Deferasirox Targeting Ferroptosis Synergistically Ameliorates Myocardial Ischemia Reperfusion Injury in Conjunction With Cyclosporine A.

BACKGROUND: Ferroptosis, an iron-dependent form of regulated cell death, is a major cell death mode in myocardial ischemia reperfusion (I/R) injury, along with mitochondrial permeability transition-driven necrosis, which is inhibited by cyclosporine A (CsA). However, therapeutics targeting ferroptosis during myocardial I/R injury have not yet been developed. Hence, we aimed to investigate the therapeutic efficacy of deferasirox, an iron chelator, against hypoxia/reoxygenation-induced ferroptosis in cultured cardiomyocytes and myocardial I/R injury. METHODS AND RESULTS: The effects of deferasirox on hypoxia/reoxygenation-induced iron overload in the endoplasmic reticulum, lipid peroxidation, and ferroptosis were examined in cultured cardiomyocytes. In a mouse model of I/R injury, the infarct size and adverse cardiac remodeling were examined after treatment with deferasirox, CsA, or both in combination. Deferasirox suppressed hypoxia- or hypoxia/reoxygenation-induced iron overload in the endoplasmic reticulum, lipid peroxidation, and ferroptosis in cultured cardiomyocytes. Deferasirox treatment reduced iron levels in the endoplasmic reticulum and prevented increases in lipid peroxidation and ferroptosis in the I/R-injured myocardium 24 hours after I/R. Deferasirox and CsA independently reduced the infarct size after I/R injury to a similar degree, and combination therapy with deferasirox and CsA synergistically reduced the infarct size (infarct area/area at risk; control treatment: 64±2%; deferasirox treatment: 48±3%; CsA treatment: 48±4%; deferasirox+CsA treatment: 37±3%), thereby ameliorating adverse cardiac remodeling on day 14 after I/R. CONCLUSIONS: Combination therapy with deferasirox and CsA may be a clinically feasible and effective therapeutic approach for limiting I/R injury and ameliorating adverse cardiac remodeling after myocardial infarction.

IFN-γ-STAT1-ERK Pathway Mediates Protective Effects of Invariant Natural Killer T Cells Against Doxorubicin-Induced Cardiomyocyte Death.

Doxorubicin (DOX)-induced cardiomyopathy has poor prognosis, and myocardial inflammation is intimately involved in its pathophysiology. The role of invariant natural killer T (iNKT) cells has not been fully determined in this disease. We here demonstrated that activation of iNKT cells by α-galactosylceramide (GC) attenuated DOX-induced cardiomyocyte death and cardiac dysfunction. αGC increased interferon (IFN)-γ and phosphorylation of signal transducers and activators of transcription 1 (STAT1) and extracellular signal-regulated kinase (ERK). Administration of anti-IFN-γ neutralizing antibody abrogated the beneficial effects of αGC on DOX-induced cardiac dysfunction. These findings emphasize the protective role of iNKT cells in DOX-induced cardiomyopathy via the IFN-γ-STAT1-ERK pathway.

Cardiac Autoantibodies Against Cardiac Troponin I in Post-Myocardial Infarction Heart Failure: Evaluation in a Novel Murine Model and Applications in Therapeutics.

BACKGROUND: Cardiac autoantibodies (cAAbs) are involved in the progression of adverse cardiac remodeling in heart failure (HF). However, our understanding of cAAbs in HF is limited owing to the absence of relevant animal models. Herein, we aimed to establish and characterize a murine model of cAAb-positive HF after myocardial infarction (MI), thereby facilitating the development of therapeutics targeting cAAbs in post-MI HF. METHODS: MI was induced in BALB/c mice. Plasma cAAbs were evaluated using modified Western blot-based methods. Prognosis, cardiac function, inflammation, and fibrosis were compared between cAAb-positive and cAAb-negative MI mice. Rapamycin was used to inhibit cAAb production. RESULTS: Common cAAbs in BALB/c MI mice targeted cTnI (cardiac troponin I). Herein, 71% (24/34) and 44% (12/27) of the male and female MI mice, respectively, were positive for cAAbs against cTnI (cTnIAAb). Germinal centers were formed in the spleens and mediastinal lymph nodes of cTnIAAb-positive MI mice. cTnIAAb-positive MI mice showed progressive cardiac remodeling with a worse prognosis (P=0.014, by log-rank test), which was accompanied by cardiac inflammation, compared with that in cTnIAAb-negative MI mice. Rapamycin treatment during the first 7 days after MI suppressed cTnIAAb production (cTnIAAb positivity, 59% [29/49] and 7% [2/28] in MI mice treated with vehicle and rapamycin, respectively; P<0.001, by Pearson χ2 test), consequently improving the survival and ameliorating cardiac inflammation, cardiac remodeling, and HF in MI mice. CONCLUSIONS: The present post-MI HF model may accelerate our understanding of cTnIAAb and support the development of therapeutics against cTnIAAbs in post-MI HF.

