Pretreatment with simvastatin attenuates myocardial dysfunction after ischemia and chronic reperfusion.

We have previously demonstrated that simvastatin attenuates myocardial cell necrosis after acute myocardial ischemia and reperfusion via induction of endothelial cell NO synthase. However, it remains unknown whether the cardioprotective effects of statins can persist after extended periods of reperfusion. Furthermore, it is unknown whether simvastatin therapy can attenuate postischemic cardiac dysfunction. Pretreatment with simvastatin attenuated myocardial injury after 30 minutes of myocardial ischemia and 24 hours of reperfusion. However, the protective effects are not recognized unless simvastatin is given at least 3 hours before myocardial ischemia. Subsequently, we pretreated mice with vehicle or simvastatin and subjected the mice to 30 minutes of myocardial ischemia and 6 months of reperfusion. Myocardial infarct size (percentage of left ventricle) was significantly reduced by 51% in the simvastatin-treated group compared with the vehicle-treated group. Left ventricular diastolic and systolic dilatation was significantly (P<0.05) reduced in simvastatin-treated mice compared with vehicle-treated mice. Additionally, the decrement in fractional shortening after 6 months of reperfusion was minimized in simvastatin-treated mice (P=NS versus baseline) compared with vehicle-treated mice (P<0.05 versus baseline). Left ventricular end-diastolic pressure was significantly (P<0.01) elevated in vehicle-treated mice (21+/-4 mm Hg) but not simvastatin-treated mice (5+/-2 mm Hg) compared with baseline values. These data demonstrate that simvastatin treatment before myocardial ischemia attenuates infarct size and preserves myocardial function after chronic reperfusion in mice.

Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function.

Up-regulation of proapoptotic genes has been reported in heart failure and myocardial infarction. To determine whether caspase genes can affect cardiac function, a transgenic mouse was generated. Cardiac tissue-specific overexpression of the proapoptotic gene Caspase3 was induced by using the rat promoter of alpha-myosin heavy chain, a model that may represent a unique tool for investigating new molecules and antiapoptotic therapeutic strategies. Cardiac-specific Caspase3 expression induced transient depression of cardiac function and abnormal nuclear and myofibrillar ultrastructural damage. When subjected to myocardial ischemia-reperfusion injury, Caspase3 transgenic mice showed increased infarct size and a pronounced susceptibility to die. In this report, we document an unexpected property of the proapoptotic gene caspase3 on cardiac contractility. Despite inducing ultrastructural damage, Caspase3 does not trigger a full apoptotic response in the cardiomyocyte. We also implicate Caspase3 in determining myocardial infarct size after ischemia-reperfusion injury, because its cardiomyocyte-specific overexpression increases infarct size.

Cardioprotective actions of endogenous IL-10 are independent of iNOS.

Myocardial ischemia-reperfusion (I/R) is a well-known stimulus for acute inflammatory responses that promote cell death and impair pump function. Interleukin-10 (IL-10) is an endogenous, potent anti-inflammatory cytokine. Recently, it has been proposed that IL-10 inhibits inducible nitric oxide synthase (iNOS) activity after myocardial I/R and consequently exerts cardioprotective effects. However, whether this actually occurs remains unclear. To test this hypothesis, we utilized iNOS-deficient (-/-), IL-10 -/-, and IL-10/iNOS -/- mice to examine the potential mechanism of IL-10-mediated cardioprotection after myocardial I/R. Wild-type, iNOS -/-, IL-10 -/-, and IL-10/iNOS -/- mice were subjected to in vivo myocardial ischemia (30 min) and reperfusion (24 h). Deficiency of iNOS alone did not significantly alter the extent of myocardial necrosis compared with wild-type mice. We found that deficiency of IL-10 resulted in a significantly (P < 0.05) larger infarct size than that in wild-type hearts. Interestingly, deficiency of both IL-10 and iNOS yielded significantly (P < 0.01) larger myocardial infarct sizes compared with wild-type animals. Histological examination of myocardial tissue samples revealed augmented neutrophil infiltration into the I/R myocardium of IL-10 -/- and IL-10/iNOS -/- mice compared with hearts of wild-type mice. These results demonstrate that 1) deficiency of endogenous IL-10 exacerbates myocardial injury after I/R; 2) the cardioprotective effects of IL-10 are not dependent on the presence or absence of iNOS; and 3) deficiency of IL-10 enhances the infiltration of neutrophils into the myocardium after I/R.

Simvastatin exerts both anti-inflammatory and cardioprotective effects in apolipoprotein E-deficient mice.

