Cardiac Cell Death Research Articles

New Insights into the Role of Ferroptosis in Cardiovascular Diseases

Cardiovascular diseases (CVDs) are the principal cause of disease burden and death worldwide. Ferroptosis is a new form of regulated cell death mainly characterized by altered iron metabolism, increased polyunsaturated fatty acid peroxidation by reactive oxygen species, depletion of glutathione and inactivation of glutathione peroxidase 4. Recently, a series of studies have indicated that ferroptosis is involved in the death of cardiac and vascular cells and has a key impact on the mechanisms leading to CVDs such as ischemic heart disease, ischemia/reperfusion injury, cardiomyopathies, and heart failure. In this article, we reviewed the molecular mechanism of ferroptosis and the current understanding of the pathophysiological role of ferroptosis in ischemic heart disease and in some cardiomyopathies. Moreover, the comprehension of the machinery governing ferroptosis in vascular cells and cardiomyocytes may provide new insights into preventive and therapeutic strategies in CVDs.

Pulsed Field Ablation for the Treatment of Atrial Fibrillation: PULSED AF Pivotal Trial.

BACKGROUND: Pulsed field ablation uses electrical pulses to cause nonthermal irreversible electroporation and induce cardiac cell death. Pulsed field ablation may have effectiveness comparable to traditional catheter ablation while preventing thermally mediated complications. METHODS: The PULSED AF pivotal study (Pulsed Field Ablation to Irreversibly Electroporate Tissue and Treat AF) was a prospective, global, multicenter, nonrandomized, paired single-arm study in which patients with paroxysmal (n=150) or persistent (n=150) symptomatic atrial fibrillation (AF) refractory to class I or III antiarrhythmic drugs were treated with pulsed field ablation. All patients were monitored for 1 year using weekly and symptomatic transtelephonic monitoring; 3-, 6-, and 12-month ECGs; and 6- and 12-month 24-hour Holter monitoring. The primary effectiveness end point was freedom from a composite of acute procedural failure, arrhythmia recurrence, or antiarrhythmic escalation through 12 months, excluding a 3-month blanking period to allow recovery from the procedure. The primary safety end point was freedom from a composite of serious procedure- and device-related adverse events. Kaplan-Meier methods were used to evaluate the primary end points. RESULTS: Pulsed field ablation was shown to be effective at 1 year in 66.2% (95% CI, 57.9 to 73.2) of patients with paroxysmal AF and 55.1% (95% CI, 46.7 to 62.7) of patients with persistent AF. The primary safety end point occurred in 1 patient (0.7%; 95% CI, 0.1 to 4.6) in both the paroxysmal and persistent AF cohorts. CONCLUSIONS: PULSED AF demonstrated a low rate of primary safety adverse events (0.7%) and provided effectiveness consistent with established ablation technologies using a novel irreversible electroporation energy to treat patients with AF.

Cerebral amyloid angiopathy-related cardiac injury: Focus on cardiac cell death.

Cerebral amyloid angiopathy (CAA) is a kind of disease in which amyloid β (Aβ) and other amyloid protein deposits in the cerebral cortex and the small blood vessels of the brain, causing cerebrovascular and brain parenchymal damage. CAA patients are often accompanied by cardiac injury, involving Aβ, tau and transthyroxine amyloid (ATTR). Aβ is the main injury factor of CAA, which can accelerate the formation of coronary artery atherosclerosis, aortic valve osteogenesis calcification and cardiomyocytes basophilic degeneration. In the early stage of CAA (pre-stroke), the accompanying locus coeruleus (LC) amyloidosis, vasculitis and circulating Aβ will induce first hit to the heart. When the CAA progresses to an advanced stage and causes a cerebral hemorrhage, the hemorrhage leads to autonomic nervous function disturbance, catecholamine surges, and systemic inflammation reaction, which can deal the second hit to the heart. Based on the brain-heart axis, CAA and its associated cardiac injury can create a vicious cycle that accelerates the progression of each other.

Cell death regulation in myocardial toxicity induced by antineoplastic drugs.

Homeostatic regulation of cardiomyocytes plays a critical role in maintaining normal physiological activity of cardiac tissue. Severe cardiotoxicity can lead to heart disease, including but not limited to arrhythmias, myocardial infarction and cardiac hypertrophy. In recent years, significant progress has been made in developing new therapies for cancer that have dramatically changed the treatment of several malignancies and continue to improve patient survival, but can also lead to serious cardiac adverse effects. Mitochondria are key organelles that maintain homeostasis in myocardial tissue and have been extensively involved in various cardiovascular disease episodes, including ischemic cardiomyopathy, heart failure and stroke. Several studies support that mitochondrial targeting is a major determinant of the cardiotoxic effects triggered by chemotherapeutic agents increasingly used in solid and hematologic tumors. This antineoplastic therapy-induced mitochondrial toxicity is due to different mechanisms, usually altering the mitochondrial respiratory chain, energy production and mitochondrial kinetics, or inducing mitochondrial oxidative/nitrosative stress, ultimately leading to cell death. This review focuses on recent advances in forms of cardiac cell death and related mechanisms of antineoplastic drug-induced cardiotoxicity, including autophagy, ferroptosis, apoptosis, pyroptosis, and necroptosis, explores and evaluates key proteins involved in cardiac cell death signaling, and presents recent advances in cardioprotective strategies for this disease. It aims to provide theoretical basis and targets for the prevention and treatment of pharmacological cardiotoxicity in clinical settings.

Mitochondria-derived damage-associated molecular patterns and inflammation in the ischemic-reperfused heart.

Cardiac cell death after myocardial infarction release endogenous structures termed damage-associated molecular patterns (DAMPs) that trigger the innate immune system and initiate a sterile inflammation in the myocardium. Cardiomyocytes are energy demanding cells and 30% of their volume are mitochondria. Mitochondria are evolutionary endosymbionts originating from bacteria containing molecular patterns similar to bacteria, termed mitochondrial DAMPs (mDAMPs). Consequently, mitochondrial debris may be particularly immunogenic and damaging. However, the role of mDAMPs in myocardial infarction is not clarified. Identifying the most harmful mDAMPs and inhibiting their early inflammatory signaling may reduce infarct size and the risk of developing post-infarct heart failure. The focus of this review is the role of mDAMPs in the immediate pro-inflammatory phase after myocardial infarction before arrival of immune cells in the myocardium. We discuss different mDAMPs, their role in physiology and present knowledge regarding their role in the inflammatory response of acute myocardial infarction.

Experimental diabetes exacerbates autophagic flux impairment during myocardial I/R injury through calpain-mediated cleavage of Atg5/LAMP2.

To explore the role of autophagic flux in the increased susceptibility of the experimental diabetic heart to ischaemia-reperfusion (I/R) injury, we established STZ-induced diabetic mice and performed I/R. In vitro, neonatal mouse cardiomyocytes were subjected to high glucose and hypoxia/reoxygenation challenge to mimic diabetic I/R injury. We found that experimental diabetes aggravated I/R-induced injury than compared with nondiabetic mice. Autophagic flux was impaired in I/R hearts, and the impairment was exacerbated in diabetic mice subjected to I/R with defective autophagosome formation and clearance. Calpains, calcium-dependent thiol proteases, were upregulated and highly activated after I/R of diabetes, while calpain inhibition attenuated cardiac function and cell death and partially restored autophagic flux. The expression levels of Atg5 and LAMP2, two crucial autophagy-related proteins, were significantly degraded in diabetic I/R hearts, alterations that were associated with calpain activation and could be reversed by calpain inhibition. Co-overexpression of Atg5 and LAMP2 reduced myocardial injury and normalized autophagic flux. In conclusion, experimental diabetes exacerbates autophagic flux impairment of cardiomyocytes under I/R stress, resulting in worse I/R-induced injury. Calpain activation and cleavage of Atg5 and LAMP2 at least partially account for the deterioration of autophagic flux impairment.

Unveiling the Vital Role of Long Non-Coding RNAs in Cardiac Oxidative Stress, Cell Death, and Fibrosis in Diabetic Cardiomyopathy.