ZBP1 Protects Against mtDNA-Induced Myocardial Inflammation in Failing Hearts.

BACKGROUND: Mitochondrial DNA (mtDNA)-induced myocardial inflammation is intimately involved in cardiac remodeling. ZBP1 (Z-DNA binding protein 1) is a pattern recognition receptor positively regulating inflammation in response to mtDNA in inflammatory cells, fibroblasts, and endothelial cells. However, the role of ZBP1 in myocardial inflammation and cardiac remodeling remains unclear. The aim of this study was to elucidate the role of ZBP1 in mtDNA-induced inflammation in cardiomyocytes and failing hearts. METHODS: mtDNA was administrated into isolated cardiomyocytes. Myocardial infarctionwas conducted in wild type and ZBP1 knockout mice. RESULTS: We here found that, unlike in macrophages, ZBP1 knockdown unexpectedly exacerbated mtDNA-induced inflammation such as increases in IL (interleukin)-1ß and IL-6, accompanied by increases in RIPK3 (receptor interacting protein kinase 3), phosphorylated NF-κB (nuclear factor-κB), and NLRP3 (nucleotide-binding domain and leucine-rich-repeat family pyrin domain containing 3) in cardiomyocytes. RIPK3 knockdown canceled further increases in phosphorylated NF-κB, NLRP3, IL-1ß, and IL-6 by ZBP1 knockdown in cardiomyocytes in response to mtDNA. Furthermore, NF-κB knockdown suppressed such increases in NLRP3, IL-1ß, and IL-6 by ZBP1 knockdown in response to mtDNA. CpG-oligodeoxynucleotide, a Toll-like receptor 9 stimulator, increased RIPK3, IL-1ß, and IL-6 and ZBP1 knockdown exacerbated them. Dloop, a component of mtDNA, but not Tert and B2m, components of nuclear DNA, was increased in cytosolic fraction from noninfarcted region of mouse hearts after myocardial infarction compared with control hearts. Consistent with this change, ZBP1, RIPK3, phosphorylated NF-κB, NLRP3, IL-1ß, and IL-6 were increased in failing hearts. ZBP1 knockout mice exacerbated left ventricular dilatation and dysfunction after myocardial infarction, accompanied by further increases in RIPK3, phosphorylated NF-κB, NLRP3, IL-1ß, and IL-6. In histological analysis, ZBP1 knockout increased interstitial fibrosis and myocardial apoptosis in failing hearts. CONCLUSIONS: Our study reveals unexpected protective roles of ZBP1 against cardiac remodeling as an endogenous suppressor of mtDNA-induced myocardial inflammation.

Doxorubicin causes ferroptosis and cardiotoxicity by intercalating into mitochondrial DNA and disrupting Alas1-dependent heme synthesis.

Clinical use of doxorubicin (DOX) is limited because of its cardiotoxicity, referred to as DOX-induced cardiomyopathy (DIC). Mitochondria-dependent ferroptosis, which is triggered by iron overload and excessive lipid peroxidation, plays a pivotal role in the progression of DIC. Here, we showed that DOX accumulated in mitochondria by intercalating into mitochondrial DNA (mtDNA), inducing ferroptosis in an mtDNA content-dependent manner. In addition, DOX disrupted heme synthesis by decreasing the abundance of 5'-aminolevulinate synthase 1 (Alas1), the rate-limiting enzyme in this process, thereby impairing iron utilization, resulting in iron overload and ferroptosis in mitochondria in cultured cardiomyocytes. Alas1 overexpression prevented this outcome. Administration of 5-aminolevulinic acid (5-ALA), the product of Alas1, to cultured cardiomyocytes and mice suppressed iron overload and lipid peroxidation, thereby preventing DOX-induced ferroptosis and DIC. Our findings reveal that the accumulation of DOX and iron in mitochondria cooperatively induces ferroptosis in cardiomyocytes and suggest that 5-ALA can be used as a potential therapeutic agent for DIC.

