Pericle Di Napoli

Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: an ex vivo study in isolated working rat hearts

OBJECTIVE We tested the hypothesis of beneficial effects of the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA)-reductase inhibitor simvastatin in a model of ischemia-reperfusion, and investigated potential mechanisms. METHODS Isolated working rat hearts were subjected to 15 min global ischemia and 22-180 min reperfusion in the presence or absence of simvastatin (10-100 microM). We evaluated creatinephosphokinase and nitrite levels in coronary effluent, heart weight changes, microvascular permeability (extravasation of fluoresceine-labeled albumin), ultrastructural alterations, and the expression of endothelial (e) and inducible (i) nitric oxide synthase (NOS) (by reverse-transcribed polymerase chain reaction and Western blotting) in the presence or absence of the transcriptional inhibitor actinomycin-D. RESULTS Simvastatin (25 microM) significantly reduced myocardial damage and vascular hyperpermeability, concomitant with a reduction in endothelial and cardiomyocyte lesions. Protection became less evident at 50 microM and reverted to increased damage at 100 microM. At 25 microM, simvastatin significantly increased eNOS mRNA and protein compared with untreated hearts, probably due to a post-transcriptional regulation since unaltered by animal pretreatment with actinomycin D. Simvastatin also significantly decreased iNOS mRNA and protein, as well as nitrite production after ischemia-reperfusion. The addition of the NOS inhibitor N(pi)-nitro-L-arginine methylester (L-NAME, 30 microM) to 25 microM simvastatin-treated hearts significantly reduced cardioprotection against ischemia-reperfusion. CONCLUSIONS In this model, in the absence of perfusing granulocytes, the acute administration of a pharmacologically relevant simvastatin concentration reduces ischemia-reperfusion injury and prevents coronary endothelial cell and cardiomyocyte damage by cholesterol-independent, NO-dependent mechanisms.

Simvastatin Attenuates Expression of Cytokine-inducible Nitric-oxide Synthase in Embryonic Cardiac Myoblasts

Cardiac stem cells or myoblasts are vulnerable to inflammatory stimulation in hearts with infarction or ischemic injury. Widely used for the prevention and treatment of atherosclerotic heart disease, the cholesterol-lowering drugs statins may exert anti-inflammatory effects. In this study, we examined the impact of inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase with simvastatin on the expression of inducible nitric-oxide synthase (iNOS) in embryonic cardiac myoblasts stimulated with the proinflammatory cytokines, interleukin-1 or tumor necrosis factor. Treatment with simvastatin significantly reduced the levels of iNOS mRNA and protein in cytokine-treated rat H9c2 cardiac embryonic myoblasts. Addition of the HMG-CoA reductase product, l-mevalonate, and the by-product of cholesterol synthesis, geranylgeranyl pyrophosphate, could reverse the statin inhibitory effect on iNOS expression. Simvastatin treatment lowered the Rho GTPase activities, whereas the Rho-associated kinase inhibitor Y27632 partially blocked the statin inhibitory effect on nitrite production in the cytokine-treated H9c2 cells. Treatment with simvastatin led to inactivation of NF-κB by elevation of the NF-κB inhibitor IκB and reduction of the NF-κB nuclear contents in the cytokine-stimulated H9c2 cells. Hence, treatment with simvastatin can attenuate iNOS expression and NO synthesis in cytokine-stimulated embryonic cardiac myoblasts. The statin inhibitory effect may occur through isoprenoid-mediated intracellular signal transduction, which involves several key signal proteins, such as Rho kinase and IκB/NF-κB. These data suggest that statin therapy may protect the cardiac myocyte progenitors against the cytotoxicity of cytokine-induced high output of NO production in infarcted or ischemic hearts with inflammation.

Left ventricular wall stress as a direct correlate of cardiomyocyte apoptosis in patients with severe dilated cardiomyopathy

BACKGROUND Apoptosis has been implicated as a possible mechanism in the development of heart failure (HF), but the mechanisms involved remain unclear. In patients with severe dilated cardiomyopathy, we evaluated cardiomyocyte apoptosis in relation to the transmural distribution of Bax and Bcl-2 proteins (2 molecules inhibiting or promoting apoptosis, respectively) and left ventricular wall stresses. METHODS We studied the presence and distribution of cardiomyocyte apoptosis in 90 tissue samples obtained from 8 patients who were undergoing left ventricular reduction with the Batista (ventricular remodeling) operation. Apoptosis was assessed in tissue samples taken from the entire left ventricular thickness (subdivided in subepicardial, midmyocardial, and subendocardial sections) with the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) technique and DNA agarose gel electrophoresis. The expression of Bcl-2 and Bax proteins were determined with both Western analysis and immunohistochemistry. RESULTS TUNEL-positive cells (apoptotic index) were 2.3% +/- 1.4%. Apoptotic cells were predominantly distributed in the subendocardium, where higher levels of Bax protein were detected. The ratio of Bax to Bcl-2 proteins (Bax/Bcl-2) was similar in the midmyocardium or subepicardium, but increased in the subendocardium, where it was directly related to systolic wall stress (y = 0.009x - 0.629; r2 = 0.85, P <.001). The apoptotic index was also directly related to systolic and end-diastolic stresses calculated from hemodynamic and echocardiographic data (r2 = 0.77, P <.001 and r2 = 0.40, P <.01, respectively). CONCLUSIONS In patients with dilated cardiomyopathy, in whom cardiomyocyte apoptosis is an important cause of cell loss, apoptosis is more extensively localized in the subendocardium and strictly related to ventricular wall stresses and the Bax/Bcl-2 ratio.

Energy Metabolism in the Normal and in the Diabetic Heart

Cardiovascular disease is a major health problem all over the world. The prevalence of type 2 diabetes mellitus has been rapidly increasing, together with the risk of cardiovascular events. Patients with diabetes, and/or with insulin resistance as well, have an impaired myocardial metabolism of glucose and free fatty acids (FFA) and accelerated and diffuse atherogenesis, with involvement of coronary artery tree. Significant metabolic alterations at heart level in diabetic patients are the decreased utilization of glucose and the increase in muscular and myocardial FFA uptake and oxidation, occurring as a consequence of the mismatch between blood supply and cardiac metabolic requirements. These metabolic changes are responsible both for the increased susceptibility of the diabetic heart to myocardial ischemia and for a greater decrease of myocardial performance for a given amount of ischemia, compared to non diabetic hearts. A therapeutic approach aimed at an improvement of cardiac metabolism, through manipulations of the utilization of metabolic substrates, may improve myocardial ischemia and left ventricular function. Modulation of myocardial FFA metabolism, in addition to optimal medical therapy, should be the key target for metabolic interventions in patients with coronary artery disease and diabetes. In diabetic patients with ischemic heart disease, the effects of modulation of FFA metabolism could even give greater benefit than in nondiabetic patients.

Effect of chronic hypoxia on inducible nitric oxide synthase expression in rat myocardial tissue.

The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in Inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (INOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of INOS. Expression of INOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). INOS expression was evaluated through immunohistochemical techniques (Ab anti-INOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of INOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for INOS in the same experimental conditions. In addition, the INOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 ± 0.01 vs 0.02 ± 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating INOS.

MK-954 (losartan potassium) exerts endothelial protective effects against reperfusion injury: evidence of an e-NOS mRNA overexpression after global ischemia

BACKGROUND the cardiac Renin-Angiotensin system (RAS) plays an important role in the regulation of coronary flow and cardiac function and structure in normal and pathological conditions such as ischemia-reperfusion (I/R) injury. The aim of this study was to investigate the effects of the Angiotensin II type 1 (AT-1) receptor antagonist MK-954 (losartan potassium) on postischemic endothelial dysfunction and NOS mRNA expression (inducible nitric oxide synthase, iNOS; endothelial nitric oxide synthase, eNOS) in isolated working rat hearts. METHODS isolated working rat hearts were subjected to 15 min global ischemia and 180 min reperfusion. MK-954 was added to perfusion buffer (a modified Krebs-Henseleit solution) at 1 microM concentration. We assessed functional parameters, creatin kinase (CK) release, heart weight changes, microvascular postischemic hyperpermeability (FITC-albumin extravasation) and morphological ultrastructural alterations. eNOS and iNOS mRNA levels were also detected by the means of multiplex RT-PCR technique using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene as internal control; results were expressed as densitometric ratio. RESULTS in Losartan-treated hearts we observed a significant reduction of postischemic contractile dysfunction, CK release and myocardial ultrastructural damage; postischemic FITC-albumin extravasation was significantly reduced respect to controls. Moreover, 1 microM Losartan produced a significant reduction of eNOS/G3PDH respect to untreated hearts submitted to I/R. Regarding iNOS/G3PDH ratio, no significant changes were detected in Losartan-treated hearts compared with controls. CONCLUSIONS our study revealed that Losartan treatment before ischemia, and during reperfusion, is able to reduce the reperfusion injury of the rat heart by reducing mechanical and microcirculatory dysfunction and necrotic cell death, ameliorating cardiac ultrastructure and endothelial protection, probably inducing eNOS over-expression and reducing post-ischemic hyperpermeability of coronary microcirculation.

Does 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Inhibitor Therapy Exert a Direct Anti-Ischemic Effect?

To the Editor: We read with great interest the recent well-written article by Andrews et al1 in Circulation , and we would like to add a comment to the editorial observation of Dr Frye.2 The most important finding of the study is that in selected patients with documented coronary artery disease associated with myocardial ischemia on ambulatory ECG monitoring, dietary treatment and lovastatin therapy induced, after 4 to 6 months, a significant reduction of ischemic events compared with the control group …

Acute Heart Failure Secondary to Myocardial Tissue Water Changes in Isolated Working Rat Hearts

Alterations in capillary interstitial liquid exchange may lead to fluid accumulation within myocardial interstitium. * An increased water content can influence tissue perfusion and myocyte vitality and probably, under certain circumstances, can contribute to myocardial dysfunction and to the onset of acute heart failure.* The aim of this study was to investigate whether myocardial tissue water changes may affect ventricular mechanical properties and myocardial structure in experimental preparations.