Adrian Szobi

Necroptotic cell death in failing heart: relevance and proposed mechanisms

As cardiomyocytes have a limited capability for proliferation, renewal, and repair, the loss of heart cells followed by replacement with fibrous tissue is considered to result in the development of ventricular dysfunction and progression to heart failure (HF). The loss of cardiac myocytes in HF has been traditionally believed to occur mainly due to programmed apoptosis or unregulated necrosis. While extensive research work is being carried out to define the exact significance and contribution of both these cell death modalities in the development of HF, recent knowledge has indicated the existence and importance of a different form of cell death called necroptosis in the failing heart. This new cell damaging process, resembling some of the morphological features of passive necrosis as well as maladaptive autophagy, is a programmed process and is orchestrated by a complex set of proteins involving receptor-interacting protein kinase 1 and 3 (RIP1, RIP3) and mixed lineage kinase domain-like protein (MLKL). Activation of the RIP1–RIP3–MLKL signaling pathway leads to disruption of cation homeostasis, plasma membrane rupture, and finally cell death. It seems likely that inhibition of any site in this pathway may prove as an effective pharmacological intervention for preventing the necroptotic cell death in the failing heart. This review is intended to describe general aspects of the signaling pathway associated with necroptosis, to describe its relationship with cardiac dysfunction in some models of cardiac injury and discuss its potential relevance in various types of HF with respect to the underlying pathologic mechanisms.

Analysis of necroptotic proteins in failing human hearts

BackgroundCell loss and subsequent deterioration of contractile function are hallmarks of chronic heart failure (HF). While apoptosis has been investigated as a participant in the progression of HF, it is unlikely that it accounts for the total amount of non-functional tissue. In addition, there is evidence for the presence of necrotic cardiomyocytes in HF. Therefore, the objective of this study was to investigate the necroptotic proteins regulating necroptosis, a form of programmed necrosis, and thereby assess its potential role in human end-stage HF.MethodsLeft ventricular samples of healthy controls (C) and patients with end-stage HF due to myocardial infarction (CAD) or dilated cardiomyopathy (DCM) were studied. Immunoblotting for necroptotic and apoptotic markers was performed. Triton X-114 fractionated samples were analyzed to study differences in subcellular localization.ResultsElevated expression of RIP1 (receptor-interacting protein), pSer227-RIP3 and its total levels were observed in HF groups compared to controls. On the other hand, caspase-8 expression, a proapoptotic protease negatively regulating necroptosis, was downregulated suggesting activation of necroptosis signaling. Total mixed-lineage kinase domain-like protein (MLKL) expression did not differ among the groups; however, active cytotoxic forms of MLKL were present in all HF samples while they were expressed at almost undetectable levels in controls. Interestingly, pThr357-MLKL unlike pSer358-MLKL, was higher in DCM than CAD. In HF, the subcellular localization of both RIP3 and pThr357-MLKL was consistent with activation of necroptosis signaling. Expression of main apoptotic markers has not indicated importance of apoptosis.ConclusionsThis is the first evidence showing that human HF of CAD or DCM etiology is positive for markers of necroptosis which may be involved in the development of HF.

Upregulation of CaMKIIδ during ischaemia-reperfusion is associated with reperfusion-induced arrhythmias and mechanical dysfunction of the rat heart: involvement of sarcolemmal Ca2+-cycling proteins.