Diabetes mellitus is a burdensome public health problem. Diabetic cardiomyopathy (DCM) is a major cause of mortality and morbidity in diabetes patients. The pathogenesis of DCM is multifactorial and involves metabolic abnormalities, the accumulation of advanced glycation end products, myocardial cell death, oxidative stress, inflammation, microangiopathy, and cardiac fibrosis. Evidence suggests that various types of cardiomyocyte death act simultaneously as terminal pathways in DCM. Long non-coding RNAs (lncRNAs) are a class of RNA transcripts with lengths greater than 200 nucleotides and no apparent coding potential. Emerging studies have shown the critical role of lncRNAs in the pathogenesis of DCM, along with the development of molecular biology technologies. Therefore, we summarize specific lncRNAs that mainly regulate multiple modes of cardiomyopathy death, oxidative stress, and cardiac fibrosis and provide valuable insights into diagnostic and therapeutic biomarkers and strategies for DCM.

Abamectin causes cardiac dysfunction in carp via inhibiting redox equilibrium and resulting in immune inflammatory response and programmed cell death.

This study aims to investigate the effects of environmentally relevant concentrations of abamectin on the cardiac function of carp and the potential mechanisms. Here, male carp were exposed to abamectin, and cardiac function-related enzymatic markers were examined. Cardiac histopathology, redox equilibrium, inflammation, and cell death were evaluated. Abamectin exposure caused cardiac dysfunction by upregulating lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK), creatine Kinase MB isoenzyme (CK-MB) and white blood cells (WBCs), and decreasing red blood cells (RBCs) and hemoglobin (Hb). DHE staining and biochemical assays revealed that abamectin caused ROS release and oxidative stress by inhibiting Nrf2-ARE pathway. Histopathological and real-time fluorescence quantitative PCR (RT-qPCR) assays revealed that abamectin caused myocardial fiber swelling and inflammatory cell infiltration, enhanced pro-inflammatory cytokines tumor necrosis factor-α (Tnf-α), interleukin-1 beta (Il-1β), and Il-6 levels and attenuated anti-inflammatory cytokines Il-10 and transforming growth factor beta 1 (Tgf-β1) through activating NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor kappa-B (NF-κB) pathway. Tunel staining showed that abamectin triggered cardiac apoptosis via activating p53-mediated mitochondrial apoptosis with elevated bcl2-associated X (Bax), reduced B-cell lymphoma-2 (Bcl-2), and activated Caspase-9 and Caspase-3. Immunoblot analysis revealed that abamectin activated autophagic flow by inhibiting mammalian target of rapamycin (mTOR), resulting in the conversion of LC3B from LC3-I to LC3-II, elevation of autophagy protein 5 (Atg5), and reduction of p62. Overall, abamectin caused cardiac dysfunction in carp via inhibiting redox equilibrium and resulting in immune inflammatory response and programmed cell death.

Bioinformatics analysis identifies ferroptosis‑related genes in the regulatory mechanism of myocardial infarction.

Since ferroptosis is considered to be a notable cause of cardiomyocyte death, inhibiting ferroptosis has become a novel strategy in reducing cardiac cell death and improving cardiopathic conditions. Therefore, the aim of the present study was to search for ferroptosis-related hub genes and determine their diagnostic value in myocardial infarction (MI) to aid in the diagnosis and treatment of the disease. A total of 10,286 DEGs were identified, including 6,822 upregulated and 3.464 downregulated genes in patients with MI compared with healthy controls. After overlapping with ferroptosis-related genes, 128 ferroptosis-related DEGs were obtained. WGCNA successfully identified a further eight functional modules, from which the blue module had the strongest correlation with MI. Blue module genes and ferroptosis-related differentially expressed genes were overlapped to obtain 20 ferroptosis-related genes associated with MI. Go and KEGG analysis showed that these genes were mainly enriched in cellular response to chemical stress, trans complex, transferring, phosphorus-containing groups, protein serine/threonine kinase activity, FoxO signaling pathway. Hub genes were obtained from 20 ferroptosis-related genes through the PPI network. The expression of hub genes was found to be down-regulated in the MI group. Finally, the miRNAs-hub genes and TFs-hub genes networks were constructed. The GSE141512 dataset and the use of RT-qPCR assays on patient blood samples were used to confirm these results. The results showed that ATM, PIK3CA, MAPK8, KRAS and SIRT1 may play key roles in the development of MI, and could therefore be novel markers or targets for the diagnosis or treatment of MI.

Cardioprotective Effect of Taxifolin against Isoproterenol-Induced Cardiac Injury through Decreasing Oxidative Stress, Inflammation, and Cell Death, and Activating Nrf2/HO-1 in Mice.

