Elena Iurova

Myocardial infarction is one of the common cardiovascular diseases. Sodium overload in the myocardium is another of the most important mechanisms underlying ischemia/reperfusion injury. Na⁺ entry into the cell through ion channels can be controlled using peptide toxins targeting ion channels. In our work, we analyze the use of mu-agatoxin-Aa1a, as a sodium channel modulator, as a cytoprotective agent in ischemia/reperfusion injury. For IRI modeling in vitro, the H9C2 culture was placed under oxygen-glucose deprivation conditions followed by reperfusion. Cell death and ion dynamics were recorded using a fluorescent plate reader. Reversible myocardial ischemia of rats in vivo was created by applying an occluder under the main branch of the left coronary artery. Histological and biochemical changes were recorded. The study demonstrated the ability of mu-agatoxin-Aa1a myocardial cells to adapt to rapidly changing extracellular conditions upon restoration of normal blood flow after a period of ischemia. Thus, the study allows us to consider mu-agatoxin-Aa1a as a biologically active molecule with a targeted effect to enhance the adaptation of cardiomyocytes to OGD/reperfusion conditions.

Ischemia-reperfusion injury (IRI) is a complex process accompanying cessation of blood supply to an organ or tissue followed by subsequent restoration of blood circulation. The IRI is especially prominent in surgery and organ transplantation. One of the strategies for reducing organ and tissue damage during transplantation is regulation of intracellular ion concentrations. Maintenance of ion concentrations in the cell during damage development can be controlled by influencing voltage-dependent ion channels with certain types of compounds. We propose the peptide toxins tropic to calcium (omega-hexatoxin-Hv1a) and sodium (mu-agatoxin-Aa1a) voltage-dependent ion channels as potential agents reducing IRI. The toxins were obtained using solid-phase peptide synthesis. The IRI modeling for evaluation of the action of toxins was carried out on a culture of epithelial cells CHO-K1 during their incubation under conditions of hypoxia and nutrient deprivation followed by subsequent replenishment of the nutrient medium. The level of cell death, concentrations of calcium, sodium, potassium ions, and pH were recorded using a multimodal plate reader and fluorescent dyes. Experiments have shown that regardless of different mechanisms of action, both toxins reduced the development of CHO-K1 cell death by changing ion concentrations and maintaining the pH level.

Ischemia–reperfusion injury (IRI) is a common phenomenon that develops both from natural causes and during major operations. Many intracellular processes mediated by calcium ions are involved in the development of IRI. Currently, chemical calcium channel blockers are used but they have a number of limitations. In this article, we study the effect of the omega-hexatoxin-Hv1a peptide toxin, an alternative to chemical calcium channel blockers, on the mechanisms of IRI development in epithelial cell culture. The toxin was produced using solid phase peptide synthesis. IRI was caused by deprivation of glucose, serum and oxygen. The data obtained demonstrate that the omega-hexatoxin-Hv1a toxin in nanomolar concentrations is able to prevent the development of apoptosis and necrosis in epithelial cells by reducing the concentration of calcium, sodium and potassium ions, as well as by delaying rapid normalization of the pH level, affecting the mitochondrial potential and oxidative stress. This toxin can be used as an alternative to chemical calcium channel blockers for preventing tissue and organ IRI due to its low-dose requirement and high bioavailability.

Ischemia-reperfusion injury (IRI) is an irreversible functional and structural injury. Restoration of normal oxygen concentration exacerbates the emergence and development of deadly cells. One of the possible moments of reperfusion damage to cells is an increase in the intracellular concentration of sodium ions. In this article, we study the mu-agatoxin-Aa1a, a modulator of sodium channels, on the processes of IRI cells damage. The toxin was synthesized using an automatic peptide synthesizer. Hypoxia was induced by reducing the content of serum and oxygen in the CHO-K1 culture. The influence of the toxin on the level of apoptosis; intracellular concentration of sodium, calcium, and potassium ions; intracellular pH; totality of reactive oxygen species (ROS), nitric oxide (NO), and ATP; and changes in the mitochondrial potential were studied. The experiments performed show that mu-agatoxin-Aa1a effectively prevents IRI of cells. Toxin reduces the level of apoptosis and prevents a decrease in the intracellular concentration of sodium and calcium ions during IRI. Mu-agatoxin-Aa1a contributes to the maintenance of elevated intracellular pH, reduces the intracellular concentration of ROS, and prevents the decrease in intracellular NO concentration and mitochondrial potential under conditions of reoxygenation/reperfusion. An analysis of experimental data shows that the mu-agatoxin-Aa1a peptide has adaptogenic properties. In the future, this peptide can be used to prevent ischemia/reperfusion tissue damage different genesis.

Apoptosis and necrosis during reperfusion after ischemia are key mechanisms at the cellular level leading to damage. The development of pathological conditions is preceded by intracellular calcium ion overload both at the stage of ischemia and at the stage of reperfusion. In this regard, one of the strategies aimed at reducing damage during ischemia/reperfusion is associated with the use of calcium channel blockers. The aim of the study was to study the effect of a peptide toxin, a calcium channel blocker omega-hexatoxin-Hv1a, on different types of epithelial cell death during in vitro reconstruction of ischemia/reperfusion conditions characteristic of organ transplantation. Materials and Methods. In this study, we used CHO-K1 epithelial cell culture. Changes in apoptosis, necrosis, cell index, and calcium ion concentration were assessed when modeling ischemia/reperfusion processes in vitro with the addition of a calcium channel blocker toxin. Ischemic and reperfusion injury was achieved by oxygen and nutrient deprivation followed by reperfusion in a complete nutrient medium. The measurements were performed using a multimodal plate reader-fluorimeter. Results. An increase in apoptosis, necrosis, and the concentration of calcium ions was recorded when modeling ischemia/reperfusion processes. A decrease in the level of apoptosis and necrosis, as well as the concentration of calcium ions to a physiological level or a level close to physiological, was noted when the toxin was added at a concentration of 50 nM at the reperfusion stage. The cell index showed a faster restoration in the presence of the toxin. Conclusion. The experimental data confirm the hypothesis of a beneficial effect of peptide calcium channel blockers on the state of epithelial cells during reperfusion after ischemia and can be considered for further study as a strategy for organ adaptation before reperfusion.