Effects of diazoxide, benzothiadiazine and benzopyrane derivatives on mitochondrial proton and electron leaks of cardiomyocytes (H9C2 cell line).

Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of adenosine triphosphate (ATP) and reactive oxygen species (ROS). In many cancer cells those organelles become dysfunctional leading to a shift of energy metabolism from oxidative phosphorylation to active glycolysis and an increase of ROS generation. According to Warberg’ theory, cancer damage might occur at the mitochondrial level, affecting tiny structures within each cell implicated in the energy production through ATP. New insight is that mitochondria might be a good therapeutic target for metabolic syndromes, ischemia/reperfusion injury and organs transplantation. Therefore, search for novel molecules able to keep mitochondria functional are of relevant interest. Methodology: Cardiomyocytes (H9C2 cells) were from ATCC (USA) and grown till confluence. The basal cellular respiratory rate, proton and electron leaks as well as ATP production were measured with the High Resolution Oxygraphy (Oroboros, Austria). All compounds: diazoxide (DIAZ), diazoxide –related analogs (1: BPDZ-259, 2: BPDZ-444), and benzopyran derivatives (3: BPDZ-490, 4: BPDZ-711) were tested at final concentration of 10-5 M, except when specified and compared to control samples (cells with or without DMSO). Results and conclusion: The basal respiratory rate of H9C2 cells (5x106/mL) was changed depending on the chemical structure of the tested compounds: e.g. compound 3 strongly enhanced the routine respiration, while 4 displayed a marked lowering effect. In contrast, the addition of similar concentration of benzothiadiazin derivatives (1, 2) had no effect on routine respiration but also on the other respiratory parameters such as oligomycin-induced leak and ATP production. Similar profile was obtained with the reference molecule: diazoxide. Overall, our findings indicate that both diazoxide-like analogues (1 and 2) and diazoxide were without significant effect on basal respiration, ATP production, even on maximal respiration. Interestingly, two derivatives show opposite effects: compound 3 behaves as a uncoupling agent and the other one (4) exhibits a real lowering effect on respiration but that was reversible. The latter effect might be of interest if this kind of molecules could be used for further use as an agent for organ conservation during transplantation. Our results also demonstrate that diazoxide, a well-known Mito-KATP opener, did not exert its effect beside of clinical situation like ischemia/reperfusion injury

Assessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases.

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Animals—5 healthy horses (5 to 15 years old). Procedures—Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding. Results—Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. Conclusions and Clinical Relevance—Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation– treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses