This review gives an overview of the mechanisms of recently described new mode of programmed cell death called necroptosis. We summarize recent studies on the role of necroptosis in the development of various diseases. In addition, the data on pathological changes at the cellular and tissue level induced by targeted deletion of genes encoding key mediators of necroptosis are presented. The limited experience on the use of pharmacological necroptosis modulators for prevention of tissue/organ injury is also described. Furthermore, the perspectives of the clinical use of necroptosis modulators are critically evaluated.

This article describes in detail the modified method of heterotopic heart transplantation in rats. The methods of animal anesthesia, surgical features of the removal of a donor heart and its implantation to a recipient rat are described in detail. The advantages and disadvantages of donor heart preservation methods are described, as well as methods for evaluating a donor heart preparation, the quality of preservation and the effectiveness of cardioprotection. Experimental approaches to the study of the effectiveness of cardioplegic and preservative solutions using this technique are proposed. The data of our own pilot study on a comparative analysis of the effectiveness of cardioprotection using preservative solutions using this experimental model are presented.

Objective.To investigate the effect of necroptosis inhibition on the morphofunctional state of the myocardium and the expression of microRNA 223 after heterotopic allogeneic heart transplantation in rat.Design and methods. Twenty Wistar rats were examined in the study. Animals were divided into the following groups: 1) control (n = 7), 2) dimethyl sulfoxide (DMSO) (n = 6), 3) necrostatin-1s (n = 7). Necrostatin-1s was used as an inhibitor of necroptosis, which was administered intraperitoneally in DMSO solution 1 hour before the start of the experiment at a dose of 1,65 mg/kg. HTK solution cooled to 4 °C was used as a preservation solution. Two hours after heart arrest, the heart was heterotopically transplanted in the abdominal cavity of recipient rat using the scheme “aorta-aorta, pulmonary trunc-posterior vena cava”. Three hours later, intracardiac hemodynamics was assessed by recording the pressure in the left ventricle, heart rate and coronary flow rate in Langendorff-perfused heart. The expression level of microRNA 223–5p and –3p in left ventricular myocardium was assessed using real-time polymerase chain reaction. The plasma levels of troponin I were assessed by enzyme immunoassay. Myocardial infarct size was measured planimetrically at the end of the experiment by staining myocardium with triphenyltetrazolium chloride.Results. Inhibition of necroptosis significantly improved the morphofunctional state of the myocardium, which manifested in a decrease of myocardial infarct size in the necrostatin-1s group compared with the control group and DMSO group. Thus, in the necrostatin-1s group, myocardial infarct size was 25 ± 8,7 %, which was smaller than in the control and DMSO groups (56 ± 9,5 and 57 ± 8,7 %, respectively; p < 0,05). Also in the necrostatin-1s group, lower diastolic intraventricular pressure was recorded, as well as higher values of pulse intraventricular pressure and coronary flow rate than in control group and DMSO group (p < 0,05). Left ventricular myocardium in the necrostatin-1s group demonstrated higher expression of the antinecroptotic miRNA 223–5p and –3p as compared with the control and DMSO groups, as well as lower plasma levels of troponin I (p < 0,05).Conclusions.Pharmacological inhibition of necroptosis in heterotopically transplanted donor heart is accompanied by marked cardioprotective effects and increases the expression of antinecroptotic microRNA 223–5p and –3p.

Objective. To compare the cardioprotective effcacy of a preservation solution based on Krebs–Henseleit buffer and HTK solution in the model of heterotopic heart transplantation in rat.Design and methods. A study was conducted on 12 Wistar rats. The animals were divided into groups depending on the preservation solution used: 1) Krebs–Henseleit buffer-based solution (n = 7), 2) HTK (n = 5). Each experiment consisted of collecting donor heart, preserving it with an appropriate cardioplegic solution, heterotopic transplantation into a recipient rat followed by explantation and evaluation of left ventricular contractility using the Langendorff model and histochemical assessment of the irreversible myocardial damage. Coronary blood flow in the donor heart was assessed in vivo using ultrasound doppler flowmetry. After 3 hours, the donor heart was explanted and connected to the Langendorff apparatus to assess left ventricular contractility, and the myocardium was subjected to histochemical staining with 1 % triphenyltetrazolium chloride for the assessment of the irreversible myocardial damage.Results. In the group of Krebs–Henseleit buffer-based cardioplegic solution, 7 experiments were performed. Myocardial infarct size was 3,5 ± 1,2%, the coronary flow rate was 4,5 ± 1,3 ml/min, and the developed left ventricular pressure of the donor heart was 70 ± 6,3 mmHg at diastolic left ventricular pressure of 10 mmHg. In the HTK solution group (n = 5), in all of the experiments after the start of blood flow, the transplanted heart did not begin to contract, and all 5 hearts remained in an asystole state. Therefore, after the end of the 3-hour reperfusion period, the assessment of the contractility of the left ventricle using the Langendorff apparatus was not carried out. Coronary flow rate was only 0,4 ± 0,1 ml/min, probably resulting from inadequate cardioprotection with HTK solution. Due to the lack of adequate reperfusion, the accurate assessment of the irreversible myocardial damage was impossible in the HTK solution group.Conclusions. The technique of heterotopic heart transplantation in rats is adequate and informative for the study of ischemia-reperfusion myocardial damage as well as for the study of the effectiveness of cardioplegic and cardiac preservation solutions. Cardioplegic solution based on Krebs– Henseleit buffer demonstrated greater cardioprotective effectiveness in our model compared to HTK solution.

