Cardioprotection of ischaemic preconditioning is associated with inhibition of translocation of MLKL within the plasma membrane

Abstract

Necroptosis, a form of cell loss involving the RIP1‐RIP3‐MLKL axis, has been identified in cardiac pathologies while its inhibition is cardioprotective. We investigated whether the improvement of heart function because of ischaemic preconditioning is associated with mitigation of necroptotic signaling, and these effects were compared with a pharmacological antinecroptotic approach targeting RIP1. Langendorff‐perfused rat hearts were subjected to ischaemic preconditioning with or without a RIP1 inhibitor (Nec‐1s). Necroptotic signaling and the assessment of oxidative damage and a putative involvement of CaMKII in this process were analysed in whole tissue and subcellular fractions. Ischaemic preconditioning, Nec‐1s and their combination improved postischaemic heart function recovery and reduced infarct size to a similar degree what was in line with the prevention of MLKL oligomerization and translocation to the membrane. On the other hand, membrane peroxidation and apoptosis were unchanged by either approach. Ischaemic preconditioning failed to ameliorate ischaemia–reperfusion‐induced increase in RIP1 and RIP3 while pSer229‐RIP3 levels were reduced only by Nec‐1s. In spite of the additive phosphorylation of CaMKII and PLN because of ditherapy, the postischaemic contractile force and relaxation was comparably improved in all the intervention groups while antiarrhythmic effects were observed in the ischaemic preconditioning group only. Necroptosis inhibition seems to be involved in cardioprotection of ischaemic preconditioning and is comparable but not intensified by an anti‐RIP1 agent. Changes in oxidative stress nor CaMKII signaling are unlikely to explain the beneficial effects.

Determination of Novel Highly Effective Necrostatin Nec-1s in Rat Plasma by High Performance Liquid Chromatography Hyphenated with Quadrupole-Time-of-Flight Mass Spectrometry

“…Nec-1s has not been studied in clinical trials so far; only in vitro and ex vivo animal studies have been performed with this agent [ 10 , 15 , 16 , 17 ]. Likewise, according to our best knowledge there are no in vivo animal studies investigating the anti-necroptotic effects of Nec-1s.…”

Simultaneous Suppression of Multiple Programmed Cell Death Pathways by miRNA-105 in Cardiac Ischemic Injury

“…However, whether all cell death mechanisms in MI affect subsequent cardiac remodeling processes remains largely unknown. Recent studies have suggested that necroptosis inhibition is involved in cardioprotection of ischemic preconditioning and is associated with inhibition of the translocation of MLKL within the plasma membrane 38 . Very recently, it was shown that Ripk3 promotes endoplasmic reticulum (ER) stress-induced necroptosis via the calcium overload/XO/ROS/mPTP opening axis in cardiac IR injury 33 …”

Antiarrhythmic Effects of Melatonin and Omega-3 Are Linked with Protection of Myocardial Cx43 Topology and Suppression of Fibrosis in Catecholamine Stressed Normotensive and Hypertensive Rats

“…TBARS levels were analyzed, as a marker of oxidative stress and lipoperoxidation, according to Shlafer and Shepard (1984) [ 38 ] with modifications [ 39 ]. 40 µL of tissue homogenates and 40 µL of 20% trichloroacetic acid solution were mixed with a 320 µL of TBARS reagent (37 mmol/L C4H 4 N 2 O 2 S, 500 mmol/L NaOH, 15% v / v CH 3 COOH) and incubated for 70 min at 100 °C.…”

“…TBARS levels were analyzed, as a marker of oxidative stress and lipoperoxidation, according to Shlafer and Shepard (1984) [38] with modifications [39]. 40 µL of tissue homogenates and 40 µL of 20% trichloroacetic acid solution were mixed with a 320 µL of TBARS reagent (37 mmol/L C4H 4 N 2 O 2 S, 500 mmol/L NaOH, 15% v/v CH 3 COOH) and incubated for 70 min at 100 • C. Subsequently, the mixture was cooled on ice for 10 min, pipetted into another tube with a mixture of n-butanol and pyridine (14:1, v/v), and centrifuged 10 min at 5000× g. The organic phase was used for measurement of absorbance at 535 nm by Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, Vermont, USA).…”