Although Ca(2+)/calmodulin-dependent protein kinase II delta (CaMKIIδ) has been implicated in development of different phenotypes of myocardial ischaemia-reperfusion injury, its involvement in arrhythmogenesis and cardiac stunning is not sufficiently elucidated. Moreover, the mechanisms by which CaMKIIδ mediates disturbances in excitation-contraction coupling, are not exactly known. To investigate this, KN-93 (0.5 µmol/L), a CaMKII inhibitor, was administered before induction of global ischaemia and reperfusion in isolated Langendorff-perfused rat hearts. Expression of CaMKIIδ and the sarcollemal Ca(2+)-cycling proteins, known to be activated during reperfusion, was analyzed using immunoblotting. KN-93 reduced reperfusion-induced ectopic activity and the incidence of ventricular fibrillation. Likewise, the severity of arrhythmias was lower in KN-treated hearts. During the pre-ischaemia phase, neither inotropic nor chronotropic effects were elicited by KN-93, whereas post-ischaemic contractile recovery was significantly improved. Ischaemia-reperfusion increased the expression of CaMKIIδ and sodium-calcium exchanger (NCX1) proteins without any influence on the protein content of alpha 1c, a pore-forming subunit of L-type calcium channels (LTCCs). On the other hand, inhibition of CaMKII normalized changes in the expression of CaMKIIδ and NCX1. Taken together, CaMKIIδ seems to regulate its own turnover and to be an important component of cascade integrating NCX1, rather than LTCCs that promote ischaemia-reperfusion-induced contractile dysfunction and arrhythmias.

Oxidative activation of CaMKIIδ in acute myocardial ischemia/reperfusion injury: A role of angiotensin AT1 receptor-NOX2 signaling axis.

During ischemia/reperfusion (IR), increased activation of angiotensin AT1 receptors recruits NADPH oxidase 2 (NOX2) which contributes to oxidative stress. It is unknown whether this stimulus can induce oxidative activation of Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ) leading into the aggravation of cardiac function and whether these effects can be prevented by angiotensin AT1 receptors blockade. Losartan, a selective AT1 blocker, was used. Its effects were compared with effects of KN-93, an inhibitor of CaMKIIδ. Global IR was induced in Langendorff-perfused rat hearts. Protein expression was evaluated by immunoblotting and lipoperoxidation was measured by TBARS assay. Losartan improved LVDP recovery by 25%; however, it did not reduce reperfusion arrhythmias. Oxidized CaMKIIδ (oxCaMKIIδ) was downregulated at the end of reperfusion compared to before ischemia and losartan did not change these levels. Phosphorylation of CaMKIIδ mirrored the pattern of changes in oxCaMKIIδ levels. Losartan did not prevent the higher lipoperoxidation due to IR and did not influence NOX2 expression. Inhibition of CaMKII ameliorated cardiac IR injury; however, this was not accompanied with changes in the levels of either active form of CaMKIIδ in comparison to the angiotensin AT1 receptor blockade. In spite of no changes of oxCaMKIIδ, increased cardiac recovery of either therapy was abolished when combined together. This study showed that oxidative activation of CaMKIIδ is not elevated at the end of R phase. NOX2-oxCAMKIIδ signaling is unlikely to be involved in cardioprotective action of angiotensin AT1 receptor blockade which is partially abolished by concomitant CaMKII inhibition.

Evidence of necroptosis in hearts subjected to various forms of ischemic insults

Long-lasting ischemia can result in cell loss; however, repeated episodes of brief ischemia increase the resistance of the heart against deleterious effects of subsequent prolonged ischemic insult and promote cell survival. Traditionally, it is believed that the supply of blood to the ischemic heart is associated with release of cytokines, activation of inflammatory response, and induction of necrotic cell death. In the past few years, this paradigm of passive necrosis as an uncontrolled cell death has been re-examined and the existence of a strictly regulated form of necrotic cell death, necroptosis, has been documented. This controlled cell death modality, resembling all morphological features of necrosis, has been investigated in different types of ischemia-associated heart injuries. The process of necroptosis has been found to be dependent on the activation of RIP1-RIP3-MLKL axis, which induces changes leading to the rupture of cell membrane. This pathway is activated by TNF-α, which has also been implicated in the cardioprotective signaling pathway of ischemic preconditioning. Thus, this review is intended to describe the TNF-α-mediated signaling leading to either cell survival or necroptotic cell death. In addition, some experimental data suggesting a link between heart dysfunction and the cellular loss due to necroptosis are discussed in various conditions of myocardial ischemia.

