Age-Dependent Effects of Remote Preconditioning in Hypertensive Rat Hearts are Associated With Activation of RISK Signaling.

Remote ischemic preconditioning (RIPC) represents one of the forms of innate cardioprotection. While being effective in animal models, its application in humans has not been always beneficial, which might be attributed to the presence of various comorbidities, such as hypertension, or being related to the confounding factors, such as patients' sex and age. RIPC has been shown to mediate its cardioprotective effects through the activation of Reperfusion Injury Salvage Kinase (RISK) pathway in healthy animals, however, scarce evidence supports this effect of RIPC in the hearts of spontaneously hypertensive (SHR) rats, in particular, in relationship with aging. The study aimed to investigate the effectiveness of RIPC in male SHR rats of different age and to evaluate the role of RISK pathway in the effect of RIPC on cardiac ischemic tolerance. RIPC was performed using three cycles of inflation/deflation of the pressure cuff placed on the hind limb of anesthetized rats aged three, five and eight months. Subsequently, hearts were excised, Langendorff-perfused and exposed to 30-min global ischemia and 2-h reperfusion. Infarct-sparing and antiarrhythmic effects of RIPC were observed only in three and five months-old animals but not in eight months-old rats. Beneficial effects of RIPC were associated with increased activity of RISK and decreased apoptotic signaling only in three and five months-old animals. In conclusion, RIPC showed cardioprotective effects in SHR rats that were partially age-dependent and might be attributed to the differences in the activation of RISK pathway and various aspects of ischemia/reperfusion injury in aging animals.

Analysis of Signaling Pathways of Necroptotic and Pyroptotic Cell Death in the Hearts of Rats With Type 2 Diabetes Mellitus.

Diabetes mellitus is known to produce various cell-damaging events and thereby underlie heart dysfunction and remodeling. However, very little is known about its inflammation-associated pathomechanisms due to necrosis-like cell death. For this purpose, we aimed to investigate signaling pathways of necroptosis and pyroptosis, known to produce plasma membrane rupture with the resultant promotion of inflammation. One-year old Zucker diabetic fatty (ZDF) rats did not exhibit significant heart dysfunction as revealed by echocardiographic measurement. On the other hand, there was a decrease in heart rate due to diabetes. Immunoblotting analysis showed that the left ventricles of ZDF rats overexpress neither the main necroptotic proteins including receptor-interacting protein kinase 3 (RIP3) and mixed lineage domain kinase-like pseudokinase (MLKL), nor the pyroptotic regulators including NLR family pyrin domain containing 3 protein (NLRP3), caspase-1, interleukin-1 beta (IL-1beta and the N-terminal gasdermin D (GSDMD-N). On the other hand, the increased activation of the RIP3 kinase due to phosphorylation was found in such hearts. In summary, we showed for the first time that the activation of cardiac RIP3 is upregulated due to disturbances in glucose metabolism which, however, did not proceed to necrosis-like cell death. These data can indicate that the activated RIP3 might also underlie other pleiotropic, non-necroptotic signaling pathways under basal conditions.

Effects of necrostatin-1, an inhibitor of necroptosis, and its inactive analogue Nec-1i on basal cardiovascular function.

Inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIP1) by necrostatin-1 (Nec-1) alleviates cardiac injury due to prevention of necroptotic cell death. Its inactive analogue necrostatin-1i (Nec-1i), lacking RIP1 activity, serves as a suitable control. It is unknown if these agents influence the heart function in the absence of damaging stimuli. For this purpose, we measured intraarterial blood pressure (systolic - sBP and diastolic - dBP) and ECG parameters after a bolus administration of Nec-1 and Nec-1i in rats during 30 min. Nec-1, unlike Nec-1i, increased sBP and dBP, as well as heart rate reaching the peak at 20 min. The P wave duration tended to be decreased and the duration of the PR interval was shortened by Nec-1 indicating faster conduction of the impulses through atria to the ventricles. The drugs did not influence the QTc interval duration and no episode of ventricular arrhythmia was observed. In summary, Nec-1 temporarily modulates blood pressure and electrical function of the healthy heart. These effects of Nec-1 are likely due to its off-target action or RIP1 has an important role in the regulation of cardiovascular function independently of its action on the necroptotic pathway.

Pleiotropic effects of simvastatin are associated with mitigation of apoptotic component of cell death upon lethal myocardial reperfusion-induced injury.

Although statins exert non-lipid cardioprotective effects, their influence on cell death is not fully elucidated. For this purpose, we investigated whether simvastatin treatment (S, 10 mg/kg, 5 days) is capable of mitigating ischemia/reperfusion-induced (IR) apoptosis in the isolated rat hearts, which was examined using immunoblotting analysis. In addition, the content of signal transducer and activator of transcription 3 (STAT3) and its active form, phosphorylated STAT3 (pSTAT3-Thr(705)), was analyzed. Simvastatin induced neither variations in the plasma lipid levels nor alterations in the baseline content of analysed proteins with the exception of upregulation of cytochrome C. Furthermore, simvastatin significantly increased the baseline levels of pSTAT3 in contrast to the control group. In the IR hearts, simvastatin reduced the expression of Bax and non-cleaved caspase-3. In these hearts, phosphorylation of STAT3 did not differ in comparison to the non-treated IR group, however total STAT3 content was slightly increased. The improved recovery of left ventricular developed pressure co-existed with the increased Bcl-2/Bax ratio. In conclusion, pleiotropic action of statins may ameliorate viability of cardiomyocytes by favouring the expression of anti-apoptotic Bcl-2 and downregulating the pro-apoptotic markers; however STAT3 does not seem to be a dominant regulator of this anti-apoptotic action of simvastatin.