BACKGROUND: Simvastatin attenuates ischemia and reperfusion in normocholesterolemic animals by stabilizing endothelial nitric oxide synthase activity and inhibiting neutrophil-mediated injury. Because endothelial dysfunction is a detrimental effect of hypercholesterolemia, we examined whether short-term treatment with simvastatin could inhibit leukocyte-endothelium interaction and attenuate myocardial ischemia-reperfusion injury in apoE-deficient (apoE(-/-)) mice fed a high-cholesterol diet. METHODS AND RESULTS: We studied leukocyte-endothelium interactions in apoE(-/-) mice fed a normal or a high-cholesterol diet after short-term (ie, 18 hours) simvastatin treatment. We also studied simvastatin treatment in myocardial ischemia-reperfusion injury by subjecting apoE(-/-) mice to 30 minutes of ischemia and 24 hours of reperfusion. ApoE(-/-) mice fed a high-cholesterol diet exhibited higher blood cholesterol levels, which were not affected by short-term simvastatin treatment. However, the increased leukocyte rolling and adherence that occurred in cholesterol-fed apoE(-/-) mice (P<0.001 versus control diet) were significantly attenuated by simvastatin treatment (P<0.01 versus vehicle). Cholesterol-fed apoE(-/-) mice subjected to myocardial ischemia-reperfusion also experienced increased myocardial necrosis (P<0.01 versus control diet), which was significantly attenuated by simvastatin (P<0.01 versus vehicle). Simvastatin therapy also significantly increased vascular nitric oxide production in apoE(-/-) mice. CONCLUSIONS: Simvastatin attenuates leukocyte-endothelial cell interactions and ameliorates ischemic injury in hypercholesterolemic mice independently of lipid-lowering actions.

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury.

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.

Organ preservation solutions increase endothelial permeability and promote loss of junctional proteins.

OBJECTIVE: To investigate the effects of the organ preservation solutions UW and Plegisol on endothelial permeability; occludin and vascular endothelial (VE)-cadherin content in human umbilical vein endothelial cells (HUVEC); and junctional localization of these proteins after exposure to these solutions. SUMMARY BACKGROUND DATA: Organ preservation for transplantation is limited by several challenges, including loss of tissue function, tissue injury, and tissue edema. Occludin and VE-cadherin are responsible for maintaining and regulating the endothelial solute barrier. Several studies have noted organ edema and dysfunction with preservation, as well as gaps between endothelial cells suggesting that disorganization of junctional proteins (e.g., occludin and VE-cadherin) is responsible for interstitial edema. METHODS: HUVEC monolayers were treated with 4 degrees C UW and Plegisol for 3 and 6 hours and then reperfused with normal buffer. Permeability was examined using FITC-dextran tracer during the reperfusion phase. Occludin and VE-cadherin content at different time points was measured by Western blotting. Treated groups were also examined by immunofluorescence for occludin, VE-cadherin, and F-actin. RESULTS: Compared with untreated controls, cold preservation for 3 and 6 hours increased endothelial permeability after rewarming, which appears to depend on the duration of cold exposure. Monolayers exposed to 3 hours of cold preservation did not have increased permeability in the first hour after rewarming but had significantly increased permeability after the first hour and all subsequent time points. Monolayers exposed to 6 hours of cold preservation had increased permeability after the first hour and at all later time points. Western blotting demonstrated that occludin content was decreased to a similar extent with all solutions after 3 hours of cold preservation. Six hours of cold preservation in Plegisol reduced the occludin content significantly compared with UW and control. VE-cadherin content was unchanged after 3 hours of cold preservation but was dramatically reduced in all groups at 6 hours. Immunofluorescent staining demonstrated junctional gap formation and discontinuous staining of occludin and VE-cadherin with all cold preservation protocols; changes in F-actin organization were observed at 3 and 6 hours after cold preservation. CONCLUSION: The changes in occludin, VE-cadherin, and F-actin content and organization and increased permeability associated with cold storage demonstrate that alterations of the tight and adherens junctions may underlie organ edema associated with cold organ preservation. These data also suggest that novel strategies to maintain the content and integrity of endothelial junctional proteins may provide an important therapeutic avenue for organ preservation.

Expression of zonula occludens and adherens junctional proteins in human venous and arterial endothelial cells: role of occludin in endothelial solute barriers.

OBJECTIVE: The purpose of this study was to correlate the expression of occludin and VE-cadherin with the solute barrier properties of arterial and venous endothelial monolayers. METHODS: Immunofluorescent confocal and traditional microscopy were used to determine junctional protein localization in endothelium in vivo and in vitro respectively, and western and northern analysis used to determine protein and gene expression levels. Permeability of endothelial monolayers was examined under normal, low calcium, and cytochalasin-D treatment conditions. Antisense oligonucleotide experiments for occludin were performed to determine the contribution of occludin to solute barrier. RESULTS: Occludin protein in endothelial monolayers is more concentrated in arterial junctions than in venous junctions both in vivo and in vitro. Arterial endothelial cells express 18-fold more occludin protein and nine times more occludin mRNA compared to venous endothelial cells. In vivo, both endothelial cells demonstrate VE-cadherin staining; and in vitro, only venous endothelial cells express VE-cadherin protein and mRNA. Occludin antisense experiments suggest that both arterial and venous barrier properties are due to these different amounts of occludin expression. Venous barrier was remarkably sensitive to low extracellular calcium, while arterial barrier was more sensitive to cytochalasin-D. CONCLUSIONS: These findings suggest strongly that arterial and venous endothelial barrier reflects the level of expression of different adhesion molecules and that modulation of these proteins, especially occludin, may regulate the level of endothelial solute barrier.

Effects of regional myocardial lidocaine infusion on high energy phosphates.

High energy phosphates [phosphocreatine (PCr) and adenosine triphosphate (ATP)] are maintained in the heart under conditions of altered myocardial contractility and under certain conditions of maintained in the heart under conditions of altered myocardial contractility and under certain conditions of myocardial ischemia (such as hibernating myocardium). However, the metabolic consequences of reduced regional contractility have not been investigated. This study was designed to test the hypotheses that (1) under conditions of normal blood flow, reduction in regional contractility does not result in changes in PCr or ATP and (2) under conditions of reduced blood flow, reduction in regional contractility prevents the expected decline in high energy phosphates usually seen in regional ischemia. An in situ open chest swine preparation was used in which regional contractility was reduced with the administration of intracoronary lidocaine. High energy phosphates were measured using phosphorus-31 magnetic resonance spectroscopy (NMR) under conditions of normal flow and reduced flow. Intracoronary lidocaine infusion in 9 animals did not change blood flow from basal levels, but significantly reduced regional segment shortening from 0.16 +/- 0.02 to 0.02 +/- 0.01. The ratio of PCr/ATP did not change with lidocaine infusion (control: 1.53 +/- 0.09; lidocaine: 1.59 +/- 0.11), but oxygen content in the anterior interventricular vein increased from 8.25 +/- 0.69 to 9.83 +/- 0.91 ml/O2/100 ml blood in parallel studies (P = 0.04). While the lidocaine infusion was maintained, subsequent coronary stenosis significantly reduced subendocardial blood flow from 0.91 +/- 0.06 to 0.41 +/- 0.06 ml/min/g without significantly altering high energy phosphates (PCr/ATP = 1.51 +/- 0.15). In contrast to the 29% decline in PCr previously seen with regional ischemia, PCr was unchanged with this degree of flow reduction in the presence of lidocaine. Thus, PCr and ATP are unchanged under conditions of reduced contractility, consistent with equilibrium of energy synthesis and utilization. In addition, factors which reduce myocardial contractility, either pharmacologically or endogenously, protect against the metabolic consequences of reduced flow by reducing MVO2.

Energy use by contractile and noncontractile processes in skeletal muscle estimated by 31P-NMR.

The goal of this study was to separately determine ATP use by contractile and noncontractile processes in stimulated skeletal muscle. ATP use by tetanically stimulated bullfrog semitendinosus muscle was monitored at room temperature with in vivo 31P-nuclear magnetic resonance. Oxidative phosphorylation was inhibited by cyanide, and ATP use could therefore be calculated by accounting for ATP derived from the creatine kinase (CK) reaction (measured from decreases in phosphocreatine) and from glycolysis (estimated from decreases of intracellular pH). In unfatigued muscles stimulated at optimal length for force production, total ATP utilization (representing both contractile and noncontractile processes) was 2.5 +/- 0.09 (SE) mM/s (n = 6; 53% ATP from glycolysis, 47% from CK). In separate experiments, cross-bridge interactions between actin and myosin filaments were eliminated by increasing sarcomere length; therefore, with stimulation, residual ATP use reflected only noncontractile processes. In stimulated stretched muscles, ATP utilization was reduced compared with unstretched muscles to 1.07 +/- 0.08 mM/s (61% ATP from glycolysis, 39% from CK). These findings suggest that, during contraction near optimum length, a large proportion (approximately 43%) of ATP is used by noncontractile processes, with more ATP derived from glycolysis than from CK.

Effect of supranormal coronary blood flow on energy metabolism and systolic function of porcine left ventricle.

