James L. Park

Mechanisms of myocardial reperfusion injury

Reperfusion of the ischemic myocardium results in irreversible tissue injury and cell necrosis, leading to decreased cardiac performance. While early reperfusion of the heart is essential in preventing further tissue damage due to ischemia, reintroduction of blood flow can expedite the death of vulnerable, but still viable, myocardial tissue, by initiating a series of events involving both intracellular and extracellular mechanisms. In the last decade, extensive efforts have focused on the role of cytotoxic reactive oxygen species, complement activation, neutrophil adhesion, and the interactions between complement and neutrophils during myocardial reperfusion injury. Without reperfusion, myocardial cell death evolves slowly over the course of hours. In contrast, reperfusion after an ischemic insult of sufficient duration initiates an inflammatory response, beginning with complement activation, followed by the recruitment and accumulation of neutrophils into the reperfused myocardium. Modulation of the inflammatory response, therefore, constitutes a potential pharmacological target to protect the heart from reperfusion injury. Recognition of the initiating factor(s) involved in myocardial reperfusion injury should aid in development of pharmacological interventions to selectively or collectively attenuate the sequence of events that mediate extension of tissue injury beyond that caused by the ischemic insult.

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression.

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.

Lupus-prone New Zealand Black/New Zealand White F1 mice display endothelial dysfunction and abnormal phenotype and function of endothelial progenitor cells:

Patients with systemic lupus erythematosus (SLE) have an impairment in phenotype and function of endothelial progenitor cells (EPCs) which is mediated by interferon α (IFN-α). We assessed whether murine lupus models also exhibit vasculogenesis abnormalities and their potential association with endothelial dysfunction. Phenotype and function of EPCs and type I IFN gene signatures in EPC compartments were assessed in female New Zealand Black/New Zealand White F1 (NZB/W), B6.MRL-Faslpr/J (B6/lpr) and control mice. Thoracic aorta endothelial and smooth muscle function were measured in response to acetylcholine or sodium nitropruside, respectively. NZB/W mice displayed reduced numbers, increased apoptosis and impaired function of EPCs. These abnormalities correlated with significant decreases in endothelium-dependent vasomotor responses and with increased type I IFN signatures in EPC compartments. In contrast, B6/lpr mice showed improvement in endothelium-dependent and endothelial-independent responses, no abnormalities in EPC phenotype or function and downregulation of type I IFN signatures in EPC compartments. These results indicate that NZB/W mice represent a good model to study the mechanisms leading to endothelial dysfunction and abnormal vasculogenesis in lupus. These results further support the hypothesis that type I IFNs may play an important role in premature vascular damage and, potentially, atherosclerosis development in SLE. Lupus (2010) 19, 288—299.

Decreased Nitric Oxide Bioavailability in a Mouse Model of Fabry Disease

Fabry disease is a lysosomal storage disorder that results in an accumulation of globotriaosylceramide in vascular tissue secondary to a deficiency in alpha-galactosidase A. The glycolipid-associated vasculopathy results in strokes and cardiac disease, but the basis for these complications is poorly understood. Recent studies in the alpha-galactosidase A-knockout mouse suggested that a decrease in nitric oxide (NO) bioavailability may play a role in the abnormal thrombosis, atherogenesis, and vasorelaxation that are characteristic of these mice. To understand better the association between impaired NO bioavailability and glycolipid accumulation, we studied alpha-galactosidase A-knockout mice or primary cultures of their aortic endothelial cells. Treatment of knockout mice with a potent inhibitor of glucosylceramide synthase reversed accumulation of globotriaosylceramide but failed to normalize the defect in vasorelaxation. Basal and insulin-stimulated endothelial NO synthase (eNOS) activities in endothelial cells derived from knockout mice were lower than those observed from wild-type mice; normalization of glycolipid only partially reversed this reduction in eNOS activity. The loss of eNOS activity associated with a decrease in high molecular weight caveolin oligomers in endothelial cells and isolated caveolae, suggesting a role for glycolipids in caveolin assembly. Finally, concentrations of ortho-tyrosine and nitrotyrosine in knockout endothelial cells were markedly elevated compared with wild-type endothelial cells. These findings are consistent with a loss of NO bioavailability, associated with eNOS uncoupling, in the alpha-galactosidase A-knockout mouse.

The Peroxisome Proliferator-Activated Receptor γ Agonist Pioglitazone Improves Cardiometabolic Risk and Renal Inflammation in Murine Lupus

Individuals with systemic lupus erythematosus (SLE) have a striking increase in the risk of premature atherosclerosis, a complication preceded by significant subclinical vascular damage. A proposed mechanism leading to accelerated vascular disease in SLE is an imbalance between vascular damage and repair, as patients with this disease display significant abnormalities in phenotype and function of endothelial progenitor cells. In addition, individuals with SLE have a higher incidence of insulin resistance which may further contribute to the increased cardiovascular risk. This study examined the role of the peroxisome proliferator activated receptor γ agonist pioglitazone in improving endothelial function, endothelial progenitor cell numbers and functional capacity, metabolic parameters, and disease activity in the lupus-prone murine model New Zealand Black/New Zealand White (NZB × NZW)F1. Ten-week-old prenephritic female NZB/NZW F1 mice were exposed to 10 or 25 mg/kg/day of oral pioglitazone or vehicle for 15 or 24 wk. Mice exposed to pioglitazone exhibited pronounced enhancement in endothelial-dependent vasorelaxation of thoracic aortas and in endothelial progenitor cell function, as assessed by the capacity of bone marrow-derived endothelial progenitor cells to differentiate into mature endothelial cells. Pioglitazone-treated mice showed improvement in insulin resistance, adipokine, and lipid profile. Kidneys from pioglitazone-treated mice showed significant decreases in immune complex deposition, renal inflammation, T cell glomerular infiltration, and intrarenal synthesis of TNF-α, IL-1β, and VCAM-1. These results indicate that peroxisome proliferator-activated receptor γ agonists could serve as important tools in the prevention of premature cardiovascular disease and organ damage in SLE.

Reduction of myocardial infarct size after ischemia and reperfusion by the glycosaminoglycan pentosan polysulfate.

Activation of the complement system contributes to the tissue destruction associated with myocardial ischemia/reperfusion. Pentosan polysulfate (PPS), a negatively charged sulfated glycosaminoglycan (GAG) and an effective inhibitor of complement activation, was studied for its potential to decrease infarct size in an experimental model of myocardial ischemia/reperfusion injury. Open-chest rabbits were subjected to 30-min occlusion of the left coronary artery followed by 5 h of reperfusion. Vehicle (saline) or PPS (30 mg/kg/h) was administered intravenously immediately before the onset of reperfusion and every hour during the reperfusion period. Treatment with PPS significantly (p < 0.05) reduced infarct size as compared with vehicle-treated animals (27.5+/-2.9% vs. 13.34+/-2.6%). Analysis of tissue demonstrated decreased deposition of membrane-attack complex and neutrophil accumulation in the area at risk. The results indicate that, like heparin and related GAGs, PPS possesses the ability to decrease infarct size after an acute period of myocardial ischemia and reperfusion. The observations are consistent with the suggestion that PPS may mediate its cytoprotective effect through modulation of the complement cascade.

VASCULAR DYSFUNCTION IN THE α-GALACTOSIDASE A-KNOCKOUT MOUSE IS AN ENDOTHELIAL CELL-, PLASMA MEMBRANE-BASED DEFECT

1 Fabry disease results from an X‐linked mutation in the lysosomal α‐galactosidase A (Gla) gene. Defective Gla results in multi‐organ accumulation of neutral glycosphingolipids (GSLs), especially in the vascular endothelium, with the major GSL accumulated being globotriaosylceramide (Gb3). Excessive endothelial Gb3 accumulation is associated with increased thrombosis, atherogenesis and endothelial dysfunction. However, the mechanism(s) by which endothelial dysfunction occurs is unclear. The purpose of the present study was to further characterize the vasculopathy associated with a murine model of Fabry disease. 2 Vascular reactivity was performed in vessels from wild‐type (Gla+/0) and Gla‐knockout (Gla−/0) mice. Conscious blood pressure and heart rate were measured in Gla+/0 and Gla−/0 mice by telemetry. 3 The present study demonstrates that vascular smooth muscle (VSM) contractions to phenylephrine and serotonin, but not to U46619, were blunted in Gla−/0 mice. Endothelium‐dependent contraction and receptor‐mediated endothelium‐dependent relaxation to acetylcholine were significantly attenuated in vessels from Gla−/0 mice. However, receptor‐independent endothelium‐dependent relaxation to the calcium ionophore ionomycin remained intact in vessels from Gla−/0 mice. Furthermore, VSM reactivity was normal in aortas from Gla−/0 mice in the absence of endothelium. These changes in vascular function were observed without changes in whole‐animal blood pressure or heart rate. 4 These results suggest that the vasculopathy associated with Fabry disease is localized to the endothelium, despite the accumulation of GSLs throughout the vasculature.

Protective Effects of Ranolazine on Ventricular Fibrillation Induced by Activation of the ATP-Dependent Potassium Channel in the Rabbit Heart.

