Ênio R. Vasques

Trisulfate Disaccharide Decreases Calcium Overload and Protects Liver Injury Secondary to Liver Ischemia/Reperfusion.

Background Ischemia and reperfusion (I/R) causes tissue damage and intracellular calcium levels are a factor of cell death. Sodium calcium exchanger (NCX) regulates calcium extrusion and Trisulfated Disaccharide (TD) acts on NCX decreasing intracellular calcium through the inhibition of the exchange inhibitory peptide (XIP). Objectives The aims of this research are to evaluate TD effects in liver injury secondary to I/R in animals and in vitro action on cytosolic calcium of hepatocytes cultures under calcium overload. Methods Wistar rats submitted to partial liver ischemia were divided in groups: Control: (n = 10): surgical manipulation with no liver ischemia; Saline: (n = 15): rats receiving IV saline before reperfusion; and TD: (n = 15): rats receiving IV TD before reperfusion. Four hours after reperfusion, serum levels of AST, ALT, TNF-α, IL-6, and IL-10 were measured. Liver tissue samples were collected for mitochondrial function and malondialdehyde (MDA) content. Pulmonary vascular permeability and histologic parameters of liver were determined. TD effect on cytosolic calcium was evaluated in BRL3A hepatic rat cell cultures stimulated by thapsigargin pre and after treatment with TD. Results AST, ALT, cytokines, liver MDA, mitochondrial dysfunction and hepatic histologic injury scores were less in TD group when compared to Saline Group (p<0.05) with no differences in pulmonary vascular permeability. In culture cells, TD diminished the intracellular calcium raise and prevented the calcium increase pre and after treatment with thapsigargin, respectively. Conclusion TD decreases liver cell damage, preserves mitochondrial function and increases hepatic tolerance to I/R injury by calcium extrusion in Ca2+ overload situations.

HEPARIN OLIGOSACCHARIDES HAVE ANTIARRHYTHMIC EFFECT BY ACCELERATING THE SODIUM-CALCIUM EXCHANGER

Background: Blockage of the Na+/Ca2+ exchanger (NCX) is used to determine the role of NCX in arrhythmogenesis. Trisulfated heparin disaccharide (TD) and Low Molecular Weight Heparins (LMWHs) can directly interact with the NCX and accelerate its activity. Objective: In this work, we investigated the antiarrhythmic effect of heparin oligosaccharides related to the NCX activity. Methods: The effects of heparin oligosaccharides were tested on the NCX current (patch clamping) and intracellular calcium transient in rat cardiomyocytes. The effects of heparin oligosaccharides were further investigated in arrhythmia induced in isolated rat atria and rats in vivo. Results: The intracellular Ca2+ concentration decreases upon treatment with either enoxaparin or ardeparin. These drugs abolished arrhythmia induction in isolated atria. The NCX antagonist KB-R7943 abolished the enoxaparin or ardeparin antiarrhythmic effects in isolated atria. In the in vivo measurements, injection of TD 15 min both before coronary occlusion or immediately after reperfusion, significantly prevented the occurrence of reperfusion-induced arrhythmias (ventricular arrhythmia and total AV block) and reduced the lethality rate. The patch clamping experiments showed that, mechanistically, TD increases the forward mode NCX current. Conclusion: Together, the data shows that heparin oligosaccharides may constitute a new class of antiarrhythmic drug that acts by accelerating the forward mode NCX under calcium overload.

Cardioprotective effect of preconditioning is more efficient than postconditioning in rats submitted to cardiac ischemia and reperfusion

PURPOSE To investigate the cardioprotective effects of ischemic preconditioning (preIC) and postconditioning (postIC) in animal model of cardiac ischemia/reperfusion. METHODS Adult rats were submitted to protocol of cardiac ischemia/reperfusion (I/R) and randomized into three experimental groups: cardiac I/R (n=33), preCI + cardiac I/R (n=7) and postCI + cardiac I/R (n=8). After this I/R protocol, the incidence of ventricular arrhythmia (VA), atrioventricular block (AVB) and lethality (LET) was evaluated using the electrocardiogram (ECG) analysis. RESULTS After reestablishment of coronary blood flow, we observed variations of the ECG trace with increased incidence of ventricular arrhythmia (VA) (85%), atrioventricular block (AVB) (79%), and increase of lethality (70%) in cardiac I/R group. The comparison between I/R + preIC group with I/R group demonstrated significant reduction in VA incidence to 28%, AVB to 0% and lethality to 14%. The comparison of I/R + postIC group with I/R group was observed significance reduction in AVB incidence to 25% and lethality to 25%. CONCLUSION The preconditioning strategies produce cardioprotection more efficient that postconditioning against myocardial dysfunctions and lethality by cardiac ischemia and reperfusion.

THE M-RNA, EXPRESSION OF SERCA2 AND NCX1 IN THE PROCESS OF PHARMACOLOGICAL CELL PROTECTION IN EXPERIMENTAL ACUTE PANCREATITIS INDUCED BY TAUROCHOLATE

ABSTRACT Background: Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. Aim: To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Methods: Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Results: Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. Conclusions: The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

A Simple and Efficient Methodology for the Study of Cardioprotective Drugs in Animal Model of Cardiac Ischemia-Reperfusion

To study cardioprotective drugs, we developed a simple and efficient methodology to evaluate effects of drugs on cardiac electrical activity using electrocardiogram (ECG) in rats submitted to cardiac ischemia-reperfusion (I/R). Using adult male Wistar rats (14 - 16-week-old) anesthetized (urethane 1.25 g/kg, i.p.) and kept under mechanical ventilation, we used surgical procedures to induce cardiac I/R by means mechanical occlusion of left anterior descendent coronary artery for 10 min (ischemia) with silk suture (tourniquet) followed by its removal to allow coronary recirculation (reperfusion). To evaluate the effects of surgical process, a group of rats (SHAM-operated) was submitted to surgical procedures previously described, but without coronary occlusion. To evaluate cardiac electrical activity in rats submitted to cardiac I/R and SHAM-operated, the ECG system was coupled to animal body to determine the incidence of ventricular arrhythmia (VA), atrio-ventricular blockade (AVB) and lethality (LET). To evaluate injury biomarkers production in rats submitted to cardiac I/R and SHAM-operated, serum concentrations of creatine kinase fraction (MB/CK-MB) and troponin I were determined by biochemical techniques. Using the methodology proposed in this work, we observed that VA, AVB and LET incidence was significantly higher in cardiac I/R group (85%, 79% and 70%, respectively) than in SHAM-operated group (0%, 0% and 0%, respectively). Serum levels of CK-MB and troponin I were also significantly higher in cardiac I/R (1,850 ± 222 U/L and 0.031 ± 0.009 ng/mL, respectively) compared to SHAM-operated group (808 ± 72 U/L and 0.200 ± 0.027 ng/mL). To evaluate efficiency of methodology proposed in this work to study the effect of cardioprotective drugs, the effects of the L-type Ca2+ channel blocker (CCB) nifedipine (30 mg/kg, intravenously - IV) in rats submitted to cardiac I/R were studied. The treatment with nifedipine (before ischemia) significantly reduced the incidence of VA (from 85% to 28%), AVB (from 79% to 14%), and LET (from 70% to 14%). These results indicate that the methodology described in the present work is simple, and efficient, to evaluate cardiac functional and biochemical alterations induced by cardiac I/R, and also the study of cardioprotective drugs in rats. This methodology could contribute to the development of new pharmacological cardioprotective strategies for to treatment of ischemic cardiac diseases in humans, such as myocardial infarction.