E. Y. Plotnikov

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and Age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke)

Effects of 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6′-plastoquinonyl) decylrhod-amine 19 (SkQR1) on rat models of H2O2- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H2O2 or by ischemia/reperfusion. Higher SkQ1 (125–250 nmol/kg per day for 2–3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2–4 days, whereas one injection of SkQ1 or SkQR1 (1 μmol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 μmol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound.

Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co‐cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria‐resembling structures were identified. Moreover, after co‐cultivation with cardiomyocytes, expression of human‐specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

New-generation Skulachev ions exhibiting nephroprotective and neuroprotective properties.

A mitochondria-targeted chimeric compound consisting of a rhodamine derivative linked to a plastoquinone molecule (10-(6′-plastoquinonyl)decylrhodamine, SkQR1) was studied under conditions of acute brain or kidney damage. A protective effect of this compound was demonstrated in a model of focal brain ischemia, rat kidney ischemia/reperfusion, myoglobinuria (rhabdomyolysis, or crush syndrome), and pyelonephritis. We found that a single intraperitoneal injection of SkQR1 diminishes the size of the ischemic zone in the brain and improves performance of a test characterizing neurological deficit in ischemic animals. An analog of SkQR1 not containing plastoquinone (C12R19) was not neuroprotective. The data show that SkQR1 is a nephroprotectant and neuroprotectant, which can be due to the antioxidative action of this Skulachev cation.

Reactive oxygen and nitrogen species: Friends or foes?

Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS)and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as “electric cables”, as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive “ROS induced ROS release”.

Interrelations of mitochondrial fragmentation and cell death under ischemia/reoxygenation and UV-irradiation: Protective effects of SkQ1, lithium ions and insulin

Mitochondria‐targeted antioxidant 10‐(6‐plastoquinonyl)decyltriphenyl‐phosphonium (SkQ1) as well as insulin and the inhibitor of glycogen‐synthase kinase, Li+ are shown to (i) protect renal tubular cells from an apoptotic death and (ii) diminish mitochondrial fission (the thread‐grain transition) induced by ischemia/reoxygenation. However, SkQ1 and LiCl protected the mitochondrial reticulum of skin fibroblasts from ultraviolet‐induced fission but were ineffective in preventing a further cell death. This means that mitochondrial fission is not essential for apoptotic cascade progression.

Role of acidosis, NMDA receptors, and acid-sensitive ion channel 1a (ASIC1a) in neuronal death induced by ischemia

This review collects data on the influence of intracellular and extracellular acidosis on neuronal viability and the effect of acidosis on neuronal damage progressing under brain ischemia/hypoxia. Particular attention is devoted to the involvement of ionotropic glutamic receptors and acid-sensitive ion channel 1a in these processes.

Nephroprotective effect of GSK-3β inhibition by lithium ions and δ-opioid receptor agonist dalargin on gentamicin-induced nephrotoxicity.

Nephrotoxicity and ototoxicity are the most considerable side effects of aminoglycoside antibiotics, such as gentamicin that seriously limits its application in medicine. The major mechanism of negative effect of gentamicin on kidney cells involves damage of mitochondria and induction of an oxidative stress that causes cell death resulting in kidney dysfunction. In this work we compared effects of the lithium ions and δ-opioid receptors agonist, dalargin on gentamicin-induced kidney injury. It was revealed that LiCl and dalargin treatment reduced renal tubular cell death and diminished kidney injury caused by gentamicin. Both LiCl and dalargin were found to enhance phosphorylation of glycogen synthase kinase 3β in the kidney which points to induction of nephroprotective signaling pathways. Thus, we conclude that lithium ions and dalargin might be considered as novel promising agents for future use to prevent negative consequences of therapy with aminoglycoside antibiotics.

Inhibition of GSK-3β decreases the ischemia-induced death of renal cells.

Pharmacological preconditioning with insulin and lithium ions prevented the death of renal cells under conditions of ischemia/reperfusion. Preincubation of cells with insulin or lithium ions decreased production of reactive oxygen species after ischemia/reoxygenation. These agents also prevented the development of mitochondrial dysfunction in renal cells induced by ischemia/reoxygenation. It was hypothesized that the protective effects of these agents are related to inhibition of glycogen synthase kinase-3(. This enzyme is inactivated upon phosphorylation of serine residue in position 9. We found that in vivo administration of lithium ions to animals before renal ischemia prevents the development of kidney failure.

Heterogeneity of Mitochondrial Potential as a Marker for Isolation of Pure Cardiomyoblast Population

Typical signs of cardiomyoblasts were determined: high mitochondrial membrane potential and high number of mitochondria in these cells compared to fibroblasts. These signs can be used for identification of these cells. Energy-dependent accumulation of highly specific mitochondrial fluorescent probes applied for visual detection of energized mitochondria allows clear-cut separation of the mixed population: cardiomyocyte population is characterized by higher transmembrane potential than concomitant cells. Using flow cytometry and cell sorting we obtained a population enriched with cardiomyocytes and cardiomyoblasts. Taking into account the fact that the dye has no toxic effect on cells and is rapidly eliminated, the risk of cell damage and death during isolation is considerably reduced compared to traditional methods. These results open possibilities for the development of a new specific method for isolation of cardiomyocyte culture from fetal and embryonic sources for their further use in clinical practice.

Dysfunction of kidney endothelium after ischemia/reperfusion and its prevention by mitochondria-targeted antioxidant

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen–glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.