Csaba Csonka

Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts

OBJECTIVE We investigated the influence of experimental hyperlipidemia on the formation of cardiac NO, superoxide, and peroxynitrite (ONOO(-)) in rat hearts. METHODS Wistar rats were fed 2% cholesterol-enriched diet or normal diet for 8 weeks. Separate groups of normal and hyperlipidemic rats were injected twice intraperitoneally with 2 x 20 micromol/kg FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), a ONOO(-) decomposition catalyst, 24 h and 1 h before isolation of the hearts. RESULTS A cholesterol diet significantly decreased myocardial NO content, however, myocardial Ca(2+)-dependent and Ca(2+)-independent NO synthase activity and NO synthase protein level did not change. Myocardial superoxide formation and xanthine oxidase activity were significantly increased; however, cardiac superoxide dismutase activity did not change in the cholesterol-fed group. Dityrosine in the perfusate, a marker of cardiac ONOO(-) formation, and plasma nitrotyrosine, a marker for systemic ONOO(-) formation, were both elevated in hyperlipidemic rats. In cholesterol-fed rats, left ventricular end-diastolic pressure (LVEDP) was significantly elevated as compared to controls. Administration of FeTPPS normalized LVEDP in the cholesterol-fed group. CONCLUSION We conclude that cholesterol-enriched diet-induced hyperlipidemia leads to an increase in cardiac ONOO(-) formation and a decrease in the bioavailability of NO which contributes to the deterioration of cardiac performance and may lead to further cardiac pathologies.

Matrix metalloproteinase activity assays: Importance of zymography.

INTRODUCTION Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. METHODS For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. RESULTS AND DISCUSSION Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

Capsaicin-sensitive local sensory innervation is involved in pacing-induced preconditioning in rat hearts: role of nitric oxide and CGRP?

Abstract Among several mediators, nitric oxide (NO) and calcitonin gene-related peptide (CGRP) were suggested to be involved in the mechanism of preconditioning. We examined the possible role of the cardiac capsaicin-sensitive sensory innervation in pacing-induced preconditioning, as well as in the cardiac NO and CGRP content. Wistar rats were treated subcutaneously with capsaicin or its solvent in the sequence of 10, 30, and 50 mg/kg increasing single daily doses for 3 days to deplete neurotransmitters of the sensory innervation. Isolated hearts from both groups were then subjected to either preconditioning induced by three consecutive periods of pacing at 600 beats per minute for 5 min with 5 min interpacing periods, or time-matched non-preconditioning perfusion, followed by a 10-min coronary occlusion. NO content of left ventricular tissue samples was assayed by electron-spin resonance, and CGRP release was determined by radioimmunoassay. CGRP immunohistochemistry was also performed. In the non-preconditioned, solvent-treated group, coronary occlusion decreased cardiac output (CO) from 68.1 to 32.1 mL/min, increased left ventricular end-diastolic pressure (LVEDP) from 0.58 to 1.90 kPa, and resulted in 200 mU/min/g LDH release. Preconditioning significantly increased ischaemic CO to 42.9 mL/min (P < 0.05), decreased ischaemic LVEDP to 1.26 kPa (P < 0.05) and decreased LDH release to 47 mU/min/g (P < 0.05) in the solvent-treated group. Preconditioning did not confer protection in the capsaicin-pretreated group (ischaemic CO: 35.6 mL/min; LVEDP: 1.76 kPa; LDH 156 mU/min/g). Capsaicin-treatment markedly decreased cardiac NO content, CGRP release, and CGRP-immunoreactivity. Conclusions: (i) The presence of an intact local sensory innervation is a prerequisite to elicit pacing-induced preconditioning in the rat heart. (ii) A significant portion of cardiac basal NO content may be of neural origin. (iii) Release of NO and CGRP from capsaicin-sensitive nerves may be involved in the mechanism of pacing-induced preconditioning.