Oxidative stress and inflammation are key components in cardiovascular diseases and heart dysfunction. Herein, we evaluated the protective effects of (+)-taxifolin (TAX), a potent flavonoid with significant antioxidant and anti-inflammatory actions, on myocardial oxidative tissue injury, inflammation, and cell death, using a mouse model of isoproterenol (ISO)-induced acute myocardial injury. Mice were given TAX (25 and 50 mg/kg, orally) for 14 days before receiving two subsequent injections of ISO (100 mg/kg, s.c.) at an interval of 24 h on the 15th and 16th days. The ISO-induced cardiac tissue injury was evidenced by increased serum creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH), along with several histopathological changes. The ISO also induced increased malondialdehyde (MDA) with concomitant declined myocardial glutathione level and antioxidant enzymes activities. Moreover, ISO-induced heart injury was accompained with elevated cardiac NF-κB p65, TNF-α, IL-1β, Bax, and caspase-3, as well as decreased Bcl-2, Nrf2, and HO-1. Remarkably, TAX reduced the severity of cardiac injury, oxidative stress, inflammation, and cell death, while enhancing antioxidants, Bcl-2, and Nrf2/HO-1 signaling in ISO-injected mice. In conclusion, TAX protects against ISO-induced acute myocardial injury via activating the Nrf2/HO-1 signaling pathway and attenuating the oxidative tissue injury and key regulators of inflammatory response and apoptosis. Thus, our findings imply that TAX may constitute a new cardioprotective therapy against acute MI, which undoubtedly deserves further exploration in upcoming human trials.

Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells

Currently, there are no treatments that ameliorate cardiac cell death, the underlying basis of cardiovascular disease. An unexplored cell type in cardiac regeneration is cardiac Purkinje cells; specialized cells from the cardiac conduction system (CCS) responsible for propagating electrical signals. Purkinje cells have tremendous potential as a regenerative treatment because they may intrinsically integrate with the CCS of a recipient myocardium, resulting in more efficient electrical conduction in diseased hearts. This study is the first to demonstrate an effective protocol for the direct reprogramming of human cardiomyocytes into cardiac Purkinje-like cells using small molecules. The cells generated were genetically and functionally similar to native cardiac Purkinje cells, where expression of key cardiac Purkinje genes such as CNTN2, ETV1, PCP4, IRX3, SCN5a, HCN2 and the conduction of electrical signals with increased velocity was observed. This study may help to advance the quest to finding an optimized cell therapy for heart regeneration.

Ischemic Preconditioning and Postconditioning Protect the Heart by Preserving the Mitochondrial Network.

Background Mitochondria fuse to form elongated networks which are more tolerable to stress and injury. Ischemic pre- and postconditioning (IPC and IPost, respectively) are established cardioprotective strategies in the preclinical setting. Whether IPC and IPost modulates mitochondrial morphology is unknown. We hypothesize that the protective effects of IPC and IPost may be conferred via preservation of mitochondrial network. Methods IPC and IPost were applied to the H9c2 rat myoblast cells, isolated adult primary murine cardiomyocytes, and the Langendorff-isolated perfused rat hearts. The effects of IPC and IPost on cardiac cell death following ischemia-reperfusion injury (IRI), mitochondrial morphology, and gene expression of mitochondrial-shaping proteins were investigated. Results IPC and IPost successfully reduced cardiac cell death and myocardial infarct size. IPC and IPost maintained the mitochondrial network in both H9c2 and isolated adult primary murine cardiomyocytes. 2D-length measurement of the 3 mitochondrial subpopulations showed that IPC and IPost significantly increased the length of interfibrillar mitochondria (IFM). Gene expression of the pro-fusion protein, Mfn1, was significantly increased by IPC, while the pro-fission protein, Drp1, was significantly reduced by IPost in the H9c2 cells. In the primary cardiomyocytes, gene expression of both Mfn1 and Mfn2 were significantly upregulated by IPC and IPost, while Drp1 was significantly downregulated by IPost. In the Langendorff-isolated perfused heart, gene expression of Drp1 was significantly downregulated by both IPC and IPost. Conclusion IPC and IPost-mediated upregulation of pro-fusion proteins (Mfn1 and Mfn2) and downregulation of pro-fission (Drp1) promote maintenance of the interconnected mitochondrial network, ultimately conferring cardioprotection against IRI.

Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.

Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα-TRAF2-NF-κB signaling in the pathogenesis of DOX cardiotoxicity. Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg-1·wk-1) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell-derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability. In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T-adeno-associated virus 9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity.

Synthesis and in vivo evaluation of new steviol derivatives that protect against cardiomyopathy by inhibiting ferroptosis

Cardiovascular diseases (CVDs) remain the leading cause of death globally. Inhibiting ferroptosis and thus preventing cardiac cell death is a promising and effective strategy for cardiomyopathy prevention and therapy. Steviol, an ent-kaurene diterpenoid, possesses broad-spectrum bioactivity. In the present study, with the aim to discover new agents for CVDs treatment, 30 derivatives of steviol, including 22 new ones, were synthesized, and evaluated their protective activity in vivo using the doxorubicin (DOX) induced zebrafish cardiomyopathy model. Our results firstly demonstrated that steviol has promising cardioprotective activity and further modification of steviol can greatly improve the activity. Among the new derivatives, 16d and 16e show the most potent activity. Both 16d (1 μM) and 16e (0.1 μM) effectively maintain the normal heart shape and prevent the cardiac dysfunction impaired by DOX in zebrafish. Their therapeutic efficacy is much superior to the parent natural product, steviol, and positive drug, levosimendan. Further study demonstrated that 16d and 16e inhibit DOX-induced ferroptosis and thus protect cardiomyopathy, by suppressing the glutathione depletion, iron accumulation, and lipid peroxidation, decreasing reactive oxygen species overaccumulation, and restoring the mitochondrial membrane potential. Consequently, due to their unique structure and significant cardioprotective activity with ferroptosis inhibition, new steviol derivatives 16d and 16e merit further research for the development of new cardioprotective drug candidates.

Novel cfDNA Methylation Biomarkers Reveal Delayed Cardiac Cell Death after Open-heart Surgery.

The use of cardiopulmonary bypass (CPB) is thought to cause delayed cardiac damage. DNA methylation-based liquid biopsies are novel biomarkers for monitoring acute cardiac cell death. We assessed cell-free DNA molecules as markers for cardiac damage after open-heart surgery. Novel cardiomyocyte-specific DNA methylation markers were applied to measure cardiac cfDNA in the plasma of 42 infants who underwent open-heart surgery. Cardiac cfDNA was elevated following surgery, reflecting direct surgery-related tissue damage, and declined thereafter in most patients. The concentration of cardiac cfDNA post-surgery correlated with the duration of CPB and aortic cross clamping. Strikingly, cardiac cfDNA at 6 h predicted duration of mechanical ventilation and maximal vasoactive-inotropic score better than did maximal troponin levels. Cardiac cfDNA reveals heart damage associated with CPB, and can be used to monitor cardiac cell death, to predict clinical outcome of surgery and to assess performance of cardioprotective interventions.

Abstract P2077: Caspase8ap2 M6a Mrna Methylation Enhances Cardiomyocyte Apoptosis Following Myocardial Ischemia

Objective: Heart failure is the leading cause of morbidity and mortality in the United States. Cardiomyocyte death is one of the critical mediators of cardiac dysfunction following ischemic injury. However, the role of posttranscriptional regulation on cell-death-associated genes is not well explored during ischemic heart failure. N6-methyladenosine (m6A) is the most abundant and conserved chemical modification found in eukaryotic mRNA and is associated with mRNA synthesis and metabolism. We have recently shown that m6A RNA methylation regulates the angiogenic potential of endothelial cells following ischemic injury. However, the role of m6A mRNA methylation in cardiac cell death following ischemic injury is not well defined. Methods: Cardiac function (using echocardiography) was measured at baseline and four weeks post-MI in mice. In the end, mice were euthanized, and hearts were excised for biochemical and histological analysis. Cardiac cell death was assessed in H9C2 cells and cardiomyocytes using FACs (Annexin V and PI) and TUNNEL assay. The Casp8AP2 stability were measured using actinomycin D assay. To demonstrate the molecular mechanism, METTL3 was inhibited using siRNA or shRNA. Results: The cardiac function was significantly reduced following MI. Enhanced m6A mRNA methylation was observed in mice hearts following MI. Histological (TUNEL assay) and biochemical (WB: Caspase 8, cleaved cas8, caspase3, cleaved caspase3, Casp8AP2) analysis showed increased myocytes apoptosis in mice following MI. At the molecular level, hypoxic stress (nutrient depiviation+1%O2) induces global m6A mRNA methylation, which leads to Cas8AP2 gene stability and activation of extrinsic apoptotic pathways in cardiomyocytes in vitro. In contrast, METTL3 inhibition significantly reduced Cas8AP2 mRNA methylation, enhanced its degradation, and ultimately reduced cardiomyocyte cell death. Conclusion: In conclusion, we have shown that increased Casp8AP2 m6A RNA methylation enhances its stability and activates caspase-3 cleavage, which eventually leads to cardiomyocyte death following myocardial ischemia. The reduction of the RNA methylation through METTL3 inhibition (methyltransferase) reduced hypoxia-induced apoptosis of cardiomyocytes.

Anti-Ischemic Effects of PIK3IP1 Are Mediated through Its Interactions with the ETA-PI3Kγ-AKT Axis.

Oxidative stress, caused by the accumulation of reactive oxygen species (ROS) during acute myocardial infarction (AMI), is one of the main factors leading to myocardial cell damage and programmed cell death. Phosphatidylinositol-3-kinase-AKT (PI3K-AKT) signaling is essential for regulating cell proliferation, differentiation, and apoptosis. Phosphoinositide-3-kinase (PI3K)-interacting protein 1 (PIK3IP1) is an intrinsic inhibitor of PI3K in various tissues, but its functional role during AMI remains unknown. In this study, the anti-ischemic role of PIK3IP1 in an in vitro AMI setting was evaluated using H9c2 cells. The MTT assay demonstrated that cell viability decreased significantly via treatment with H2O2 (200–500 μM). The TUNEL assay results revealed substantial cellular apoptosis following treatment with 200 μM H2O2. Under the same conditions, the expression levels of hypoxia-inducible factor (HIF-1α), endothelin-1 (ET-1), bcl-2-like protein 4 (BAX), and cleaved caspase-3 were elevated, whereas those of PIK3IP1, LC3II, p53, and Bcl-2 decreased significantly. PIK3IP1 overexpression inhibited H2O2-induced and PI3K-mediated apoptosis; however, PIK3IP1 knockdown reversed this effect, suggesting that PIK3IP1 functions as an anti-apoptotic molecule. To identify both the upstream and downstream molecules associated with PIK3IP1, ET-1 receptor type-specific antagonists (BQ-123 and BQ-788) and PI3K subtype-specific antagonists (LY294002 and IPI-549) were used to determine the participating isoforms. Co-immunoprecipitation was performed to identify the binding partners of PIK3IP1. Our results demonstrated that ROS-induced cardiac cell death may occur through the ETA-PI3Kγ-AKT axis, and that PIK3IP1 inhibits binding with both ETA and PI3Kγ. Taken together, these findings reveal that PIK3IP1 plays an anti-ischemic role by reducing the likelihood of programmed cell death via interaction with the ETA-PI3Kr-AKT axis.

Low doses of advanced glycation end-products and fructosilation products promotes premature cell death of human cardiac cells and increases drug resistance of human breast cancer cells exposed to doxorubicin through NLRP3 and MyD88 pathways.

e24055 Background: Advanced glycosylation end-products (AGEs) are non-enzyme, protein glycosylation products caused by glucose-induced metabolic disorder, and play an important role in aging, cancer, metabolic syndrome and cardiovascular diseases. We aimed to assess whether AGEs and fructosilation products, such as methylglyoxal (MG) and N-carboxymethyllysine (N-CML) could increase susceptibility to cell damages induced by doxorubicin. Methods: Human cardiomyocytes were pre-exposed for 24h with low doses (50 mmol/L) of methylglyoxal (MG) or N-carboxymethyllysine (N-CML). After, cells were exposed to subclinical concentration of doxorubicin (at 100 and 200 nM) for 48 and 72h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Expression of p53 was also performed through western blot method. Results: Pre-exposure to methylglyoxal (MG) but especially to N-carboxymethyllysine (N-CML) increase cell mortality to doxorubicin of 48-53% compared to control. Pre-exposure to N-CML promotes premature death of cardiomyocytes to doxorubicin through NLRP3-driven pathways. Notably, MG and N-CML increased significantly the cardiotoxicity through NLRP3 inflammasome, Myd88 myddosome and cytochrome C-mediated apoptosis. Pre-exposure to N-CML and MG increase the secretion of interleukin-6 that increase cell apoptosis through a paracrine and autocrine mechanism. Induction of IL-6 reduces the expression of p53 resulting in the induction of the apoptotic process. Conclusions: These observations suggest that AGEs and fructosilation producs promotes premature cardiotoxicity of human cardiac cells exposed to doxorubicin by activation of NLRP3, Myd88-related pathways and downregulation of p53.

