RESUMO
Succinate accumulates during myocardial ischemia and is rapidly oxidized during reperfusion, leading to reactive oxygen species (ROS) production through reverse electron transfer (RET) from mitochondrial complex II to complex I, and favoring cell death. Given that connexin 43 (Cx43) modulates mitochondrial ROS production, we investigated whether Cx43 influences RET using inducible knock-out Cx43Cre-ER(T)/fl mice. Oxygen consumption, ROS production, membrane potential and coenzyme Q (CoQ) pool were analyzed in subsarcolemmal (SSM, expressing Cx43) and interfibrillar (IFM) cardiac mitochondria isolated from wild-type Cx43fl/fl mice and Cx43Cre-ER(T)/fl knock-out animals treated with 4-hydroxytamoxifen (4OHT). In addition, infarct size was assessed in isolated hearts from these animals submitted to ischemia-reperfusion (IR), and treated or not with malonate, a complex II inhibitor attenuating RET. Succinate-dependent ROS production and RET were significantly lower in SSM, but not IFM, from Cx43-deficient animals. Mitochondrial membrane potential, a RET driver, was similar between groups, whereas CoQ pool (2.165 ± 0.338 vs. 4.18 ± 0.55 nmol/mg protein, p < 0.05) and its reduction state were significantly lower in Cx43-deficient animals. Isolated hearts from Cx43Cre-ER(T)/fl mice treated with 4OHT had a smaller infarct size after IR compared to Cx43fl/fl, despite similar concentration of succinate at the end of ischemia, and no additional protection by malonate. Cx43 deficiency attenuates ROS production by RET in SSM, but not IFM, and was associated with a decrease in CoQ levels and a change in its redox state. These results may partially explain the reduced infarct size observed in these animals and their lack of protection by malonate.
Assuntos
Conexina 43 , Camundongos Knockout , Mitocôndrias Cardíacas , Espécies Reativas de Oxigênio , Ubiquinona , Animais , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Ubiquinona/metabolismo , Ubiquinona/deficiência , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Conexina 43/metabolismo , Conexina 43/genética , Espécies Reativas de Oxigênio/metabolismo , Camundongos , Transporte de Elétrons/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/genética , MasculinoRESUMO
Introduction: Atypical hemolytic uremic syndrome (aHUS) is a complement system (CS)-mediated ultrarare disease that manifests as thrombotic microangiopathy (TMA) with preferential small kidney vessels involvement. Transient CS activation is also observed in secondary TMA or in patients at risk of developing aHUS. There is no gold standard test to monitor disease activity; however, the ex vivo C5b-9 deposition test seems to be a good approach. Methods: We assessed the C5b-9 deposition induced by serum samples of patients with aHUS (n = 8) and with TMA associated with kidney (n = 2), lung (n = 1) or hematopoietic stem cell (HSC) transplantation (HSCT, n = 2) during the acute phase of the disease or in remission. As control for transplant-associated TMA (TA-TMA), we analyzed samples of clinically stable kidney and HSC-transplanted patients without signs of TMA. In addition, we studied 1 child with genetic risk of aHUS during an acute infection. Results: In the acute disease phase or in patients with disease activity despite C5 blockade, a significant increase of C5b-9 deposition was detected. In all patients with clinical response to C5 blockade but one, levels of C5b-9 deposition were within the normal range. Finally, we detected increased C5b-9 deposition levels in an asymptomatic child with genetic risk of aHUS when a concomitant otitis episode was ongoing. Conclusion: The ex vivo C5b-9 deposition test is an auspicious tool to monitor CS activity in aHUS and TA-TMA. In addition, we demonstrate that the test may be useful to detect subclinical increase of CS activity, which expands the spectrum of patients that would benefit from a better CS activity assessment.