Cardiomyocyte Cell Death Research Articles

Brite Adipocyte FGF21 Attenuates Cardiac Ischemia/Reperfusion Injury in Rat Hearts by Modulating NRF2.

Although the optimal therapy for myocardial infarction includes reperfusion to restore blood flow to the ischemic area, myocardial injury after ischemia/reperfusion usually leads to an inflammatory response, oxidative stress, and cardiomyocyte apoptosis. In this study, rat adipose-derived stem cells were differentiated into low-thermogenic beige adipocytes (LBACs) and high-thermogenic beige adipocytes (HBACs) to study the different cardioprotective effects of heterogeneous expression of brown adipocytes. We found that antioxidant and antiapoptotic factors in H9c2 cardiomyocytes were upregulated by high levels of secreted FGF21 in HBAC conditioned medium (HBAC-CM), whereas FGF21 in HBAC-CM did not affect antioxidative or antiapoptotic cell death in H9c2 cardiomyocytes with Nrf2 knockdown. These results show that NRF2 mediates antioxidative and antiapoptotic effects through the HBAC-secreted factor FGF21. Consistent with this finding, the expression of antioxidant and antiapoptotic genes was upregulated by highly secreted FGF21 after HBAC-CM treatment compared to LBAC-CM treatment in H9c2 cardiomyocytes via NRF2 activation. Furthermore, HBAC-CM significantly attenuated ischemic rat heart tissue injury via NRF2 activation. Based on these findings, we propose that HBAC-CM exerts beneficial effects in rat cardiac ischemia/reperfusion injury by modulating NRF2 and has potential as a promising therapeutic agent for myocardial infarction.

CaMKII-δ9 Induces Cardiomyocyte Death to Promote Cardiomyopathy and Heart Failure.

Heart failure is a syndrome in which the heart cannot pump enough blood to meet the body's needs, resulting from impaired ventricular filling or ejection of blood. Heart failure is still a global public health problem and remains a substantial unmet medical need. Therefore, it is crucial to identify new therapeutic targets for heart failure. Ca2+/calmodulin-dependent kinase II (CaMKII) is a serine/threonine protein kinase that modulates various cardiac diseases. CaMKII-δ9 is the most abundant CaMKII-δ splice variant in the human heart and acts as a central mediator of DNA damage and cell death in cardiomyocytes. Here, we proved that CaMKII-δ9 mediated cardiomyocyte death promotes cardiomyopathy and heart failure. However, CaMKII-δ9 did not directly regulate cardiac hypertrophy. Furthermore, we also showed that CaMKII-δ9 induced cell death in adult cardiomyocytes through impairing the UBE2T/DNA repair signaling. Finally, we demonstrated no gender difference in the expression of CaMKII-δ9 in the hearts, together with its related cardiac pathology. These findings deepen our understanding of the role of CaMKII-δ9 in cardiac pathology and provide new insights into the mechanisms and therapy of heart failure.

MiR-210 Regulates Apoptotic Cell Death during Cellular Hypoxia and Reoxygenation in a Diametrically Opposite Manner.

Apoptotic cell death of cardiomyocytes is a characteristic hallmark of ischemia–reperfusion (I/R) injury. The master hypoxamiR, microRNA-210 (miR-210), is considered the primary driver of the cellular response to hypoxic stress. However, to date, no consensus has emerged with regards to the polarity of the miR-210-elicited cellular response, as miR-210 has been shown to exacerbate as well as attenuate hypoxia-driven apoptotic cell death. Herein, in AC-16 cardiomyocytes subjected to hypoxia-reoxygenation (H-R) stress, we unravel novel facets of miR-210 biology and resolve the biological response mediated by miR-210 into the hypoxia and reoxygenation temporal components. Using transient overexpression and decoy/inhibition vectors to modulate miR-210 expression, we elucidated a Janus role miR-210 in the cellular response to H-R stress, wherein miR-210 mitigated the hypoxia-induced apoptotic cell death but exacerbated apoptotic cell death during cellular reoxygenation. We further delineated the underlying cellular mechanisms that confer this diametrically opposite effect of miR-210 on apoptotic cell death. Our exhaustive biochemical assays cogently demonstrate that miR-210 attenuates the hypoxia-driven intrinsic apoptosis pathway, while significantly augmenting the reoxygenation-induced caspase-8-mediated extrinsic apoptosis pathway. Our study is the first to unveil this Janus role of miR-210 and to substantiate the cellular mechanisms that underlie this functional duality.

545 Cardiac protection by pirfenidone after myocardial infarction: a bioinformatic analysis

Abstract Aims Left ventricular (LV) remodelling after myocardial infarction (MI) is promoted by an intense fibrotic response, which could be targeted by an anti-fibrotic drug such as pirfenidone. Methods and results We explored the relationship between protein modulation by pirfenidone and post-MI remodelling, based on publicly available molecular information and transcriptomic data from a swine model of MI. We also compared the effects of pirfenidone and angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (ACEi/ARB), mineralocorticoid receptor blockers (MRA) and beta-blockers. We identified six causative motives of post-MI remodelling (cardiomyocyte cell death, impaired myocyte contractility, extracellular matrix remodelling and fibrosis, hypertrophy, renin–angiotensin–aldosterone system activation, and inflammation), 4 pirfenidone targets and 21 bioflags (indirect effectors). When considering both targets and bioflags, pirfenidone showed a broad relationship encompassing all six motives. p38γ-MAPK12 blockade inhibits cardiomyocyte apoptosis, cardiomyocyte hypertrophy and inflammation. Furthermore, pirfenidone can modulate extracellular matrix remodelling and cardiac fibrosis by targeting the TGFβ1-SMAD2/3 pathway and other effector proteins such as matrix metalloproteases 2 and 14, PDGFA/B, and IGF1, which promote myocardial fibrosis, cardiomyocyte hypertrophy and impaired contractility. All the gold standard drugs were found to be important for specific clinical motives, but pirfenidone had a more widespread action on the molecular pathways active in the post-MI setting. Conclusions A bioinformatic approach allowed to identify several possible mechanisms of action of pirfenidone with beneficial effects in the post-MI LV remodelling, and suggests additional effects over guideline-recommended therapies. These findings support clinical studies evaluating the beneficial effects of pirfenidone in patients with MI.

Increase in Plasma Oxidized Phosphatidylcholines (OxPCs) in Patients Presenting With ST-Elevation Myocardial Infarction (STEMI).

Objective: ST-segment Elevation Myocardial Infarction (STEMI) occurs as a result of acute occlusion of the coronary artery. Despite successful reperfusion using percutaneous coronary intervention (PCI), a large percentage of myocardial cells die after reperfusion which is recognized as ischemia/reperfusion injury (I/R). Oxidized phosphatidylcholines (OxPCs) are a group of oxidized lipids generated through non-enzymatic oxidation and have pro-inflammatory properties. This study aimed to examine the roles of OxPCs in a clinical setting of myocardial I/R.Methods: Blood samples were collected from STEMI patients at presentation prior to primary PCI (PPCI) (Isch) and at 4 time-points post-PPCI, including 2 h (R-2 h), 24 h (R-24 h), 48 h (R-48 h), and 30 days (R-30 d) post-PPCI. As controls, blood samples were collected from patients with non-obstructive coronary artery disease after diagnostic coronary angiography. Aspiration thrombectomy was also performed in selected STEMI patients. High-performance lipid chromatography-electrospray mass spectrometry (LC-MS/MS) was used for OxPCs analysis.Results: Twenty-two distinct OxPC species were identified and quantified in plasma samples in patients presenting with STEMI. These compounds were categorized as fragmented and non-fragmented species. Total levels of OxPCs did not significantly differ between Isch and control groups. However, total levels of fragmented OxPCs increased significantly in the ischemic period compared with controls (Isch: 4.79 ± 0.94, Control: 1.69 ± 0.19 ng/μl of plasma, P < 0.05). Concentrations of non-fragmented OxPCs had significant reductions during ischemia compared to the control group (Isch: 4.84 ± 0.30, Control: 6.6 ± 0.51 ng/μl, P < 0.05). Levels of total OxPCs in patients with STEMI were not significantly different during reperfusion periods. However, fragmented OxPCs levels were elevated at 48 h post-reperfusion and decreased at 30 days following MI, when compared to R-2 h and R-24 h time points (Isch: 4.79 ± 0.94, R-2 h: 5.33 ± 1.17, R-24 h: 5.20 ± 1.1, R-48 h: 4.18 ± 1.07, R-30 d: 1.87 ± 0.31 ng/μl, P < 0.05). Plasma levels of two fragmented OxPCs, namely, POVPC and PONPC were significantly correlated with peak creatine kinase (CK) levels (P < 0.05). As with plasma levels, the dominant OxPC species in coronary aspirated thrombus were fragmented OxPCs, which constituted 77% of total OxPC concentrations.Conclusion: Biologically active fragmented OxPC were elevated in patients presenting with STEMI when compared to controls. PONPC concentrations were subsequently increased after PPCI resulting in reperfusion. Moreover, levels of POVPC and PONPC were also associated with peak CK levels. Since these molecules are potent stimulators for cardiomyocyte cell death, therapeutics attenuating their activities can result in a novel therapeutic pathway for myocardial salvage for patients undergoing reperfusion therapy.