Exogenous growth differentiation factor 15 (GDF15) exerts direct cardioprotective properties towards myocardial ischemia reperfusion injury

Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine which can be produced under certain pathological situations, mainly related to inflammatory stress, aging and disease. While clinical data suggest that GDF15 is a powerful risk factor in several high-risk cardiovascular situations such as myocardial infarction and coronary artery disease, experimental studies rather consider this cytokine as a cardioprotective molecule. We aimed to clarify the direct cardiac effects of GDF15 during ischemia-reperfusion (I-R) injury, at concentrations found in patients with high cardiovascular risk, such as these of patients presenting with acute myocardial infarction or stroke. Wistar male rats (200–400 g) were used for both in vivo and ex vivo ischemia-reperfusion and anesthetized with isoflurane. In vivo rats were intubated before a left thoracotomy was performed between the 3rd and the 4th intercostal space. Rats were injected with either saline (Control groups) or 2.5 μg/kg of GDF15 (GDF15 groups), 20 minutes before the transient ligation of the left anterior descending artery for 30 min followed by 24 h of reperfusion. Ex vivo rat's hearts were isolated and perfused on a Langendorff model with either saline (Control groups) or 2000 ng/L of GDF15 upstream the coronary bed (GDF15 groups) for 10 minutes, then subjected to either 20 or 30 minutes of global normothermic total ischemia, followed by 2 h of reperfusion. GDF15 decreased the infarct size in vivo as well as in ex vivo. Moreover, in our ex vivo model, GDF15 induced a better recovery of most contractile parameters after ischemia ( P < 0.05 for all parameters). These results demonstrate for the first time that exogenous preischemic short-term administration of GDF15 decreased cardiac cell death in vivo but also ex vivo, the latter situation involving that inflammatory, endocrine, and nervous systems are not interfering. These original results suggest that GDF15 exerts direct cardioprotective properties towards ischemia-reperfusion injury.

The role of Bcl11b in the onset of cardiac myopathies

B cell lymphoma 11b (Bcl11b) is a zinc finger transcription factor with dual action (repression/activation). It is involved in various physiological responses under healthy and pathological conditions. Genetic variations in the locus harboring this gene are associated with increased risk for cardiovascular disease making it relevant to analyze its role in the heart. Our aims are: (1) To analyze cardiac function in the absence of Bcl11b. (2) To investigate the role of Bcl11b in the onset of pathological cardiac hypertrophy. To achieve the goals, we conducted experiments on both in vivo and in vitro models. In vivo, the impact of Bcl11b on cardiac function, morphology, and molecular pathways were analyzed in cardiac-specific Bcl11b knockout mice induced by tamoxifen administration. In vitro, Bcl11b deletion was induced by recombinase Cre via adenovirus infection in isolated newborn primary cardiac cell. Cardiomyocytes were stimulated by angiotensin II or phenylephrin to induce cell hypertrophy and then analyzed. Invalidation of Bcl11b specifically in cardiomyocytes (Bcl11b HKO mice) results in cardiac cell death accompanied by inflammation, fibrosis, a moderate cardiac hypertrophy and decreased contractile function. Fibrosis was observed as soon as week and increased over time. Further examination of 1-week mutant hearts revealed the presence of “dead cell” zones and macrophage invasion. This was preceded by the detection of IgG positive cardiomyocytes that suggest compromised membrane integrity. At the molecular level, the expression of genes related to inflammation/repair and cell death was increased in the mutant heart. Finally, echocardiographic analysis showed a 10% decrease in ejection fraction and fractional shortening in mutant mice as compared to control. The cardiac specific Bcl11b KO mice exhibit diminished heart function, increased fibrosis and cell death. Thus, Bcl11b could play a role in the maintenance of cardiac homeostasis and in the cardiac damage response.