Cholesterol diet-induced hyperlipidemia impairs the cardioprotective effect of postconditioning: role of peroxynitrite

The aim of the present study was to investigate if hyperlipidemia interferes with the infarct size-limiting effect of postconditioning and to study the involvement of peroxynitrite in this phenomenon. Rats were fed a 2% cholesterol-enriched or normal diet for 12 wk. Infarct size by triphenyltetrazolium chloride staining was measured in hearts isolated from both groups and subjected to 30 min coronary occlusion followed by 120 min reperfusion with or without the postconditioning protocol induced by six cycles of 10 s coronary occlusion and 10 s reperfusion at the onset of the reperfusion. Postconditioning significantly decreased infarct size in the normolipidemic but not in the hyperlipidemic group. Postconditioning increased cardiac 3-nitrotyrosine concentration (a marker for peroxynitrite formation) in the normal but not in the cholesterol-fed group when measured at the 5th min of reperfusion. Next, we tested if the postconditioning-induced acute increase in peroxynitrite is involved in the cardioprotection in normolipidemic animals in separate experiments. Postconditioning failed to decrease infarct size in the presence of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron [III] (20 mg/l) in normolipidemic animals. We conclude that an early increase in peroxynitrite after postconditioning plays a role in cardioprotection. Furthermore, hyperlipidemia blocks the cardioprotective effect of postconditioning at least in part via deterioration of the postconditioning-induced early increase in peroxynitrite formation.

Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts

Little is known concerning the effect of oxidative stress on the expression of antioxidative enzymes in the decompensated cardiac hypertrophy of spontaneously hypertensive rats (SHR), considered as a model of dilative cardiomyopathy in man. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) were characterized in isolated perfused hearts of 18 month old SHR and the age-matched normotensive control Wistar-Kyoto (WKY) rats, before and after 30 min infusion of 25 microM H(2)O(2). After infusion of H(2)O(2), aortic flow decreased in WKY from 26.2 +/- 2.2 to 16.0 +/- 0.8 ml/min (p <.05) but not in SHR (18.2 +/- 1.9 vs. 20.7 +/- 2.2 ml/min). This protection was related to the higher myocardial activities of GPx, MnSOD and CuZnSOD in SHR, compared with those of the WKY group. Although total SOD activity in the SHR fell after H(2)O(2) exposure (to 1.81 +/- 0.13 from 3.56 +/- 0.49 U/mg of protein), catalase activity increased (to 2.46 +/- 0.34 from 1.56 +/- 0.29 k min(-1)mg(-1)protein), compared with the pre-infusion period (p <.05 in each case). In additional studies, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The results obtained in ischemic/reperfused hearts show the same changes in enzyme activities measured as it was observed in H(2)O(2) perfused hearts, indicating that oxidative stress is independent of the way it was induced. The higher catalase activity derived from elevated mRNA synthesis. The antioxidative system in dilative cardiomyopathic hearts of SHR is induced, probably due to episodes of oxidative stress, during the process of decompensation. This conditioning of the antioxidative potential may help overcome acute stress situations caused by reactive oxygen species in the failing myocardium.

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2.

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.

MicroRNAs associated with ischemia-reperfusion injury and cardioprotection by ischemic pre- and postconditioning: protectomiRs

We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. Hearts isolated from male Wistar rats were subjected to 1) time-matched nonischemic perfusion, 2) ischemia-reperfusion (30 min of coronary occlusion and 120 min of reperfusion), 3) preconditioning (3 × 5 min of coronary occlusion) followed by ischemia-reperfusion, or 4) ischemia-reperfusion with postconditioning (6 × 10 s of global ischemia-reperfusion at the onset of reperfusion). Infarct size was significantly reduced by both interventions. Of 350 different microRNAs assessed by microarray analysis, 147-160 microRNAs showed detectable expression levels. Compared with microRNA alterations induced by ischemia-reperfusion versus time-matched nonischemic controls, five microRNAs were significantly affected by both pre- and postconditioning (microRNA-125b*, microRNA-139-3p, microRNA-320, microRNA-532-3p, and microRNA-188), four microRNAs were significantly affected by preconditioning (microRNA-487b, microRNA-139-5p, microRNA-192, and microRNA-212), and nine microRNAs were significantly affected by postconditioning (microRNA-1, microRNA let-7i, microRNA let-7e, microRNA let-7b, microRNA-181a, microRNA-208, microRNA-328, microRNA-335, and microRNA-503). Expression of randomly selected microRNAs was validated by quantitative real-time PCR. By a systematic comparison of the direction of microRNA expression changes in all groups, we identified microRNAs, specific mimics, or antagomiRs that may have pre- and postconditioning-like cardioprotective effects (protectomiRs). Transfection of selected protectomiRs (mimics of microRNA-139-5p, microRNA-125b*, microRNA let-7b, and inhibitor of microRNA-487b) into cardiac myocytes subjected to simulated ischemia-reperfusion showed a significant cytoprotective effect. This is the first demonstration that the ischemia-reperfusion-induced microRNA expression profile is significantly influenced by both pre- and postconditioning, which shows the involvement of microRNAs in cardioprotective signaling. Moreover, by analysis of microRNA expression patterns in cardioprotection by pre- and postconditioning, specific protectomiRs can be revealed as potential therapeutic tools for the treatment of ischemia-reperfusion injury.

Measurement of myocardial infarct size in preclinical studies.

Ischemic heart disease is a major cause of morbidity and mortality worldwide. Myocardial ischemia followed by reperfusion results in tissue injury termed ischemia/reperfusion injury which is characterized by decreased myocardial contractile function, occurrence of arrhythmias, and development of tissue necrosis (infarction). These pathologies are all relevant as clinical consequences of myocardial ischemia/reperfusion injury and they are also important as experimental correlates and endpoints. The most critical determinant of acute and long-term mortality after myocardial infarction is the volume of the infarcted tissue. Therefore, development of cardioprotective therapies aims at reducing the size of the infarct developing due to myocardial ischemia/reperfusion injury. Different techniques are available to measure myocardial infarct size in humans and in experimental settings, however, accurate determination of the extent of infarction is necessary to evaluate interventions that may delay the onset of necrosis and/or limit the total extent of infarct size during ischemia/reperfusion. This paper highlights recent advances of the different techniques to measure infarct size.

Cholesterol diet‐induced hyperlipidemia influences gene expression pattern of rat hearts: a DNA microarray study

To profile gene expression patterns involved in the direct myocardial effect of cholesterol‐enriched diet‐induced hyperlipidemia, we monitored global gene expression changes by DNA microarray analysis of 3200 genes in rat hearts. Twenty‐six genes exhibited significant up‐regulation and 25 showed down‐regulation in hearts of rats fed a 2% cholesterol‐enriched diet for 8 weeks as compared to age‐matched controls. The expression changes of 12 selected genes were also assessed by real‐time quantitative polymerase chain reaction. Genes with altered expression in the heart due to hyperlipidemia included procollagen type III, cofilin/destrin, tensin, transcription repressor p66, synaptic vesicle protein 2B, Hsp86, chaperonin subunit 5ϵ, metallothionein, glutathione S‐transferase, protein kinase C inhibitor, ATP synthase subunit c, creatine kinase, chloride intracellular channel 4, NADH oxidoreductase and dehydrogenase, fibronectin receptor β chain, CD81 antigen, farnesyltransferase, calreticulin, disintegrin, p120 catenin, Smad7, etc. Although some of these genes have been suspected to be related to cardiovascular diseases, none of the genes has been previously shown to be involved in the mechanism of the cardiac effect of hyperlipidemia.

HEME OXYGENASE AND CARDIAC FUNCTION IN ISCHEMIC/REPERFUSED RAT HEARTS

We investigated whether the expression of heme oxygenase (HO) isozymes was related to the occurrence of ventricular fibrillation (VF) induced by ischemia/reperfusion in nondiabetic and diabetic myocardium. To study the role of HO-1 and HO-2 mRNA expression in VF, isolated hearts obtained from nondiabetic and 8-week diabetic rats were subjected to 30 min of ischemia followed by 2 h of reperfusion. Expression of HO-1 and HO-2 mRNA was studied in fibrillated and nonfibrillated myocardium using Northern blotting and reverse transcription polymerase chain reaction (RT-PCR). The effect of zinc protoporphyrin IX (Zn-PPIX), a potent inhibitor of HO activity, on HO activity was also studied in ischemic/reperfused hearts. Upon reperfusion, an expression of HO-1 was observed in nonfibrillated myocardium. HO-1 mRNA expression was significantly reduced in hearts showed VF. Zn-PPIX (5 microM) treatment reduced HO activity from its control values of 398+/-27 (in nondiabetics) and 370+/-20 pmol bilirubin/h (in diabetics) to 69+/-14 (in nondiabetics, p<.05) and 60+/-11 pmol bilirubin/h (in diabetics, p<.05), respectively, and all hearts, upon reperfusion, showed VF in both nondiabetic and diabetic subjects. HO-2 expression was unchanged in nonfibrillated and fibrillated myocardium. Postischemic function showed no correlation with the expression of these genes. Our data show that the mechanism(s) of ischemia/reperfusion-induced VF involves the downregulation of HO-1 mRNA and a reduction in HO activity. Furthermore, the mechanism(s) of VF at molecular level involving HO isozymes does not show a significant difference between nondiabetics and diabetics.