Melatonin Alleviates LPS-Induced Pyroptotic Cell Death in Human Stem Cell-Derived Cardiomyocytes by Activating Autophagy.

Endotoxemia in sepsis remains a problem due to a lack of effective strategies. Our previous studies have demonstrated that melatonin (Mel) protects against ischemic heart injury and arteriosclerosis. However, its role in endotoxemia-exposed cardiomyocytes remains poorly understood. This study explored, for the first time, the protective effect of Mel on the pyroptosis of human stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to lipopolysaccharide (LPS). Our results showed that treatment with 1 μM or 10 μM Mel for 12 h significantly improved 1 μg/ml LPS-induced hiPSC-CM injuries, as reflected by drastically decreased LDH release and increased cell viability, which was accompanied by the overt induction of autophagy. Specifically, Mel profoundly alleviated LPS-induced cell pyroptosis, as evidenced by decreased propidium iodide (PI) and active caspase-1 double-positive cell rates; suppressed the expression of NLRP3, cleaved caspase-1 (activated form of caspase-1), and GSDMD-NT (functional N-terminal fragment of GSDMD) expression; and inhibited the production of the cleaved IL-1β and cleaved IL-18 cytokines. Additionally, double-membrane autophagosomes were observed in LPS-injured hiPSC-CMs treated with 1 μM or 10 μM Mel. The hiPSC-CMs treated with LPS exhibited considerably fewer acidic vesicles (as revealed by LAMP1 staining) and autophagosomes (as revealed by LC3-II staining); however, Mel reversed this outcome in a dose-dependent manner. Furthermore, coincubation with rapamycin (an autophagy activator) or 3-MA (an autophagy inhibitor) accentuated and attenuated the antipyroptotic actions of Mel, respectively. Collectively, our findings demonstrate that Mel shields hiPSC-CMs against pyroptosis during endotoxemia by activating autophagy.

Abstract 11928: Perm1 Promotes Cardiomyocyte Mitochondrial Biogenesis and Protects Against Hypoxia/Reoxygenation-Induced Damage in Mice

Introduction: Dysregulation of cardiac energy metabolism can result in immature heart development and disrupt the ability of the adult myocardium to adapt to stress, potentially leading to heart failure. In contrast, restoration of abnormal mitochondrial function can have beneficial effects on cardiac dysfunction. Previously, we identified a novel protein termed Perm1 (PGC-1 and ERR induced regulator, muscle 1) that is enriched in skeletal and cardiac muscle and transcriptionally regulated by PGC-1 (Peroxisome proliferator-activated receptor gamma coactivator 1) and ERR (Estrogen-related receptor). The role of Perm1 in the heart is poorly understood and was studied here. Hypothesis: We hypothesize that Perm1 plays a crucial role in cardiac metabolism and ischemic injury. Methods: To directly study the effect of Perm1 on cardiomyocyte oxidative metabolism, Perm1 or LacZ (control) was expressed in mouse neonatal cardiomyocytes (NCM) using an adenovirus system. We analyzed genes and proteins that play essential roles in cardiac metabolism. NCM were also subjected to hypoxia/reoxygenation (H/R). Finally, to study the mechanism underlying Perm1’s action, we investigated the effect of Perm1 on the activity of ERR and the interaction of Perm1 and PGC-1α. Results: Cardiac Perm1 expression was down-regulated in mice with heart failure and human patients with dilated cardiomyopathy. Perm1 selectively regulated PGC-1/ERR targets, such as Ckmt2 and Sirt3. Perm1 interacted with PGC-1α, enhanced activation of PGC-1 and ERRs, and increased recruitment of PGC-1α to the target gene promoters (Ckmt2 and Sirt3), possibly creating a feedforward loop of Perm1 and PGC-1α/ERRs. Perm1 also increased mitochondrial DNA copy number and augmented oxidative capacity in NCM. Moreover, we found that Perm1 reduced cellular damage induced by H/R and mitigated cell death in cardiomyocytes. Conclusions: Perm1 promotes mitochondrial biogenesis in mouse cardiomyocytes through PGC-1/ERR and protects cellular damage induced by H/R. Future studies can assess the potential of Perm1 to be used as a novel therapeutic to restore cardiac dysfunction caused by ischemic injury. An NIH grant, R01HL151239, supports this work (To Y. C. and R. S. R.).