Objective . To investigate the effects of various necroptosis inhibitors on the morphological and functional characteristics of the myocardium in the model of prolonged cold preservation of the donor rat heart. Design and methods. Thirty eight Wistar rats were divided into the following groups: 1) control (n = 7), 2) dimethylsulfoxide (DMSO) (n = 7), 3) necrostatin-1s (n = 6), 4) necrosulfonamide (n = 6), 5) necrostatin-5 (n = 6), 6) necrostatin-1 (n = 6). Necroptosis inhibitors were administered by intraperitoneal injection 1 hour prior to the start of the experiment, previously dissolved in DMSO. HTK solution cooled to 4 °C was used as a preservative solution. The functional state of the myocardium was assessed by continuous recording of intraventricular pressure, heart rate and coronary flow velocity. Morphological changes in the myocardium were assessed by the size of myocardial necrosis, measured planimetrically after the end of 8-hour ischemia and 2-hour reperfusion. Results. All investigated necroptosis inhibitors significantly improved the morphological and functional state of the myocardium, which manifested itself in a significantly smaller amount of myocardial necrosis in the groups of necroptotic inhibitors in comparison with control groups and DMSO. Thus, in the groups of necrosulfonamide and necrostatin-1s, myocardial necrosis size was 31 ± 8,7 % and 27 ± 8,2 %, respectively; in necrostatin-5 and necrostatin-1 groups — 37 ± 9,2 % and 34 ± 7,8, respectively, which is lower than in control groups and DMSO (58 ± 8,6 % and 60 ± 7,6 %, respectively, p < 0,05). Also, diastolic intraventricular pressure was lower and pulse intraventricular pressure and coronary flow velocity were higher during the reperfusion period in the groups of necroptosis inhibitors compared to control group and DMSO group (p < 0,05). We did not find any significant differences between necroptosis inhibitors regarding cardioprotective activity in the model of prolonged cold preservation of the donor heart. Conclusions. All inhibitors of necroptosis equally improved the morphological and functional state of the myocardium in the model of prolonged static cold storage of the donor heart.

Objective . To investigate the effects of necroptosis inhibitors — necrosulfonamide and necrostatin‑1s — on morphofunctional characteristics of the myocardium in the model of prolonged cold preservation of the donor rat heart. Design and methods . The study was performed on 27 Wistar rats. The animals were divided into the following groups: 1) control (n = 7), 2) dimethylsulfoxide (DMSO, n = 6), 3) necrostatin‑1s (n = 8), 4) necrosulfonamide (n = 6). All test compounds were administered intraperitoneally 1 hour before the start of the experiment. Histidine-tryptophan-ketoglutarate (HTK) solution cooled to 4 °C was used as a preservative agent. DMSO was used as a solvent for necrostatin‑1s and necrosulfonamide. During the experiment, left ventricular pressure, heart rate and coronary flow were recorded. At the end of 8‑hour of ischemia and 2‑hour of reperfusion, myocardial infarct size was planimetrically evaluated. Results . Necrostatin-1s and necrosulfonamide show cardioprotective effects, as evidenced by the significantly lower levels of myocardial necrosis in these groups, as well as the best indices of intracardiac hemodynamics during reperfusion. In the groups of necrosulfonamide and necrostatin‑1s, myocardial infarct size was 32,2 ± 9,6 and 29,0 ± 9,2 %, respectively, which is significantly lower than in control groups and DMSO (54,4 ± 6,6 and 59,2 ± 5,6 %, respectively, p < 0,05). During reperfusion in necrosulfonamide and necrostatin‑1s groups, higher values of pulse intraventricular pressure and coronary flow rates were recorded, as well as lower diastolic intraventricular pressure, compared to control group and DMSO (p < 0,05). Conclusions . Necroptosis inhibitors necrostatin‑1s and necrosulfonamide improve morphofunctional characteristics of the myocardium state during prolonged cold preservation of the donor heart.

