in each specimen and compared with clinical, echocardiographic, hemodynamic, angiographic, and histologic parameters. ANP-present myocytes were noted in all of the 21 patients with dilated cardiomyopathy, in 11 of the 18 patients with postmyocarditis, in four of the five patients with volume overload, and in zero of the five controls. The mean percentage of ANP-present myocytes was significantly greater in the left-side specimens (35 +/- 37%) than in the right-side ones (2 +/- 4%). The percentage of ANP-present myocytes in the left-side specimens significantly correlated with peak systolic or end-diastolic wall stress (r = 0.67 and 0.58), left ventricular end-systolic or end-diastolic volume index (r = 0.75 and 0.69), or left ventricular end-diastolic pressure (r = 0.42) and inversely correlated with ejection fraction (r = -0.73), systolic left ventricular wall thickness (r = -0.58), or cardiac index (r = -0.30). Expression of ANP was rarely seen in the cases with normal wall stresses, normal ejection fraction, normal volume, or normal myocyte size. However, it was seen frequently even in hearts with normal levels of left ventricular end-diastolic pressure and cardiac index (compensated hearts). The percent of ANP-present myocytes in both sides significantly correlated with size of myocytes (r = 0.48 at right and r = 0.57 at left side) or degree of fibrosis (r = 0.45 at right and r = 0.48 at left side). These results suggest that ANP expression is augmented in the dilated ventricles regardless of the causes of dilatation and that the augmentation is a compensatory mechanism as prevention against decompensation responding to reduced contractility, excess of wall stresses, or both, concomitantly occurring with cardiac dilatation and myocardial hypertrophy.

We compared the histologic picture of myocardial fiber disarray in thin (4 mu) and thick (25 mu) sections of tissues obtained at autopsy from 18 adults and eight infants with clinically normal hearts, from 10 hearts with concentric hypertrophy (hypertensive patients), nine with myocardial infarction and four with hypertrophic cardiomyopathy (HCM). In the thick sections, so-called bizarre myocardial fiber disarray in thin sections was seen as a bizarre fascicle disarray. Therefore, the usual fiber disarray reported in cases of HCM is actually a fascicle disarray with a three-dimensional complex network. There was no marked difference in distribution and frequency of fascicle disarray among normal adult and infant hearts, and diseased hearts with hypertension and myocardial infarction. In the four hearts with HCM, diffuse bizarre fascicle disarray in the thick section was detected in the septum and anterior and posterior walls of the left ventricles in all cases, and in the lateral walls in one case. In the portions without the diffuse fascicle disarray, the distribution of disarray was the same as that in hearts with no HCM. Such fascicle disarray, including that of HCM, is probably congenital.

To assess the importance of contraction band necrosis (CBN) in patients with acute myocardial infarction (AMI) treated with selective intracoronary thrombolysis, CBN, coagulation necrosis, and infarct size (expressed as CBN + coagulation necrosis) were analyzed quantitatively in 16 autopsied hearts. Intracoronary thrombolysis was performed from 2 to 6 hr after the onset of AMI, and the time from the onset of AMI to death was 7 to 168 hr. Cineangiography revealed no evidence of good collateral circulation in any of the patients. The 16 patients were classified into three groups: six patients with successful thrombolysis (100% to 99% stenosis, group I), five patients with unsuccessful thrombolysis (100% to 100%, group II), and five patients with 99% stenosis before thrombolysis (group III). Among the three groups, there were no significant differences in the time from the onset of AMI to thrombolysis, the time from the onset of AMI to death, the cause of death, or the degree of collateral circulation. The percentage of the risk area involved by the infarct in group I (82 +/- 6%) was similar to that in group II (80 +/- 11%). Infarct size was not reduced in group I because collateral circulation was not good and because the degree of recanalization after thrombolysis was 1%. However, the percentage of the infarct area with CBN was significantly higher in group I (20 +/- 9%) then in group II (3 +/- 3%). This finding shows that diffuse CBN occurred after reperfusion in patients with AMI treated with thrombolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemorrhagic acute myocardial infarction (AMI) was studied after selective intracoronary thrombolysis (SICT) in 30 patients undergoing autopsy. Urokinase, 240,000 to 1,200,000 U, was selectively injected into the infarct-related coronary artery at 2 to 9 hr (4 +/- 2 hr) after the onset of AMI. The infarct-related coronary artery showed complete occlusion in 21, 99% stenosis in eight, and 90% stenosis in one patient before SICT. After SICT, complete occlusion was seen in only five, 99% stenosis in 22, and 90% stenosis in three patients. Twenty-eight patients had transmural infarction and the other two had subendocardial infarction. Macroscopically and microscopically, the degree of hemorrhage was classified as no, slight, moderate, or marked bleeding and the hemorrhagic infarction was defined as moderate or marked diffuse bleeding in the infarct area. According to the interval from SICT to death, patients were also classified into stage I (early acute stage, 1 to 4 hr after SICT and 4 to 13 hr after the onset of AMI; n = 7), stage II (late acute stage, 9 hr to 11 days after SICT and 15 hr to 11 days after the onset of AMI; n = 18), or stage III (old infarction stage, over 17 days after AMI and SICT; n = 5). There were no significant differences with respect to the frequency of recanalization, the time from the onset of AMI to SICT, the dose of urokinase, or other clinical parameters among patients at the three stages. Only the hearts of patients in stage II showed hemorrhagic infarction, and it was found in 15 of 18 of these hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial fiber diameters were measured to determine their distribution throughout the ventricular wall in normal adult hearts, hypertensive hearts and hearts with hypertrophic cardiomyopathy (HCM). In normal adult hearts and hypertensive hearts, the diameter decreased from the inner to the outer third of the left ventricular free wall and from the left ventricular side to the right ventricular side of the septum. In HCM, these regional differences were preserved in the left ventricular free wall, but not in the septum. The diameter was greatest in the middle third of the septum, where myocardial fiber disarray was widely distributed. The diameters of the fibers in the right ventricular side of the septum were significantly larger than those of the fibers in the left ventricular side of the septum in HCM. This finding, in contrast to that in normal adult hearts or hypertensive hearts, was considered to be related to the inward convex curvature of the left ventricular chamber. Although there was no significant difference in the diameter of myocardial fibers in the left ventricular free wall between hypertensive hearts and hearts with HCM, the diameters of those in the right ventricular free wall, in the right ventricular side of the septum and in the middle third of the septum were significantly larger in HCM than in hypertensive hearts. We conclude that there is a transmural variation of myocardial fiber diameter in the left ventricular free wall and the ventricular septum, and such transmural variation in HCM is clearly different from that in hypertensive hearts.

To clarify the pathophysiologic role of intramyocardial small artery (IMSA) diseases in hypertrophied hearts, narrowings of the IMSA were quantitatively evaluated in 39 autopsied hearts, 10 from patients with typical hypertrophic cardiomyopathy (HCM), four from patients with HCM showing features mimicking dilated cardiomyopathy (DCM-like HCM), 10 from patients with hypertension, and 15 from normal adults. The relations of narrowings of the IMSA to myocytic hypertrophy, myocardial fiber disarray, and fibrosis were also examined. The external caliber and the ratio of the luminal area to the total vascular area (percent luminal area, % lumen) were calculated by an image analyzer in 85 to 203 IMSAs from each patient. The external calibers of the IMSAs were similar among groups of hearts with HCM, hypertensive hearts, and normal hearts but were greater in those with DCM-like HCM. The mean % lumen of the IMSAs was similarly reduced in the hearts with HCM (29 +/- 5% in the ventricular septum and 31 +/- 5% in the left ventricular free wall) and in hypertensive hearts (30 +/- 8% and 31 +/- 7%) compared with that in normal hearts (40 +/- 5% and 38 +/- 5%) and was the lowest in the ventricular septum of hearts with DCM-like HCM (17 +/- 3%). The mean % lumen of the IMSA was inversely correlated with heart weight (r = -.59), the mean size of myocytes (r = -.66 in the ventricular septum, r = -.63 in the free wall), and percent fibrotic area in the septum (r = -.68). The mean % lumen values of the IMSAs in the tissues with and without disarray in the hearts with HCM were similar. Thus IMSA disease is of pathophysiologic importance in patients with HCM, DCM-like HCM in particular, or with hypertension.

Coronary arteries in six children who had Kawasaki disease but lacked coronary arterial aneurysms were examined. Four children died of myocarditis at the acute stage, and two children died of bacterial sepsis or as a result of an occurrence during cineangiography at the healed stage. Twenty-one children without Kawasaki disease were examined as controls. The six children with Kawasaki disease had no thrombi, recanalization, or stenosis greater than 50% in the major coronary arteries. Three patients had dilatation of the major coronary arteries at the acute stage. Two of the three patients died during the acute stage, and autopsy showed slight dilatation of coronary arteries and abnormal intimal thickening due to panvasculitis. In the third child, who died at the healed stage, dilatation of the coronary arteries detected by two-dimensional echocardiography at the acute stage had disappeared at the healed stage. No dilatation of the major coronary arteries was seen at autopsy. However, abnormal fibrous intimal thickening of the major coronary arteries without inflammatory changes was found. The other three patients had no dilatation of the major coronary arteries at the acute stage. Two patients died at the acute stage, and slight inflammation without abnormal intimal thickening was seen in the intima and the adventitial area. In the third patient, who died during the healed stage, two-dimensional echocardiography revealed no dilatation during the clinical course, and there was no inflammatory changes or abnormal intimal thickening at autopsy.(ABSTRACT TRUNCATED AT 250 WORDS)

To assess the importance of contraction band necrosis (CBN) in reperfusion, CBN, coagulation necrosis (CN), and infarct size, expressed as CBN + CN, were quantitatively analyzed in 25 porcine hearts without collateral circulation. The left anterior descending coronary artery was ligated for 20, 30, 60, and 120 min and then reperfused for 8 hr (groups 1 to 4, respectively). Five hearts were not reperfused (group 5). The areas of CBN and CN were traced at a magnification of X 100 under an inverted microscope and quantified with use of an image analyzer. There was no change in hemodynamics with either occlusion or reperfusion. Regional myocardial blood flow, measured by the generated hydrogen gas clearance method, decreased to almost zero after occlusion but recovered during reperfusion. Percent of risk area infarcted in groups 1 to 4 was 0 +/- 0%, 11 +/- 7%, 80 +/- 9%, and 96 +/- 2%, respectively, and the percent of risk area infarcted in group 4 was the same as that in hearts subjected to permanent occlusion (95 +/- 3%). The percent area of CBN was 100 +/- 0% in group 2, 68 +/- 11% in group 3, 2 +/- 1% in group 4, and 2 +/- 2% in group 5. In group 3, the inner third of the ischemic left ventricular wall showed CN and the middle and outer third CBN. These findings show that in pig hearts without collateral circulation, the transmural infarct, two-thirds of which is occupied by CBN, is evident even when reperfusion is achieved after 1 hr occlusion. Therefore, protection against CBN might reduce infarct size after reperfusion.