Analysis of the serum levels of RIP3 and Drp1 in patients with heart failure.

AIMS: As necroptosis involving receptor-interacting protein kinase 3 (RIP3) and dynamin-related protein 1 (Drp1)-mediated signalling is a crucial mechanism of cell loss in heart failure (HF), we aimed to determine the potential diagnostic use of these molecules. METHODS AND RESULTS: The serum samples of the healthy subjects (n = 8) and patients with HF with reduced ejection fraction (n = 31), being subdivided according to the aetiology and New York Heart Association (NYHA) class, were used to measure RIP3 and Drp1 levels by enzyme-linked immunosorbent assay. Although the serum levels of Drp1 in the patients with HF were comparable with those seen in healthy individuals, we found a trend of increase in the levels of RIP3 (P = 0.0697) in the diseased group. These changes were unlikely dependent on the HF aetiology or NYHA class. The circulating RIP3 correlated with neither the main parameters assessing cardiac function (left ventricular ejection fraction, left ventricular end-diastolic diameter, and N-terminal pro-brain natriuretic peptide) nor the marker of inflammation (C-reactive protein). CONCLUSIONS: In this pilot study, findings on serum RIP3 supported the importance of necroptosis in HF pathomechanisms. The potential diagnostic use of circulating RIP3, unlike Drp1, as an additional biomarker of HF has also been indicated; however, further large studies are needed to prove this concept.

Behavior of Hypertrophied Right Ventricle during the Development of Left Ventricular Failure Due to Myocardial Infarction.

In order to determine the behavior of the right ventricle, we have reviewed the existing literature in the area of cardiac remodeling, signal transduction pathways, subcellular mechanisms, ß-adrenoreceptor-adenylyl cyclase system and myocardial catecholamine content during the development of left ventricular failure due to myocardial infarction. The right ventricle exhibited adaptive cardiac hypertrophy due to increases in different signal transduction pathways involving the activation of protein kinase C, phospholipase C and protein kinase A systems by elevated levels of vasoactive hormones such as catecholamines and angiotensin II in the circulation at early and moderate stages of heart failure. An increase in the sarcoplasmic reticulum Ca2+ transport without any changes in myofibrillar Ca2+-stimulated ATPase was observed in the right ventricle at early and moderate stages of heart failure. On the other hand, the right ventricle showed maladaptive cardiac hypertrophy at the severe stages of heart failure due to myocardial infarction. The upregulation and downregulation of ß-adrenoreceptor-mediated signal transduction pathways were observed in the right ventricle at moderate and late stages of heart failure, respectively. The catalytic activity of adenylate cyclase, as well as the regulation of this enzyme by Gs proteins, were seen to be augmented in the hypertrophied right ventricle at early, moderate and severe stages of heart failure. Furthermore, catecholamine stores and catecholamine uptake in the right ventricle were also affected as a consequence of changes in the sympathetic nervous system at different stages of heart failure. It is suggested that the hypertrophied right ventricle may serve as a compensatory mechanism to the left ventricle during the development of early and moderate stages of heart failure.

Is Intrinsic Cardioprotection a Laboratory Phenomenon or a Clinically Relevant Tool to Salvage the Failing Heart?

Cardiovascular diseases, especially ischemic heart disease, as a leading cause of heart failure (HF) and mortality, will not reduce over the coming decades despite the progress in pharmacotherapy, interventional cardiology, and surgery. Although patients surviving acute myocardial infarction live longer, alteration of heart function will later lead to HF. Its rising incidence represents a danger, especially among the elderly, with data showing more unfavorable results among females than among males. Experiments revealed an infarct-sparing effect of ischemic "preconditioning" (IPC) as the most robust form of innate cardioprotection based on the heart's adaptation to moderate stress, increasing its resistance to severe insults. However, translation to clinical practice is limited by technical requirements and limited time. Novel forms of adaptive interventions, such as "remote" IPC, have already been applied in patients, albeit with different effectiveness. Cardiac ischemic tolerance can also be increased by other noninvasive approaches, such as adaptation to hypoxia- or exercise-induced preconditioning. Although their molecular mechanisms are not yet fully understood, some noninvasive modalities appear to be promising novel strategies for fighting HF through targeting its numerous mechanisms. In this review, we will discuss the molecular mechanisms of heart injury and repair, as well as interventions that have potential to be used in the treatment of patients.

Novel, non-conventional pathways of necroptosis in the heart and other organs: Molecular mechanisms, regulation and inter-organelle interplay.

Necroptosis, a cell death modality that is defined as a necrosis-like cell death depending on the receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL), has been found to underlie the injury of various organs. Nevertheless, the molecular background of this cell loss seems to also involve, at least under certain circumstances, some novel axes, such as RIPK3-PGAM5-Drp1 (mitochondrial protein phosphatase 5-dynamin-related protein 1), RIPK3-CaMKII (Ca2+/calmodulin-dependent protein kinase II) and RIPK3-JNK-BNIP3 (c-Jun N-terminal kinase-BCL2 Interacting Protein 3). In addition, endoplasmic reticulum stress and oxidative stress via the higher production of reactive oxygen species produced by the mitochondrial enzymes and the enzymes of the plasma membrane have been implicated in necroptosis, thereby depicting an inter-organelle interplay in the mechanisms of this cell death. However, the role and relationship between these novel non-conventional signalling and the well-accepted canonical pathway in terms of tissue- and/or disease-specific prioritisation is completely unknown. In this review, we provide current knowledge on some necroptotic pathways being not directly associated with RIPK3-MLKL execution and report studies showing the role of respective microRNAs in the regulation of necroptotic injury in the heart and in some other tissues having a high expression of the pro-necroptotic proteins.

Intermittent Hypoxic Preconditioning Plays a Cardioprotective Role in Doxorubicin-Induced Cardiomyopathy.

Intermittent hypoxic preconditioning (IHP) is a well-established cardioprotective intervention in models of ischemia/reperfusion injury. Nevertheless, the significance of IHP in different cardiac pathologies remains elusive. In order to investigate the role of IHP and its effects on calcium-dependent signalization in HF, we employed a model of cardiomyopathy induced by doxorubicin (Dox), a widely used drug from the class of cardiotoxic antineoplastics, which was i.p. injected to Wistar rats (4 applications of 4 mg/kg/week). IHP-treated group was exposed to IHP for 2 weeks prior to Dox administration. IHP ameliorated Dox-induced reduction in cardiac output. Western blot analysis revealed increased expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) while the expression of hypoxia inducible factor (HIF)-1-α, which is a crucial regulator of hypoxia-inducible genes, was not changed. Animals administered with Dox had further decreased expression of TRPV1 and TRPV4 (transient receptor potential, vanilloid subtype) ion channels along with suppressed Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. In summary, IHP-mediated improvement in cardiac output in the model of Dox-induced cardiomyopathy is likely a result of increased SERCA2a expression which could implicate IHP as a potential protective intervention in Dox cardiomyopathy, however, further analysis of observed effects is still required.

Facts and ideas on statins with respect to their lipophilicity: a focus on skeletal muscle cells and bone besides known cardioprotection.

Statins are known to block cholesterol synthesis in the liver. They also exhibit non-lipid pleiotropic effects due to the inhibition of protein prenylation, thereby modulating various signaling pathways of cellular homeostasis and integrity. Both lipid control and pleiotropic action of statins are clinically used, mainly for treatment of hypercholesterolemia and primary and secondary prevention of cardiovascular diseases. Because the prescription of statins is increasing and statin therapy is often lifelong, in particular in patients with other risk factors, safety issues being associated with polymorbidity and polypragmasia as well as the persistence with and adherence to statins are specific points of attention of clinicians and clinical pharmacologists. Furthermore, because skeletal myocytes have a cholesterol inhibitory sensitivity greater than hepatocytes, a choice of an appropriate statin based on its lipophilicity and the associated likelihood of its side effects on skeletal muscle cells and bone is warranted in such polymorbid patients. These approaches can effectively modulate the risk: benefit ratio and highlight a need for personalized therapy as much as possible, thereby minimizing risk of discontinuation of therapy and poor compliance.

Quercetin alleviates diastolic dysfunction and suppresses adverse pro-hypertrophic signaling in diabetic rats.

Introduction: Quercetin (Que) is a potent anti-inflammatory and antioxidant flavonoid with cardioprotective potential. However, very little is known about the signaling pathways and gene regulatory proteins Que may interfere with, especially in diabetic cardiomyopathy. Therefore, we aimed to study the potential cardioprotective effects of Que on the cardiac phenotype of type 2 diabetes mellitus (T2DM) accompanied by obesity. Methods: For this experiment, we used Zucker Diabetic Fatty rats (fa/fa) and their age-matched lean controls (fa/+) that were treated with either vehicle or 20 mg/kg/day of Que for 6 weeks. Animals underwent echocardiographic (echo) examination before the first administration of Que and after 6 weeks. Results: After the initial echo examination, the diabetic rats showed increased E/A ratio, a marker of left ventricular (LV) diastolic dysfunction, in comparison to the control group which was selectively reversed by Que. Following the echo analysis, Que reduced LV wall thickness and exhibited an opposite effect on LV luminal area. In support of these results, the total collagen content measured by hydroxyproline assay was decreased in the LVs of diabetic rats treated with Que. The follow-up immunoblot analysis of proteins conveying cardiac remodeling pathways revealed that Que was able to interfere with cardiac pro-hypertrophic signaling. In fact, Que reduced relative protein expression of pro-hypertrophic transcriptional factor MEF2 and its counter-regulator HDAC4 along with pSer246-HDAC4. Furthermore, Que showed potency to decrease GATA4 transcription factor, NFAT3 and calcineurin, as well as upstream extracellular signal-regulated kinase Erk5 which orchestrates several pro-hypertrophic pathways. Discussion: In summary, we showed for the first time that Que ameliorated pro-hypertrophic signaling on the level of epigenetic regulation and targeted specific upstream pathways which provoked inhibition of pro-hypertrophic signals in ZDF rats. Moreover, Que mitigated T2DM and obesity-induced diastolic dysfunction, therefore, might represent an interesting target for future research on novel cardioprotective agents.

Role of Oxidative Stress in Cardiac Dysfunction and Subcellular Defects Due to Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury is well-known to be associated with impaired cardiac function, massive arrhythmias, marked alterations in cardiac metabolism and irreversible ultrastructural changes in the heart. Two major mechanisms namely oxidative stress and intracellular Ca2+-overload are considered to explain I/R-induced injury to the heart. However, it is becoming apparent that oxidative stress is the most critical pathogenic factor because it produces myocardial abnormalities directly or indirectly for the occurrence of cardiac damage. Furthermore, I/R injury has been shown to generate oxidative stress by promoting the formation of different reactive oxygen species due to defects in mitochondrial function and depressions in both endogenous antioxidant levels as well as regulatory antioxidative defense systems. It has also been demonstrated to adversely affect a wide variety of metabolic pathways and targets in cardiomyocytes, various resident structures in myocardial interstitium, as well as circulating neutrophils and leukocytes. These I/R-induced alterations in addition to myocardial inflammation may cause cell death, fibrosis, inflammation, Ca2+-handling abnormalities, activation of proteases and phospholipases, as well as subcellular remodeling and depletion of energy stores in the heart. Analysis of results from isolated hearts perfused with or without some antioxidant treatments before subjecting to I/R injury has indicated that cardiac dysfunction is associated with the development of oxidative stress, intracellular Ca2+-overload and protease activation. In addition, changes in the sarcolemma and sarcoplasmic reticulum Ca2+-handling, mitochondrial oxidative phosphorylation as well as myofibrillar Ca2+-ATPase activities in I/R hearts were attenuated by pretreatment with antioxidants. The I/R-induced alterations in cardiac function were simulated upon perfusing the hearts with oxyradical generating system or oxidant. These observations support the view that oxidative stress may be intimately involved in inducing intracellular Ca2+-overload, protease activation, subcellular remodeling, and cardiac dysfunction as a consequence of I/R injury to the heart.

Analysis of necroptosis and its association with pyroptosis in organ damage in experimental pulmonary arterial hypertension.

In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group-sacrificed 4 weeks after MCT administration and ptMCT group-prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr231 /Ser232 -RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr231 /Ser232 -RIP3 levels in both PAH subgroups were associated rather with GSDMD-mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH-induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr231 /Ser232 -RIP3 upregulation resulting in two different necrosis-like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.

Involvement of Oxidative Stress in the Development of Subcellular Defects and Heart Disease.

It is now well known that oxidative stress promotes lipid peroxidation, protein oxidation, activation of proteases, fragmentation of DNA and alteration in gene expression for producing myocardial cell damage, whereas its actions for the induction of fibrosis, necrosis and apoptosis are considered to result in the loss of cardiomyocytes in different types of heart disease. The present article is focused on the discussion concerning the generation and implications of oxidative stress from various sources such as defective mitochondrial electron transport and enzymatic reactions mainly due to the activation of NADPH oxidase, nitric oxide synthase and monoamine oxidase in diseased myocardium. Oxidative stress has been reported to promote excessive entry of Ca2+ due to increased permeability of the sarcolemmal membrane as well as depressions of Na+-K+ ATPase and Na+-Ca2+ exchange systems, which are considered to increase the intracellular of Ca2+. In addition, marked changes in the ryanodine receptors and Ca2+-pump ATPase have been shown to cause Ca2+-release and depress Ca2+ accumulation in the sarcoplasmic reticulum as a consequence of oxidative stress. Such alterations in sarcolemma and sarcoplasmic reticulum are considered to cause Ca2+-handling abnormalities, which are associated with mitochondrial Ca2+-overload and loss of myofibrillar Ca2+-sensitivity due to oxidative stress. Information regarding the direct effects of different oxyradicals and oxidants on subcellular organelles has also been outlined to show the mechanisms by which oxidative stress may induce Ca2+-handling abnormalities. These observations support the view that oxidative stress plays an important role in the genesis of subcellular defects and cardiac dysfunction in heart disease.

Interplay of Oxidative Stress and Necrosis-like Cell Death in Cardiac Ischemia/Reperfusion Injury: A Focus on Necroptosis.

Extensive research work has been carried out to define the exact significance and contribution of regulated necrosis-like cell death program, such as necroptosis to cardiac ischemic injury. This cell damaging process plays a critical role in the pathomechanisms of myocardial infarction (MI) and post-infarction heart failure (HF). Accordingly, it has been documented that the modulation of key molecules of the canonical signaling pathway of necroptosis, involving receptor-interacting protein kinases (RIP1 and RIP3) as well as mixed lineage kinase domain-like pseudokinase (MLKL), elicit cardioprotective effects. This is evidenced by the reduction of the MI-induced infarct size, alleviation of myocardial dysfunction, and adverse cardiac remodeling. In addition to this molecular signaling of necroptosis, the non-canonical pathway, involving Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated regulation of mitochondrial permeability transition pore (mPTP) opening, and phosphoglycerate mutase 5 (PGAM5)-dynamin-related protein 1 (Drp-1)-induced mitochondrial fission, has recently been linked to ischemic heart injury. Since MI and HF are characterized by an imbalance between reactive oxygen species production and degradation as well as the occurrence of necroptosis in the heart, it is likely that oxidative stress (OS) may be involved in the mechanisms of this cell death program for inducing cardiac damage. In this review, therefore, several observations from different studies are presented to support this paradigm linking cardiac OS, the canonical and non-canonical pathways of necroptosis, and ischemia-induced injury. It is concluded that a multiple therapeutic approach targeting some specific changes in OS and necroptosis may be beneficial in improving the treatment of ischemic heart disease.

Inhibition of Cardiac RIP3 Mitigates Early Reperfusion Injury and Calcium-Induced Mitochondrial Swelling without Altering Necroptotic Signalling.

Receptor-interacting protein kinase 3 (RIP3) is a convergence point of multiple signalling pathways, including necroptosis, inflammation and oxidative stress; however, it is completely unknown whether it underlies acute myocardial ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts subjected to 30 min ischemia followed by 10 min reperfusion exhibited compromised cardiac function which was not abrogated by pharmacological intervention of RIP3 inhibition. An immunoblotting analysis revealed that the detrimental effects of I/R were unlikely mediated by necroptotic cell death, since neither the canonical RIP3-MLKL pathway (mixed lineage kinase-like pseudokinase) nor the proposed non-canonical molecular axes involving CaMKIIδ-mPTP (calcium/calmodulin-dependent protein kinase IIδ-mitochondrial permeability transition pore), PGAM5-Drp1 (phosphoglycerate mutase 5-dynamin-related protein 1) and JNK-BNIP3 (c-Jun N-terminal kinase-BCL2-interacting protein 3) were activated. Similarly, we found no evidence of the involvement of NLRP3 inflammasome signalling (NOD-, LRR- and pyrin domain-containing protein 3) in such injury. RIP3 inhibition prevented the plasma membrane rupture and delayed mPTP opening which was associated with the modulation of xanthin oxidase (XO) and manganese superoxide dismutase (MnSOD). Taken together, this is the first study indicating that RIP3 regulates early reperfusion injury via oxidative stress- and mitochondrial activity-related effects, rather than cell loss due to necroptosis.

Natural and synthetic antioxidants targeting cardiac oxidative stress and redox signaling in cardiometabolic diseases.

Cardiometabolic diseases (CMDs) are metabolic diseases (e.g., obesity, diabetes, atherosclerosis, rare genetic metabolic diseases, etc.) associated with cardiac pathologies. Pathophysiology of most CMDs involves increased production of reactive oxygen species and impaired antioxidant defense systems, resulting in cardiac oxidative stress (OxS). To alleviate OxS, various antioxidants have been investigated in several diseases with conflicting results. Here we review the effect of CMDs on cardiac redox homeostasis, the role of OxS in cardiac pathologies, as well as experimental and clinical data on the therapeutic potential of natural antioxidants (including resveratrol, quercetin, curcumin, vitamins A, C, and E, coenzyme Q10, etc.), synthetic antioxidants (including N-acetylcysteine, SOD mimetics, mitoTEMPO, SkQ1, etc.), and promoters of antioxidant enzymes in CMDs. As no antioxidant indicated for the prevention and/or treatment of CMDs has reached the market despite the large number of preclinical and clinical studies, a sizeable translational gap is evident in this field. Thus, we also highlight potential underlying factors that may contribute to the failure of translation of antioxidant therapies in CMDs.

The Molecular Mechanisms of Iron Metabolism and Its Role in Cardiac Dysfunction and Cardioprotection.

Iron is an essential mineral participating in different functions of the organism under physiological conditions. Numerous biological processes, such as oxygen and lipid metabolism, protein production, cellular respiration, and DNA synthesis, require the presence of iron, and mitochondria play an important role in the processes of iron metabolism. In addition to its physiological role, iron may be also involved in the adaptive processes of myocardial "conditioning". On the other hand, disorders of iron metabolism are involved in the pathological mechanisms of the most common human diseases and include a wide range of them, such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease, and accelerate the development of atherosclerosis. Furthermore, iron also exerts potentially deleterious effects that may be manifested under conditions of ischemia/reperfusion (I/R) injury, myocardial infarction, heart failure, coronary artery angioplasty, or heart transplantation, due to its involvement in reactive oxygen species (ROS) production. Moreover, iron has been recently described to participate in the mechanisms of iron-dependent cell death defined as "ferroptosis". Ferroptosis is a form of regulated cell death that is distinct from apoptosis, necroptosis, and other types of cell death. Ferroptosis has been shown to be associated with I/R injury and several other cardiac diseases as a significant form of cell death in cardiomyocytes. In this review, we will discuss the role of iron in cardiovascular diseases, especially in myocardial I/R injury, and protective mechanisms stimulated by different forms of "conditioning" with a special emphasis on the novel targets for cardioprotection.

Programmed Cell Death in the Left and Right Ventricle of the Late Phase of Post-Infarction Heart Failure.

While necroptosis has been shown to contribute to the pathogenesis of post-infarction heart failure (HF), the role of autophagy remains unclear. Likewise, linkage between these two cell death modalities has not been sufficiently investigated. HF was induced by 60-min left coronary occlusion in adult Wistar rats and heart function was assessed 6 weeks later followed by immunoblotting analysis of necroptotic and autophagic proteins in both the left (LV) and right ventricle (RV). HF had no effect on RIP1 and RIP3 expression. PhosphoSer229-RIP3, acting as a pro-necroptotic signal, was increased in LV while deceased in RV of failing hearts. Total MLKL was elevated in RV only. Decrease in pSer555-ULK1, increase in pSer473-Akt and no significant elevation in beclin-1 and LC3-II/I ratio indicated rather a lowered rate of autophagy in LV. No beclin-1 upregulation and decreased LC3 processing also suggested the inhibition of both autophagosome formation and maturation in RV of failing hearts. In contrast, p89 PARP1 fragment, a marker of executed apoptosis, was increased in RV only. This is the first study showing a different signaling in ventricles of the late phase of post-infarction HF, highlighting necroptosis itself rather than its linkage with autophagy in LV, and apoptosis in RV.

Role of Oxidative Stress in the Genesis of Ventricular Arrhythmias.

Ventricular arrhythmias, mainly lethal arrhythmias, such as ventricular tachycardia and fibrillation, may lead to sudden cardiac death. These are triggered as a result of cardiac injury due to chronic ischemia, acute myocardial infarction and various stressful conditions associated with increased levels of circulating catecholamines and angiotensin II. Several mechanisms have been proposed to underlie electrical instability of the heart promoting ventricular arrhythmias; however, oxidative stress which adversely affects ion homeostasis due to changes in the ion channel structure and function, seems to play a critical role in eliciting different types of ventricular arrhythmias. Prevention or mitigation of the severity of ventricular arrhythmias due to antioxidants has been indicated as the fundamental contribution in the field of preventive cardiology; however, novel interventions have to be developed for greater effectiveness and specificity in attenuating the adverse effects of oxidative stress. In this review, we have attempted to discuss proarrhythmic effects of oxidative stress differing in time and concentration dependence and highlight a molecular and cellular concept how it alters cardiac cell automaticity and conduction velocity sensitizing the probability of ventricular arrhythmias with resultant sudden cardiac death due to ischemic heart disease and other stressful situations. It is concluded that pharmacological approaches targeting multiple mechanisms besides oxidative stress might be more effective in the treatment of ventricular arrhythmias than current antiarrhythmic therapy.

Mitochondrial and mitochondrial-independent pathways of myocardial cell death during ischaemia and reperfusion injury.

Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial-independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled 'Mitochondria as targets of acute cardioprotection' and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.

Hyperlipidaemia and cardioprotection: Animal models for translational studies.

Hyperlipidaemia is a well-established risk factor for cardiovascular diseases and therefore, many animal model have been developed to mimic the human abnormal elevation of blood lipid levels. In parallel, extensive research for the alleviation of ischaemia/reperfusion injury has revealed that hyperlipidaemia is a major co-morbidity that attenuates the cardioprotective effect of conditioning strategies (preconditioning, postconditioning and remote conditioning) and that of pharmacological interventions by interfering with cardioprotective signalling pathways. In the present review article, we summarize the existing data on animal models of hypercholesterolaemia (total, low density and HDL abnormalities) and hypertriglyceridaemia used in ischaemia/reperfusion injury and protection from it. We also provide recommendations on preclinical animal models to be used for translations of the cardioprotective strategies into clinical practice. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

Guidelines for evaluating myocardial cell death.

Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms.

Different signalling in infarcted and non-infarcted areas of rat failing hearts: A role of necroptosis and inflammation.

Necroptosis has been recognized in heart failure (HF). In this study, we investigated detailed necroptotic signalling in infarcted and non-infarcted areas separately and its mechanistic link with main features of HF. Post-infarction HF in rats was induced by left coronary occlusion (60 minutes) followed by 42-day reperfusion. Heart function was assessed echocardiographically. Molecular signalling and proposed mechanisms (oxidative stress, collagen deposition and inflammation) were investigated in whole hearts and in subcellular fractions when appropriate. In post-infarction failing hearts, TNF and pSer229-RIP3 levels were comparably increased in both infarcted and non-infarcted areas. Its cytotoxic downstream molecule p-MLKL, indicating necroptosis execution, was detected in infarcted area. In non-infarcted area, despite increased pSer229-RIP3, p-MLKL was present in neither whole cells nor the cell membrane known to be associated with necroptosis execution. Likewise, increased membrane lipoperoxidation and NOX2 levels unlikely promoted pro-necroptotic environment in non-infarcted area. Collagen deposition and the inflammatory csp-1-IL-1ß axis were active in both areas of failing hearts, while being more pronounced in infarcted tissue. Although apoptotic proteins were differently expressed in infarcted and non-infarcted tissue, apoptosis was found to play an insignificant role. p-MLKL-driven necroptosis and inflammation while inflammation only (without necroptotic cell death) seem to underlie fibrotic healing and progressive injury in infarcted and non-infarcted areas of failing hearts, respectively. Upregulation of pSer229-RIP3 in both HF areas suggests that this kinase, associated with both necroptosis and inflammation, is likely to play a dual role in HF progression.