OBJECTIVE: The goal was to determine if supranormal coronary blood flow increases myocardial oxygen consumption, high energy phosphate levels, and systolic function in the in situ autoperfused heart. METHODS: Thirteen anaesthetised open chest pigs with an intact, autoperfused coronary circulation, weight 30-40 kg, were studied. Measurements were made under basal conditions and during regional hyperperfusion of the anterior left ventricle produced by intracoronary infusion of adenosine (mean dose 3.3 mumol.min-1). Doppler coronary blood flow velocity in the anterior descending coronary artery, arterial and anterior interventricular venous blood oxygen content, high energy phosphates (by transmurally localised 31P NMR), and myocardial wall thickening (by sonomicrometry) were measured. RESULTS: With adenosine, coronary flow was increased to 355(SEM 59)% of control. Supranormal coronary flow produced no significant changes in anterior left ventricular oxygen consumption [99(12)% of control]. 31P NMR spectroscopy revealed no significant changes in the peak intensities of phosphocreatine or ATP in either the subendocardium or subepicardium (90-97% of control). Systolic anterior left ventricular wall thickening also did not change [107(13)% of control]. CONCLUSIONS: Supranormal coronary flow does not augment myocardial oxygen consumption, high energy phosphates, or systolic function in the in situ autoperfused heart. Myocardial oxygen delivery does not limit oxidative metabolism under normal conditions.

Response of high-energy phosphates and lactate release during prolonged regional ischemia in vivo.

BACKGROUND: The functional impairment of persistently ischemic, or "hibernating," myocardium may serve to maintain myocardial cell viability through a reduction of energy requirements. Although previous studies have, in a variety of experimental models, independently shown variable responses in lactate metabolism and intracellular phosphates during prolonged ischemia, the responses of these metabolites under identical flow conditions have not been adequately described. METHODS AND RESULTS: To examine the responses of high-energy phosphates and lactate metabolism to prolonged ischemia induced by partial coronary artery stenosis, 12 open-chest pigs were studied using 31P nuclear magnetic resonance spectroscopy. Concurrent measurements of blood flow, segment shortening, high-energy phosphates, and lactate release (in nine animals) were made during 2 hours of regional ischemia. Subendocardial blood flow and segment shortening were persistently depressed during ischemia, with parallel reductions in ATP, phosphocreatine (PCr), and the ratio of phosphocreatine to inorganic phosphate (PCr/Pi). Pi was persistently elevated during the ischemic period. In contrast, lactate release increased significantly from 0.23 +/- 0.04 to 1.34 +/- 0.28 mumol/ml after 15 minutes of ischemia (p less than 0.05) but then decreased to 0.73 +/- 0.17 mumol/ml at 2 hours (p less than 0.05 versus 15 minutes, p = NS versus control). Similarly, pH increased significantly from a nadir of 6.82 +/- 0.07 at 30 minutes of ischemia to 6.98 +/- 0.05 at 2 hours. CONCLUSIONS: Changes in high-energy phosphates parallel changes in blood flow and function during prolonged ischemia, whereas there is a partial amelioration in lactate production and acidosis. These data support the concept that reduction of myocardial energy requirements during prolonged flow reduction results in signs of reduced ischemia.

Metabolic and functional consequences of blunted myocardial reactive hyperemia.

This study determined whether the rapidity of myocardial metabolic and contractile recovery after brief coronary occlusion depends upon the intensity of reactive hyperemia. We also tested the hypothesis that coronary flow rate modulates contractility after brief myocardial ischemia, independent of changes in phosphorus metabolites. Eight open-chest pigs were studied with phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy with 14 s time resolution. After a 29-s anterior descending coronary occlusion, peak Doppler coronary flow velocity was alternately unrestricted (normal hyperemia, 443 +/- 40% of control) or limited to 159 +/- 9% of control. During 29 s coronary occlusion, phosphocreatine-to-inorganic phosphate ratio (PCr/Pi) and systolic segment shortening in the ischemic region fell to 28 +/- 4 and 7 +/- 7% of control, respectively. With normal hyperemia, PCr/Pi and segment shortening recovered within 29 s. With blunted hyperemia, recovery of both parameters was delayed an additional 29-43 s, associated with reduced subendocardial blood flow (measured with radioactive microspheres) and persistent intracellular acidosis. However, the relationship between segment shortening and PCr/Pi was unaffected by the intensity of reactive hyperemia. Thus blunted reactive hyperemia significantly delays metabolic and contractile recovery from brief ischemia, probably via transient maldistribution of transmural perfusion. However, coronary blood flow rate does not independently modulate contractility after brief reversible ischemia.