Background: The authors studied the antifibrillatory effects of the adenosine-triphosphate (ATP)-sparing metabolic modulator ranolazine in a rabbit isolated heart model in which ventricular fibrillation occurs under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener pinacidil. Methods and Results: Ten minutes after ranolazine or vehicle administration, addition of pinacidil (1.25 μM) to the buffer was followed by a 12-minute hypoxic period and 40 minutes of reoxygenation. At a reduced concentration of ranolazine (10 μM), ventricular fibrillation occurred in 60% of the hearts. compared to 89% in the control group (P = NS). In contrast, only three of nine hearts (33%) treated with 20 μM ranolazine developed ventricular fibrillation (P <.05 vs vehicle). Hemodynamic parameters including coronary perfusion pressure, left ventricular developed pressure, and ±dP/dt were not affected by the presence of ranolazine in the perfusion medium. Ranolazine did not prevent or modify the negative inotropic or coronary vasodilator actions of pinacidil, suggesting a mechanism of action independent of potassium channel antagonism. Conclusions: Ranolazine significantly reduced the incidence of ventricular fibrillation in the hypoxic/reoxygenated heart exposed to the ATP-dependent potassium channel opener, pinacidil. The reported ability of ranolazine to prevent the decrease in cellular ATP during periods of a reduced oxygen supply may account for its observed antifibrillatory action. By maintaining intracellular ATP, ranolazine may modulate or prevent further opening of the ATP-dependent potassium channel in response to hypoxia and/or pinacidil.

GLUT4 Facilitative Glucose Transporter Specifically and Differentially Contributes to Agonist-Induced Vascular Reactivity in Mouse Aorta

Objective—We hypothesized that GLUT4 is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs), and GLUT4 is necessary for agonist-induced VSMC contraction. Methods and Results—Glucose deprivation and indinavir, a GLUT4 antagonist, were used to assess the role of GLUT4 and non-GLUT4 transporters in vascular reactivity. In isolated endothelium-denuded mouse aorta, ≈50% of basal glucose uptake was GLUT4-dependent. Norepinephrine-mediated contractions were dependent on both GLUT4 and non-GLUT4 transporters, serotonin (5-HT)-mediated contractions were mainly GLUT4-dependent, and prostaglandin (PG) F2&agr;-mediated contractions were dependent on non-GLUT4 transporters, whereas indinavir had no effect in GLUT4 knockout vessels. We also observed a 46% decrease in GLUT4 expression in aortas from angiotensin II hypertensive mice. Indinavir caused a less profound attenuation of maximal 5-HT–mediated contraction in these vessels, corresponding to the lower GLUT4 levels in the hypertensive aortas. Finally, and somewhat surprisingly, chronic GLUT4 knockout was associated with increased vascular reactivity compared with that in wild-type animals, suggesting that chronic absence or reduction of GLUT4 expression in VSMCs leads to opposite effects observed with acute inhibition of GLUT4. Conclusions—Thus, we conclude that GLUT4 is constitutively expressed in large arteries and likely participates in basal glucose uptake. In addition, GLUT4, as well as other non-GLUT4 facilitative glucose transporters, are necessary for agonist-induced contraction, but each transporter participates in VSMC contraction selectively, depending on the agonist, and changes in GLUT4 expression may account for some of the functional changes associated with vascular diseases like hypertension.

Effects of tedisamil (KC-8857) on cardiac electrophysiology and ventricular fibrillation in the rabbit isolated heart.

1 The direct cardiac electrophysiological and antifibrillatory actions of tedisamil (KC‐8857) were studied in rabbit isolated hearts. 2 Tedisamil (1, 3, and 10 μm), prolonged the ventricular effective refractory period (VRP) from 120±18ms (baseline) to 155±19, 171±20, and 205±14 ms, respectively. Three groups of isolated hearts (n = 6 each) were used to test the antifibrillatory action of tedisamil. Hearts were perfused with 1.25 μm pinacidil, a KATP channel activator. Hearts were subjected to hypoxia for 12 min followed by 40 min of reoxygenation. Ventricular fibrillation (VF) developed during hypoxia and reoxygenation in both the control and 1 μm tedisamil‐treated groups (5/6 and 4/6, respectively). Tedisamil (3 μm) reduced the incidence of VF (0/6, P = 0.007 vs. control). 3 In a separate group of hearts, VF was initiated by electrical stimulation. The administration of 0.3 ml of 10 mM tedisamil, via the aortic cannula, terminated VF in all hearts, converting them to normal sinus rhythm. 4 Tedisamil (3 μm) reversed pinacidil‐induced negative inotropic effects in rabbit isolated atrial muscle which were equilibrated under normoxia, as well as in atrial muscle subjected to hypoxia and reoxygenation. 5 The results demonstrate a direct antifibrillatory action of tedisamil in vitro. The mechanism responsible for the observed effects may involve modulation by tedisamil of the cardiac ATP‐regulated potassium channel, in addition to its antagonism of IK and Ito.