Abstract 12366: Intracoronary Delivery of E06 Antibody Directed to Oxidized Phospholipids Decreases Infarct Size in a Porcine Model of Ischemia/Reperfusion Injury

Introduction: Myocardial ischemia/reperfusion injury (IRI) results in additional myocardial cell loss post reperfusion. Oxidized phosphocholine(PC)-containing phospholipids (OxPL) have been shown to result in cardiomyocyte cell death during IRI. E06 is a natural murine monoclonal IgM antibody which binds to the PC headgroup of OxPL. Transgenic mice expressing a single-chain Fv E06 fragment have recently been shown to have smaller infarct sizes post myocardial IRI. The objective of this study was to determine if directly infused E06 IgM reduced IRI in a porcine model of reperfusion injury that more closely reflects human physiology. Methods: Male pigs, 45 to 50 kg, were randomized equally to IRI in two groups: 1) control with saline coronary infusion (n=7). 2) IgM E06 coronary infusion over 1 hour (n=7). The left anterior descending (LAD) artery was occluded distal to the second diagonal branch for 60 minutes using an angioplasty balloon. Following balloon deflation, saline or IgM E06 was infused over 60 min through a microcatheter. Following 120 min of total reperfusion, the area at risk (AAR) and infarct size (IS) were determined by triphenyltetrazolium chloride staining technique (TTC), and myocardial salvage index determined. Results: All animals survived post reperfusion and no differences were present in hemodynamics at the baseline and 2 h post reperfusion between groups. A 51.3% reduction in infarct size was noted in animals receiving intracoronary E06 compared to controls (IS/AAR 21.7% ± 3.4 vs 42.3% ± 3.7 p&lt;0.0005). IS/LV size (7.7 ± 3.2 vs 11.9 ± 1.2 p&lt;0.004) and myocardial salvage index (82.6% vs 57.6% in controls) were significantly improved in animals receiving E06 compared to controls. Conclusion: Intracoronary delivery of E06 antibody at the time of reperfusion leads to a significant reduction in infarct size, suggesting that generation of OxPL during reperfusion leads to cardiomyocyte cell death. Translation of this approach using humanized anti-OxPL antibodies may allow a novel approach to treat myocardial IRI in humans.

Abstract 14182: Diverse microRNA Cargo is Essential for the Cardioprotective Effects of Therapeutic Extracellular Vesicles

Background: Extracellular vesicles (EVs) deliver their protein and RNA cargo across the plasma membrane of target cells to exert a functional response. Injection of cardiosphere-derived cell (CDC) EVs have been shown to reduce cardiomyocyte (CM) cell death and improve cardiac function in large and small animal models of myocardial infarction (MI) and ischemia-reperfusion. While several EV miRNAs (miRs) have been identified to play a role in cardioprotection, it has not yet been established whether miR cargo is necessary for the therapeutic benefit of EVs. We hypothesized that miRNA cargo is essential in CDC-EV mediated cardioprotection post-MI. Methods: CDCs were transfected with siRNA against Drosha, the initial endonuclease in the miR biogenesis pathway. EVs were isolated from serum-free media and characterized by size, morphology, and protein/miR expression as previously described. We examined the role of EV miRNA on target cell apoptosis, proliferation, angiogenesis, and gene expression using a series of in vitro assays. To determine if miR cargo is essential for the therapeutic efficacy of CDC-EVs in vivo , we induced acute MI in adult C57BL/6 mice and randomized them to receive 1) EVs, 2) miR depleted EVs or 3) vehicle control. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of histological endpoints. Results: Transfection of CDCs with siRNA against Drosha reduces miRNA in CDCs and their EVs by 81±5% and 65±4%, respectively (mean ± SEM, t-test, p&lt;0.001). miR depleted EVs failed to recapitulate the reduction in cardiomyocyte apoptosis, decrease in fibroblast proliferation, and enhancement in angiogenesis seen with control EVs. Furthermore, while intramyocardial administered CDC-derived EVs improved LVEF compared with vehicle control treated animals 28 days post-MI (62 ± 4% vs 44 ± 4%), mice treated with miR-depleted EVs demonstrated no significant difference in LVEF (42 ± 6%) compared with vehicle control (n=8 mice/group, mean ± SEM, p&lt;0.05, one-way ANOVA). Conclusions: miR deficient human CDC-EVs are unable to recapitulate the cardioprotective effects seen with control extracellular vesicles, providing important insight into the mechanism of action of therapeutic EVs.