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

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

Necrostatins have been shown to retard necroptosis, a programmed necrotic-like cell death, which has been shown to underlie pathophysiology of various diseases. Nec-1s, a novel highly effective necrostatin, overcomes some drawbacks of former necrostatin analogues. The determination of Nec-1s in biological system, however, has not been carried out so far. Therefore, this study was undertaken to optimize and validate the HPLC-DAD-Q-TOF method for the assessment of Nec-1s levels in the plasma what is the necessity for designing its proper dosing regimen for in vivo studies. Benefits of the proposed analytical protocol include: (i) simple sample preparation (precipitation of plasma proteins, evaporation of acetonitrile, reconstitution in mobile phase), (ii) fast, selective and sensitive analysis due to a highly orthogonal LC-MS system providing less than 8 min analysis time, (iii) detection of Nec-1s without any matrix interferences, and quantitation of very low concentration levels of Nec-1s (LLOQ ~ 20 ng/mL), (iv) high reliability of Nec-1s determination with precision and accuracy values meeting the FDA criteria for biomedical analysis. The proposed analytical protocol is suitable for routine use in relevant biological studies, and, in this work, it was successfully applied for monitoring of Nec-1s plasma levels in rats providing reproducible and consistent results. Based on pharmacokinetic features, which can also be assessed due to the results of this study, there will be efforts to perform both acute and chronic in vivo studies and potential clinical safety studies first.

Consecutive Isoproterenol and Adenosine Treatment Confers Marked Protection against Reperfusion Injury in Adult but Not in Immature Heart: A Role for Glycogen

Consecutive treatment of adult rat heart with isoproterenol and adenosine (Iso/Aden), known to consecutively activate PKA/PKC signaling, is cardioprotective against ischemia and reperfusion (I/R). Whether this is cardioprotective in an immature heart is unknown. Langendorff–perfused hearts from adult and immature (60 and 14 days old) male Wistar rats were exposed to 30 min ischemia and 120 min reperfusion, with or without prior perfusion with 5 nM Iso for 3 min followed by 30 μM Aden for 5 min. Changes in hemodynamics (developed pressure and coronary flow) and cardiac injury (Lactate Dehydrogenase (LDH) release and infarct size) were measured. Additional hearts were used to measure glycogen content. Iso induced a similar inotropic response in both age groups. Treatment with Iso/Aden resulted in a significant reduction in time to the onset of ischemic contracture in both age groups whilst time to peak contracture was significantly shorter only in immature hearts. Upon reperfusion, the intervention reduced cardiac injury and functional impairment in adults with no protection of immature heart. Immature hearts have significantly less glycogen content compared to adult. This work shows that Iso/Aden perfusion confers protection in an adult heart but not in an immature heart. It is likely that metabolic differences including glycogen content contribute to this difference.

Erratum to: Hypercholesterolemia downregulates autophagy in the rat heart

Erratum Following publication of the original article [1], it came to the attention of authors that the definition of the term “isolated hypercholesterolemia” used frequently in the article is missing. In this paper, authors define isolated hypercholesterolemia as a hypercholesterolemia without an accompanying hypertriglyceridemia. Therefore, we would like to amend the Background paragraph of the Abstract with the following sentence: “However, it is unknown whether isolated hypercholesterolemia (i.e., hypercholesterolemia without hypertriglyceridemia) disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways.” In addition, the second sentence of Background section was also incorrectly phrased; which we would like to correct as follows: “The role of atherosclerosis is well studied in these pathologies; however, myocardial effects of hypercholesterolemia is less well understood.”

Erratum to: Analysis of necroptotic proteins in failing human hearts

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Erratum to: J Transl Med (2017) 15:86 DOI 10.1186/s12967‐017‐1189‐5 In the original version of this article [1], published on 28 April 2017, the name of author ‘Zoltán V. Varga’ was wrongly displayed. Originally the author name has been published as: