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.

Effect of cardioplegic arrest and reperfusion on left and right ventricular proteome/phosphoproteome in patients undergoing surgery for coronary or aortic valve disease.

Our earlier work has shown inter­disease and intra­disease differences in the cardiac proteome between right (RV) and left (LV) ventricles of patients with aortic valve stenosis (AVS) or coronary artery disease (CAD). Whether disease remodeling also affects acute changes occuring in the proteome during surgical intervention is unknown. This study investigated the effects of cardioplegic arrest on cardiac proteins/phosphoproteins in LV and RV of CAD (n=6) and AVS (n=6) patients undergoing cardiac surgery. LV and RV biopsies were collected during surgery before ischemic cold blood cardioplegic arrest (pre) and 20 min after reperfusion (post). Tissues were snap frozen, proteins extracted, and the extracts were used for proteomic and phosphoproteomic analysis using Tandem Mass Tag (TMT) analysis. The results were analysed using QuickGO and Ingenuity Pathway Analysis softwares. For each comparision, our proteomic analysis identified more than 3,000 proteins which could be detected in both the pre and Post samples. Cardioplegic arrest and reperfusion were associated with significant differential expression of 24 (LV) and 120 (RV) proteins in the CAD patients, which were linked to mitochondrial function, inflammation and cardiac contraction. By contrast, AVS patients showed differential expression of only 3 LV proteins and 2 RV proteins, despite a significantly longer duration of ischaemic cardioplegic arrest. The relative expression of 41 phosphoproteins was significantly altered in CAD patients, with 18 phosphoproteins showing altered expression in AVS patients. Inflammatory pathways were implicated in the changes in phosphoprotein expression in both groups. Inter­disease comparison for the same ventricular chamber at both timepoints revealed differences relating to inflammation and adrenergic and calcium signalling. In conclusion, the present study found that ischemic arrest and reperfusion trigger different changes in the proteomes and phosphoproteomes of LV and RV of CAD and AVS patients undergoing surgery, with markedly more changes in CAD patients despite a significantly shorter ischaemic period.

Pathology-related changes in cardiac energy metabolites, inflammatory response and reperfusion injury following cardioplegic arrest in patients undergoing open-heart surgery.

Introduction: Changes in cardiac metabolites in adult patients undergoing open-heart surgery using ischemic cardioplegic arrest have largely been reported for non-ventricular tissue or diseased left ventricular tissue, with few studies attempting to assess such changes in both ventricular chambers. It is also unknown whether such changes are altered in different pathologies or linked to the degree of reperfusion injury and inflammatory response. The aim of the present work was to address these issues by monitoring myocardial metabolites in both ventricles and to establish whether these changes are linked to reperfusion injury and inflammatory/stress response in patients undergoing surgery using cold blood cardioplegia for either coronary artery bypass graft (CABG, n = 25) or aortic valve replacement (AVR, n = 16). Methods: Ventricular biopsies from both left (LV) and right (RV) ventricles were collected before ischemic cardioplegic arrest and 20 min after reperfusion. The biopsies were processed for measuring selected metabolites (adenine nucleotides, purines, and amino acids) using HPLC. Blood markers of cardiac injury (Troponin I, cTnI), inflammation (IL- 6, IL-8, Il-10, and TNFα, measured using Multiplex) and oxidative stress (Myeloperoxidase, MPO) were measured pre- and up to 72 hours post-operatively. Results: The CABG group had a significantly shorter ischemic cardioplegic arrest time (38.6 ± 2.3 min) compared to AVR group (63.0 ± 4.9 min, p = 2 x 10-6). Cardiac injury (cTnI release) was similar for both CABG and AVR groups. The inflammatory markers IL-6 and Il-8 were significantly higher in CABG patients compared to AVR patients. Metabolic markers of cardiac ischemic stress were relatively and significantly more altered in the LV of CABG patients. Comparing diabetic and non-diabetic CABG patients shows that only the RV of diabetic patients sustained major ischemic stress during reperfusion and that diabetic patients had a significantly higher inflammatory response. Discussion: CABG patients sustain relatively more ischemic stress, systemic inflammatory response and similar injury and oxidative stress compared to AVR patients despite having significantly shorter cross-clamp time. The higher inflammatory response in CABG patients appears to be at least partly driven by a higher incidence of diabetes amongst CABG patients. In addition to pathology, the use of cold blood cardioplegic arrest may underlie these differences.

Bicuspid Aortic Valve Alters Aortic Protein Expression Profile in Neonatal Coarctation Patients.

Coarctation of the aorta is a form of left ventricular outflow tract obstruction in paediatric patients that can be presented with either bicuspid (BAV) or normal tricuspid (TAV) aortic valve. The congenital BAV is associated with hemodynamic changes and can therefore trigger different molecular remodelling in the coarctation area. This study investigated the proteomic and phosphoproteomic changes associated with BAV for the first time in neonatal coarctation patients. Aortic tissue was collected just proximal to the coarctation site from 23 neonates (BAV; n = 10, TAV; n = 13) that were matched for age (age range 4-22 days). Tissue from half of the patients was frozen and used for proteomic and phosphoproteomic analysis whilst the remaining tissue was formalin fixed and used for analysis of elastin content using Elastic Van-Gieson (EVG) staining. A total of 1796 protein and 75 phosphoprotein accession numbers were detected, of which 34 proteins and one phosphoprotein (SSH3) were differentially expressed in BAV patients compared to TAV patients. Ingenuity Pathway Analysis identified the formation of elastin fibres as a significantly enriched function (p = 1.12 × 10-4) due to the upregulation of EMILIN-1 and the downregulation of TNXB. Analysis of paraffin sections stained with EVG demonstrated increased elastin content in BAV patients. The proteomic/phosphoproteomic analysis also suggested changes in inositol signalling pathways and reduced expression of the antioxidant SOD3. This work demonstrates for the first time that coarcted aortic tissue in neonatal BAV patients has an altered proteome/phosphoproteome consistent with observed structural vascular changes when compared to TAV patients.

Changes in contractile protein expression are linked to ventricular stiffness in infants with pulmonary hypertension or right ventricular hypertrophy due to congenital heart disease.

Background: The right ventricle (RV) is not designed to sustain high pressure leading to failure. There are no current medications to help RV contraction, so further information is required on adaption of the RV to such hypertension. Methods: The Right Ventricle in Children (RVENCH) study assessed infants with congenital heart disease undergoing cardiac surgery with hypertensive RV. Clinical and echocardiographic data were recorded, and samples of RV were taken from matched infants, analysed for proteomics and compared between pathologies and with clinical and echocardiographic outcome data. Results: Those with tetralogy of Fallot (TOF) were significantly more cyanosed than those with ventricular septal defect (median oxygen saturation 83% vs 98%, P=0.0038), had significantly stiffer RV (tricuspid E wave/A wave ratio 1.95 vs 0.84, P=0.009) and had most had restrictive physiology. Gene ontology in TOF, with enrichment analysis, demonstrated significant increase in proteins of contractile mechanisms and those of calmodulin, actin binding and others associated with contractility than inventricular septal defect. Structural proteins were also found to be higher in association with sarcomeric function: Z-disc, M-Band and thin-filament proteins. Remaining proteins associated with actin binding, calcium signalling and myocyte cytoskeletal development. Phosphopeptide enrichment led to higher levels of calcium signalling proteins in TOF. Conclusion: This is the first demonstration that those with an RV, which is stiff and hypertensive in TOF, have a range of altered proteins, often in calcium signalling pathways. Information about these alterations might guide treatment options both in terms of individualised therapy or inotropic support for the Right ventricle when hypertensive due to pulmoanry hypertension or congenital heart disease.

Remote ischemic preconditioning triggers changes in autonomic nervous system activity: implications for cardioprotection.

Cardioprotective efficacy of remote ischemic preconditioning (RIPC) remains controversial. Experimental studies investigating RIPC have largely monitored cardiovascular changes during index ischemia and reperfusion with little work investigating changes during RIPC application. This work aims to identify cardiovascular changes associated with autonomic nervous system (ANS) activity during RIPC and prior to index ischemia. RIPC was induced in anesthetized male C57/Bl6 mice by four cycles of 5 min of hindlimb ischemia using inflated cuff (200 mmHg) followed by 5 min reperfusion. Electrocardiography (ECG) and microcirculatory blood flow in both hindlimbs were recorded throughout RIPC protocol. Heart rate variability (HRV) analysis was performed using ECG data. Hearts extracted at the end of RIPC protocol were used either for measurement of myocardial metabolites using high-performance liquid chromatography or for Langendorff perfusion to monitor function and injury during 30 min index ischemia and 2 h reperfusion. Isolated-perfused hearts from RIPC animals had significantly less infarct size after index ischemia and reperfusion (34 ± 5% vs. 59 ± 7%; mean ± SE P < 0.05). RIPC protocol was associated with increased heart rate measured both in ex vivo and in vivo. Frequency ratio of HRV spectra was altered in RIPC compared to control. RIPC was associated with a standard hyperemic response in the cuffed-limb but there was a sustained reduction in blood flow in the uncuffed contralateral limb. RIPC hearts (prior to index ischemia) had significantly lower phosphorylation potential and energy charge compared to the control group. In conclusion, RIPC is associated with changes in ANS activity (heart rate, blood flow, HRV) and mild myocardial ischemic stress that would contribute to cardioprotection.

Cardiac phosphoproteomics during remote ischemic preconditioning: a role for the sarcomeric Z-disk proteins.

Remote ischemic preconditioning (RIPC) induced by brief ischemia/reperfusion cycles of remote organ (e.g., limb) is cardioprotective. The myocardial cellular changes during RIPC responsible for this phenomenon are not currently known. The aim of this work was to identify the activation by phosphorylation of cardiac proteins following RIPC. To achieve our aim we used isobaric tandem mass tagging (TMT) and reverse phase nanoliquid chromatography tandem spectrometry using a Linear Trap Quadropole (LTQ) Orbitrap Velos mass spectrometer. Male C57/Bl6 mice were anesthetized by an intraperitoneal injection of Tribromoethanol. A cuff was placed around the hind limb and inflated at 200 mmHg to prevent blood flow as confirmed by Laser Doppler Flowmetry. RIPC was induced by 4 cycles of 5 min of limb ischemia followed by 5 min of reperfusion. Hearts were extracted for phosphoproteomics. We identified approximately 30 phosphoproteins that were differentially expressed in response to RIPC protocol. The levels of several phosphoproteins in the Z-disk of the sarcomere including phospho-myozenin-2 were significantly higher than control. This study describes and validates a novel approach to monitor the changes in the cardiac phosphoproteome following the cardioprotective intervention of RIPC and prior to index ischemia. The increased level of phosphorylated sarcomeric proteins suggests they may have a role in cardiac signaling during RIPC.

The teratogenicity and behavioral teratogenicity of di(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) in a chick model.

Phthalates are industrial chemicals widely used in consumer products, plastics and children toys, and the risk of exposure to phthalates, especially prenatal exposure, is a growing concern justifying the development of an animal model to better understand their effect. The present study was designed to evaluate the suitability of a chick model for phthalate DEHP teratogenicity and neurobehavioral teratogenicity, a model which is simple and devoid of potential confounding factors such as maternal toxicity, maternal-fetal unit and maternal-neonatal interactions; major findings were confirmed in the DBP study. Prehatch exposure to DEHP in doses ranging from 20 to 100 mg/kg, reduced the percent hatching from 80% in control eggs to 65%, and increased late hatchings from 12.5% in control eggs to 29.4%. In addition it induced developmental defects characterized by an opening or weakening of abdominal muscles allowing internal organs to protrude externally with or without a sac, omphalocele or gastroschisis, respectively. The effect was dose dependent ranging from 8% with DEHP (20 mg/kg) to 22% (100 mg/kg). Similar treatment with DBP 100mg/kg has reduced percentage hatching to 57% and increased late hatching to 37.5%, with a 14% increase in gastroschisis. Biochemical evaluation revealed elevated levels of alkaline phosphatase, which reflects non-specific toxicity of DEHP at such a high dose. Behavioral evaluation using an imprinting test and locomotor activity on chicks pretreated with DEHP (100 mg/kg) has shown an abolishment of imprinting performance from the control (0.65) preference ratio. DNA damage measurements of the metabolite 8-hydroxydeoxyguanosine (8-OH-dG) in blood samples showed an increase of 39.7% after prehatch exposure to phthalates. This was statistically significant for DEHP and indicates genetic toxicity, since part of the teratogenic activity is associated with oxidative stress and DNA damage.