Name: CLEYDIANNE LUISA VIEIRA PEREIRA
Publication date: 24/08/2017
Advisor:
Name |
Role |
|---|---|
| IVANITA STEFANON | Advisor * |
| JONES BERNARDES GRACELI | Co-advisor * |
Examining board:
| Name |
Role |
|---|---|
| AURÉLIA ARAÚJO FERNANDES | External Examiner * |
| IVANITA STEFANON | Advisor * |
| JONES BERNARDES GRACELI | Co advisor * |
| JOSE GERALDO MILL | Internal Examiner * |
Summary: Tributyltin (TBT) is an organotin environmental contaminant used in farming and antifouling paints. Its release directly into the water, from ships, shipyards and ports, was the cause of environmental impacts on aquatic ecosystems. Numerous studies indicate that organotin compounds produce neuro, cito and genotoxic effects in various systems. However, its toxic effect on the cardiovascular system has not yet been fully elucidated. The aim of this study was to analyze the acute effect of TBT on myocardial contractility Wistar rats weighing 200 -250 g were anesthetized with intraperitoneal injection of ketamine (40 mg / kg) and Xilazine (8 mg / kg). The heart was isolated and perfused by the Langendorff Technique in Krebs solution, pH 7.4, 37° C. The left ventricular isovolumetric systolic pressure (LVISP) was assessed by insertion of a latex balloon in the LV which was stretched to measure left ventricular diastolic pressure (DP). Experimental animals were randomly grouped into: Control group (N = 7) and group perfused for 5 minutes with TBT solution (50 μM) (TBT group N = 8). To evaluate the participation of reactive oxygen species (ROS) on the effects of TBT, hearts were perfused with anti-oxidants: Tiron (500 μM, N = 5), Tempol (100 μM, N = 5), Apocynin 100 μM, N = 4) and angiotensin receptor blocker, Losartan (10 μM, N = 5). Myocardial contractility was evaluated by homeometric stimulus: calcium and β-adrenergic agonist isoproterenol (injection in bolus, 100 μL, 10-4 M). The heterometric response was assessed using the Frank Starling mechanism by increasing the DP from 0 to 30 mmHg in 5 mmHg intervals. The acute effect of TBT on ROS was evaluated by dihydroethidium technique (Arbitrary Units, AU). In another group of rats, cardiomyocytes were isolated using collagenase in order to measure Ca2+ sparks frequency and amplitude during rest condition and Ca2+ transient in cells stimulated at 0.5 Hz. The Ca2+ content of the sarcoplasmic reticulum (SR) was evaluated using caffeine (10 mM) in intact cardiomyocytes. The results were presented as mean ± SEM. The statistical analysis used was ANOVA 1 or 2 ways with Tukey post hoc, p <0.05. All protocols were approved by CEUA/UFES (27/2016). Perfusion with TBT induced a negative inotropic effect evidenced by the lower contractile response to calcium increase (Control = 115 ± 9 vs TBT = 66 ± 4 mmHg, 1.25 mM CaCl2, p <0.05). Acute exposure to TBT resulted in a reduction in the pressure developed in the Frank Starling curve, in 1.25 mM calcium in all DP (Control= 88 ± 6 vs TBT = 45 ± 2 mmHg, DP = 10 mmHg, p <0.05) and only the antioxidant tiron reversed this reduction in lower DP (TBT = 45 ± 2 vs TBT + tiron = 79 ± 6 mmHg, p <0.05). TBT significantly reduced the response to isoproterenol (Control = 132.78 ± 22 vs TBT = 24,14 ± 13.08 mmHg, p <0.05). Total exposure to TBT increased in situ production of O2‾ (Control group = 0.065 ± 0.002 vs TBT group 0.094 ± 0.004 AU p <0.05) and only Tempol and Losartan were able to reverse (TBT + Tempol 0.074 ± 0.003 and TBT+Losartan 0.071 ± 0.004 AU, p <0.05). TBT increased the sparks frequency (Control= 9 ± 1 vs TBT 10-7 M = 16 ± 1.1 μm/s), decreased its amplitude (Control = 0.582 ± 0.08 vs TBT 10-7 M = 0.342 ± 0.05, p <0, 05), decreased the Ca2+ transient and the SR Ca2+ content. The results demonstrate that TBT induced an important negative inotropic effect that may depend on the Ca2+ regulatory proteins destabilizing the RyR2 receptor and reducing the activity of the SR Ca2+ pump, SERCA2a, which appear to be modulated, at least in part, by ROS.