Abstract 12442: Extracellular Vesicles Engineered to Target Cardiomyocytes Demonstrate Improved Cardioprotection in a Mouse Model of Myocardial Infarction

Background: Cardiomyocyte (CM) cell death plays a significant role in left ventricular (LV) remodeling and cardiac dysfunction following myocardial infarction (MI). While extracellular vesicles (EVs) secreted by cardiosphere-derived cells (CDCs) have shown efficacy in promoting cardiac repair in large animal models of MI, their translational potential is limited by their biodistribution. To improve cardiac uptake, we previously engineered CDC-derived EVs to express a CM-specific binding peptide (CMP) on their surface. CMP-EVs demonstrated improved cardiac retention in vivo and decreased CM apoptosis in vitro when compared with non-targeted EVs. We hypothesized that targeting therapeutic EVs to CMs would result in further reduction of CM cell death in vivo and improvement in cardiac function post-MI. Methods: EVs were isolated from serum-free media as previously described and characterized by established protocols for size, morphology, and protein expression. To determine if CMP-EVs could enhance cardioprotection in vivo , we induced acute MI in adult C57BL/6 mice and randomized them to receive 1) EVs, 2) CMP-EVs or 3) vehicle control by IV and intramyocardial delivery according to approved institutional protocols. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of EV biodistribution and histological endpoints. Results: CMP-EVs demonstrated superior cardiac targeting (IV route) and retention (intramyocardial route) when compared with control EVs 24 hours post MI (fold change of 1.8 ± 0.23 and 4.8 ± 2.3 respectively; n=8-10 mice/group, mean ± SEM, p&lt;0.05, t-test). Intramyocardial administered CDC-derived EVs improved LVEF compared with vehicle control treated animals 4 weeks post-MI (55 ± 2% vs 44 ± 2%). Mice treated with CMP-EVs demonstrated a statistically significant improvement in LVEF (64 ± 5%) compared with non-targeted EVs (n=6-8 mice/group, mean ± SEM, p&lt;0.05, one-way ANOVA). Conclusions: Targeting CMP-EVs to CMs post-MI improved cardiac function compared with control EVs, highlighting the potential importance of CMs as a target cell for CDC-derived EV therapy and demonstrating a strategy to further optimize therapeutic EV delivery to increase efficacy and decease off-target effects.

CFLIPL Alleviates Myocardial Ischemia-Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress.

Endoplasmic reticulum stress (ERS) plays a crucial role in myocardial ischemia-reperfusion injury (MIRI). Cellular FLICE-inhibitory protein (cFLIP) is an essential regulator of apoptosis and plays a major role in regulating ERS. The present study aimed to investigate the effects of long isoform cFLIP (cFLIPL) on endogenous apoptosis and the mechanism of ERS in MIRI. The cFLIPL recombinant adenovirus vector was used to infect H9c2 cells and Sprague-Dawley (SD) rats. After infection for 72 h, ischemia was induced for 30 min, and reperfusion was then performed for 2 h to establish the MIRI model in SD rats. H9c2 cells were hypoxic for 4 h and then reoxygenated for 12 h to simulate ischemia/reperfusion (I/R) injury. Model parameters were evaluated by assessing cardiomyocyte viability, cell death (apoptosis), and ERS-related protein expression. In addition, tunicamycin (TM), an ERS agonist, was also added to the medium for pretreatment. Coimmunoprecipitation (Co-IP) of cFLIPL and p38 MAPK protein was performed. cFLIPL expression was decreased in I/R injury and hypoxia/reoxygenation (H/R) injury, and cFLIPL overexpression reduced myocardial infarction in vivo and increased the viability of H9c2 cells in vitro. I/R and H/R upregulated the protein expression of GRP78, IRE-1, and PERK to induce ERS and apoptosis. Interestingly, overexpression of cFLIPL significantly inhibited ERS and subsequent apoptosis, which was reversed by an agonist of ERS. Moreover, Co-IP showed that cFLIPL attenuated ERS and was associated with inhibiting the activation of p38 protein. The expression of cFLIPL is significantly downregulated in MIRI, and it is accompanied by excessive ERS and apoptosis. Upregulated cFLIPL suppresses ERS to reduce myocardial apoptosis, which is associated with inhibiting the activity of p38 MAPK. Therefore, cFLIPL may be a potential intervention target for MIRI.

Vascular endothelial growth factor ameliorated palmitate-induced cardiomyocyte injury via JNK pathway

Enhanced apoptosis of cardiomyocytes in suffering overloaded saturated fatty acids (SFAs) can result in myocardial infarction and cardiac dysfunction. The function of vascular endothelial growth factor (VEGF) in cardiomyocyte protection was not clearly described. To investigate the preservative effects of VEGF sensitization on ceramide-mediated programmed cell death of cardiomyocytes, palmitate-induced injury in H9c2 cells was established as an in vitro model. Results revealed that 0.5 mM palmitate application effectively led to debased viability and activated apoptotic factors. A significant time-dependent relation between PAL and cardiomyocyte injury was observed. The apoptosis rate was increased greatly after 16 h of treatment with 0.5 mM PAL. In addition, cell viability was restored by VEGF overexpression during treatment with 0.5 mM PAL. Reduced apoptosis rate and expression of caspase 3, Bax, and NF-κB p65 were observed in this process, while boosted Bcl-2, p-JNK/JNK expression and activity of caspase 3 were checked. However, p-ERK/ERK levels did not exhibit a significant change. These findings indicated the protective effects of VEGF in confronting the ceramide-induced cardiomyocyte apoptosis, and would devote therapeutic targets for cardiovascular safeguard in dealing with fatty acid stress.

The circRNA CNEACR regulates necroptosis of cardiomyocytes through Foxa2 suppression.

Circular RNAs (circRNAs) are differentially expressed in various cardiovascular disease including myocardial ischemia-reperfusion (I/R) injury. However, their functional impact on cardiomyocyte cell death, in particular, in necrotic forms of death remains elusive. In this study, we found that the level of mmu_circ_000338, a cardiac- necroptosis-associated circRNA (CNEACR), was reduced in hypoxia-reoxygenation (H/R) exposed cardiomyocytes and I/R-injured mice hearts. The enforced expression of CNEACR attenuated the necrotic form of cardiomyocyte death caused by H/R and suppressed of myocardial necrosis in I/R injured mouse heart, which was accompanied by a marked reduction of myocardial infarction size and improved cardiac function. Mechanistically, CNEACR directly binds to histone deacetylase (HDAC7) in the cytoplasm and interferes its nuclear entry. This leads to attenuation of HDAC7-dependent suppression of forkhead box protein A2 (Foxa2) transcription, which can repress receptor-interacting protein kinase 3 (Ripk3) gene by binding to its promoter region. In addition, CNEACR-mediated upregulation of FOXA2 inhibited RIPK3-dependent necrotic/necroptotic death of cardiomyocytes. Our study reveals that circRNAs such as CNEACR can regulate the cardiomyocyte necroptosis associated activity of HDACs, promotes cell survival and improves cardiac function in I/R-injured heart. Hence, the CNEACR/HDAC7/Foxa2/ RIPK3 axis could be an efficient target for alleviating myocardial damage caused by necroptotic death in ischemia heart diseases.

Selenium Deficiency Induces Apoptosis and Necroptosis Through ROS/MAPK Signal in Human Uterine Smooth Muscle Cells.

Selenium (Se) is one of the essential trace elements; its deficiency induces ROS production and cell death in cardiomyocytes, skeletal muscle cells, and vascular smooth muscle cells, but it is still not clear the impact of Se deficiency on human uterine smooth muscle cells (HUSMCs). To investigate the effect of low Se on the mRNA expression of selenoproteins, the mRNA and protein expression of apoptosis and necroptosis of HUSMCs and their mechanism, Se deficient HUSMCs mode was established through culturing with 1% FBS containing 0ng/mL, 0.7ng/mL, and 7ng/mL Se, and 10% FBS was as the control group. Then, the apoptosis and necroptosis rates, intracellular ROS content and the expression levels of selenoproteins, apoptosis, necroptosis, MAPK pathway-related genes were examined under different Se concentrations. The results showed that Se deficiency led to the augment of cell apoptosis and necroptosis in HUSMCs (p < 0.05), downregulated (p < 0.05) 19 selenoproteins (GPX1, GPX2, GPX3, GPX4, GPX6, Dio3, Txnrd2, Txnrd3, SEPHS2, SEL15, SELH, SELI, SELM, SELN, SELO, SELS, SELT, SELV, and SELW), while Dio2, SELK, Txnrd1, and MSRB1 were not affected by Se deficiency (p ≥ 0.05). In addition, Se deficiency led to increased intracellular ROS content, p-P38 and p-JNK gene expression levels (p < 0.05), the mitochondrial apoptosis pathway Bax, Casp9 and Cle-Casp3 protein expression levels (p < 0.05), and decreased Bcl2 protein expression level (p < 0.05), simultaneously, increased necroptosis marker genes RIP1, RIP3, and MLKL protein expression levels (p < 0.05) with a dose-dependent pattern. The above results indicate that Se deficiency induces HUSMCs apoptosis and necroptosis through the ROS/MAPK pathway and is closely related to selenoproteins.

RIP1/RIP3/MLKL Mediates Myocardial Function Through Necroptosis in Experimental Autoimmune Myocarditis.

Cardiomyopathy often leads to dilated cardiomyopathy (DCM) when caused by viral myocarditis. Apoptosis is long considered as the principal process of cell death in cardiomyocytes, but programmed necrosis or necroptosis is recently believed to play an important role in cardiomyocyte cell death. We investigated the role of necroptosis and its interdependency with other processes of cell death, autophagy, and apoptosis in a rat system of experimental autoimmune myocarditis (EAM). We successfully created a rat model system of EAM by injecting porcine cardiac myosin (PCM) and showed that in EAM, all three forms of cell death increase considerably, resulting in the deterioration of cardiac conditions with an increase in inflammatory infiltration in cardiomyocytes. To explore whether necroptosis occurs in EAM rats independent of autophagy, we treated EAM rats with a RIP1/RIP3/MLKL kinase-mediated necroptosis inhibitor, Necrostatin-1 (Nec-1). In Nec-1 treated rats, cell death proceeds through apoptosis but has no significant effect on autophagy. In contrast, autophagy inhibitor 3-Methyl Adenine (3-MA) increases necroptosis, implying that blockage of autophagy must be compensated through necroptosis. Caspase 8 inhibitor zVAD-fmk blocks apoptosis but increases both necroptosis and autophagy. However, all necroptosis, apoptosis, and autophagy inhibitors independently reduce inflammatory infiltration in cardiomyocytes and improve cardiac conditions. Since apoptosis or autophagy is involved in many important cellular aspects, instead of suppressing these two major cell death processes, Nec1 can be developed as a potential therapeutic target for inflammatory myocarditis.

MicroRNA-377 Signaling Modulates Anticancer Drug-Induced Cardiotoxicity in Mice.

Doxorubicin (DOX, an anthracycline) is a widely used chemotherapy agent against various forms of cancer; however, it is also known to induce dose-dependent cardiotoxicity leading to adverse complications. Investigating the underlying molecular mechanisms and strategies to limit DOX-induced cardiotoxicity might have potential clinical implications. Our previous study has shown that expression of microRNA-377 (miR-377) increases in cardiomyocytes (CMs) after cardiac ischemia-reperfusion injury in mice, but its specific role in DOX-induced cardiotoxicity has not been elucidated. In the present study, we investigated the effect of anti-miR-377 on DOX-induced cardiac cell death, remodeling, and dysfunction. We evaluated the role of miR-377 in CM apoptosis, its target analysis by RNA sequencing, and we tested the effect of AAV9-anti-miR-377 on DOX-induced cardiotoxicity and mortality. DOX administration in mice increases miR-377 expression in the myocardium. miR-377 inhibition in cardiomyocyte cell line protects against DOX-induced cell death and oxidative stress. Furthermore, RNA sequencing and Gene Ontology (GO) analysis revealed alterations in a number of cell death/survival genes. Intriguingly, we observed accelerated mortality and enhanced myocardial remodeling in the mice pretreated with AAV9-anti-miR-377 followed by DOX administration as compared to the AAV9-scrambled-control-pretreated mice. Taken together, our data suggest that in vitro miR-377 inhibition protects against DOX-induced cardiomyocyte cell death. On the contrary, in vivo administration of AAV9-anti-miR-377 increases mortality in DOX-treated mice.

Cardiomyocyte-Specific Circulating Cell-Free Methylated DNA in Esophageal Cancer Patients Treated with Chemoradiation

Thoracic high dose radiation therapy (RT) for cancer has been associated with early and late cardiac toxicity. To assess altered rates of cardiomyocyte cell death due to RT we monitored changes in cardiomyocyte-specific, cell-free methylated DNA (cfDNA) shed into the circulation. Eleven patients with distal esophageal cancer treated with neoadjuvant chemoradiation to 50.4 Gy (RT) and concurrent carboplatin and paclitaxel were enrolled. Subjects underwent fasting blood draws prior to the initiation and after completion of RT as well as 4–6 months following RT. An island of six unmethylated CpGs in the FAM101A locus was used to identify cardiomyocyte-specific cfDNA in serum. After bisulfite treatment this specific cfDNA was quantified by amplicon sequencing at a depth of >35,000 reads/molecule. Cardiomyocyte-specific cfDNA was detectable before RT in the majority of patient samples and showed some distinct changes during the course of treatment and recovery. We propose that patient-specific cardiac damages in response to the treatment are indicated by these changes although co-morbidities may obscure treatment-specific events.

WITHDRAWN: Inhibition of Malt1 protease induces apoptosis and cell death in cardiomyocytes

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Molecular mechanisms of anthracycline cardiovascular toxicity.

Anthracyclines are effective chemotherapeutic agents, commonly used in the treatment of a variety of hematologic malignancies and solid tumors. However, their use is associated with a significant risk of cardiovascular toxicities and may result in cardiomyopathy and heart failure. Cardiomyocyte toxicity occurs via multiple molecular mechanisms, including topoisomerase II-mediated DNA double-strand breaks and reactive oxygen species (ROS) formation via effects on the mitochondrial electron transport chain, NADPH oxidases (NOXs), and nitric oxide synthases (NOSs). Excess ROS may cause mitochondrial dysfunction, endoplasmic reticulum stress, calcium release, and DNA damage, which may result in cardiomyocyte dysfunction or cell death. These pathophysiologic mechanisms cause tissue-level manifestations, including characteristic histopathologic changes (myocyte vacuolization, myofibrillar loss, and cell death), atrophy and fibrosis, and organ-level manifestations including cardiac contractile dysfunction and vascular dysfunction. In addition, these mechanisms are relevant to current and emerging strategies to diagnose, prevent, and treat anthracycline-induced cardiomyopathy. This review details the established and emerging data regarding the molecular mechanisms of anthracycline-induced cardiovascular toxicity.

Perm1 promotes cardiomyocyte mitochondrial biogenesis and protects against hypoxia/reoxygenation-induced damage in mice

Normal contractile function of the heart depends on a constant and reliable production of ATP by cardiomyocytes. Dysregulation of cardiac energy metabolism can result in immature heart development and disrupt the ability of the adult myocardium to adapt to stress, potentially leading to heart failure. Further, restoration of abnormal mitochondrial function can have beneficial effects on cardiac dysfunction. Previously, we identified a novel protein termed Perm1 (PGC-1 and estrogen-related receptor (ERR)-induced regulator, muscle 1) that is enriched in skeletal and cardiac-muscle mitochondria and transcriptionally regulated by PGC-1 (peroxisome proliferator-activated receptor gamma coactivator 1) and ERR. The role of Perm1 in the heart is poorly understood and is studied here. We utilized cell culture, mouse models, and human tissue, to study its expression and transcriptional control, as well as its role in transcription of other factors. Critically, we tested Perm1's role in cardiomyocyte mitochondrial function and its ability to protect myocytes from stress-induced damage. Our studies show that Perm1 expression increases throughout mouse cardiogenesis, demonstrate that Perm1 interacts with PGC-1α and enhances activation of PGC-1 and ERR, increases mitochondrial DNA copy number, and augments oxidative capacity in cultured neonatal mouse cardiomyocytes. Moreover, we found that Perm1 reduced cellular damage produced as a result of hypoxia and reoxygenation-induced stress and mitigated cell death of cardiomyocytes. Taken together, our results show that Perm1 promotes mitochondrial biogenesis in mouse cardiomyocytes. Future studies can assess the potential of Perm1 to be used as a novel therapeutic to restore cardiac dysfunction induced by ischemic injury.