Introduction and purpose. A number of landmark trials have demonstrated clear benefits of metformin therapy in the prevention of macrovascular outcomes. Nevertheless, there is a lack of robust evidence to suggest whether metformin therapy will have similar beneficial outcomes in one of the most serious type 2 diabetes-related renal microvascular complications known as diabetic nephropathy. The study aimed to evaluate the effects of ten-week metformin treatment on renal morphofunctional changes in rats with non-genetic type 2 diabetic nephropathy. Materials and methods. Starting at 3 weeks after unilateral nephrectomy, adult male Wistar rats were fed the high-fat diet for 5 weeks, and then successively received nicotinamide (230 mg/kg) and streptozotocin (65 mg/kg) intraperitoneally in 15-min interval. Results. Starting at 11 weeks after confirmation of diabetes, metformin treatment did not attenuate routine renal dysfunction markers such as creatinine, creatinine clearance and albuminuria compared to placebo-treated diabetic group, and glomerulosclerosis index and glomerular expression of type IV collagen didn't significantly change either. Nevertheless, level of urinary kidney injury molecule-1, considered to be the marker of tubular damage in diabetes, was significantly lower in metformin-treated animals. Moreover, reduction of tubulointerstitial lesion tended to be significant. Conclusions. Under conditions of diabetic nephropathy modeling, metformin has shown direct protective effects against diabetic tubular disturbance. To assess long-term renal outcomes of these findings, more pre-clinical studies and clinical trials are required.

Minimization of irreversible myocardial damage after ischemia-reperfusion episode remains valid and unsolved problem. Among of many pharmacological agents have been effective in experimental studies, only a few of them are evidence of its efficacy in clinical trials. This is largely explained by the mechanism of action of these compounds, aimed primarily at preventing reperfusion injury, and do not affect myocardial cells subjected to prolonged ischemia without reperfusion. In recent years, the interest of researchers confined to the mechanisms of programmed necrosis or necroptosis, which morphologically has no different to necrosis, but has molecular targets for suppression. In this paper, on the model of global ischemia-reperfusion in rats we have studied cardioprotective effects of high-activity and low-toxicity necroptosis inhibitors - necrosulfonamide and necrostatin-1s. We demonstrated the infarct-limiting effect of these compounds, as well as the best parameters of intracardiac hemodynamics after an episode of global ischemia-reperfusion. We believe these compounds are interesting for further preclinical studies.

The cardioprotective effects of necroptosis inhibitor necrostatin-7 were studied in the rat model of permanent coronary occlusion. It was found that intraperitoneal injection of necrostatin-7 at a dose of 14.5 mg/kg 60 minutes prior to permanent left coronary artery occlusion reduced the amount of scar tissue and scar length in the left ventricle on the 21st day after surgery. In addition, pretreatment with necrostatin-7 resulted in decreased plasma level of N-terminal pro-brain natriuretic peptide, which points to the improvement in left ventricular function.

Cardioprotective effects of necrostatin-1 (necroptosis inhibitor) and 3-methyladenine (autophagy inhibitor) were studied on the model of long-term cold preservation of rat heart. Addition of necrostatin-1 (490 nmol/liter) or 3-methyladenine (4.5 mmol/liter) to custodiol preserving solution reduced myocardial infarction size and improved left-ventricular function during reperfusion after 8-h preservation at 4°C. Inhibition of necroptosis and autophagy contributed to the cardioprotective effect under conditions of cold preservation of the donor heart.

One of the most important aims of modern transplantation is the preservation of the morphofunctional state of donor organs for as long as possible time after removing them from the donor prior to implantation into a patient. To solve this problem, heart preservation solutions are used. However, despite the large number of prescriptions of preservation solutions, the time, limited to six hours, after removing donor heart is still a limiting factor in determining the possibility of implanting heart into a patient. In this study, cardioprotective effects of widely used in clinical practice preservation solution HTK and new Krebs-Henseleit buffer-based preservation solution were studied in the rat model of prolonged static cold storage. It was shown that new Krebs-Henseleit buffer-based preservation solution protected the heart significantly even after an eight-hour global ischemia and has the advantage over Custodiol in the infarct-limiting action.

The cardioprotective effects of necroptosis inhibitors necrostatin-1 and necrostatin-5 were studied on the isolated heart model in rats. Intraperitoneal injection of necrostatin-1 (1.65 mg/kg) or necrostatin-5 (2.46 mg/kg) 60 min before reperfusion of the isolated heart reduced the infarction zone caused by 30-min global ischemia and 120-min reperfusion. Intracoronary injection of necrostatin-1 (44.5 μmol/liter) caused an increase of left-ventricular systolic pressure, that is produced a positive inotropic effect, but did not reduce the infarction zone.

Cardioprotective properties of dimethyl sulfoxide (DMSO) were studied in the isolated rat heart model. Intraperitoneal administration of DMSO to animals for 3 days before the experiment, but not addition of DMSO to the perfusate, reduced infarction size. Both intraperitoneal and intracoronary administration of DMSO reduced the severity of postischemic left-ventricular dysfunction.