Iron Overload via Heme Degradation in the Endoplasmic Reticulum Triggers Ferroptosis in Myocardial Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury is a promising therapeutic target to improve clinical outcomes after acute myocardial infarction. Ferroptosis, triggered by iron overload and excessive lipid peroxides, is reportedly involved in I/R injury. However, its significance and mechanistic basis remain unclear. Here, we show that glutathione peroxidase 4 (GPx4), a key endogenous suppressor of ferroptosis, determines the susceptibility to myocardial I/R injury. Importantly, ferroptosis is a major mode of cell death in I/R injury, distinct from mitochondrial permeability transition (MPT)-driven necrosis. This suggests that the use of therapeutics targeting both modes is an effective strategy to further reduce the infarct size and thereby ameliorate cardiac remodeling after I/R injury. Furthermore, we demonstrate that heme oxygenase 1 up-regulation in response to hypoxia and hypoxia/reoxygenation degrades heme and thereby induces iron overload and ferroptosis in the endoplasmic reticulum (ER) of cardiomyocytes. Collectively, ferroptosis triggered by GPx4 reduction and iron overload in the ER is distinct from MPT-driven necrosis in both in vivo phenotype and in vitro mechanism for I/R injury. The use of therapeutics targeting ferroptosis in conjunction with cyclosporine A can be a promising strategy for I/R injury.

Ethoxyquin is a Competent Radical-Trapping Antioxidant for Preventing Ferroptosis in Doxorubicin Cardiotoxicity.

ABSTRACT: Doxorubicin (DOX) is an effective anti-cancer agent for various malignancies. Nevertheless, it has a side effect of cardiotoxicity, referred to as doxorubicin-induced cardiomyopathy (DIC), that is associated with a poorer prognosis. This cardiotoxicity limits the clinical use of DOX as a therapeutic agent for malignancies. Recently, ferroptosis, a form of regulated cell death induced by the accumulation of lipid peroxides, has been recognized as a major pathophysiology of DIC. Ethoxyquin is a lipophilic antioxidant widely used for food preservation and thus may be a potential therapeutic drug for preventing DIC. However, the efficacy of ethoxyquin against ferroptosis and DIC remains to be fully elucidated. Here, we investigated the inhibitory action of ethoxyquin against GPx4-deficient ferroptosis and its therapeutic efficacy against DOX-induced cell death in cultured cardiomyocytes and cardiotoxicity in a murine model of DIC. In cultured cardiomyocytes, ethoxyquin treatment effectively prevented GPx4-deficient ferroptosis. Ethoxyquin also prevented DOX-induced cell death, accompanied by the suppression of malondialdehyde (MDA) and mitochondrial lipid peroxides, which were induced by DOX. Furthermore, ethoxyquin significantly prevented DOX-induced cell death without any suppression of caspase cleavages representing apoptosis. In DIC mice, ethoxyquin treatment ameliorated cardiac impairments, such as contractile dysfunction and myocardial atrophy, and lung congestion. Ethoxyquin also suppressed serum lactate dehydrogenase and creatine kinase activities, decreased the levels of lipid peroxides such as MDA and acrolein, inhibited cardiac fibrosis, and reduced TUNEL-positive cells in the hearts of DIC mice. Collectively, ethoxyquin is a competent antioxidant for preventing ferroptosis in DIC and can be its prospective therapeutic drug.

Heart Rate Reduction with Ivabradine Prevents Cardiac Rupture after Myocardial Infarction in Mice.

PURPOSE: Cardiac rupture is a fatal complication following myocardial infarction (MI). An increase in heart rate (HR) is reportedly an independent risk factor for cardiac rupture during acute MI. However, the role of HR reduction in cardiac rupture after MI remains to be fully elucidated. We aimed to evaluate the therapeutic efficacy of HR reduction with ivabradine (IVA) on post-MI cardiac rupture in mice. METHODS: We induced MI in mice by ligating the left anterior descending coronary artery. Subsequently, we subcutaneously implanted osmotic pumps filled with IVA solution or vehicle (Veh) in the surviving MI mice at 24 h postoperatively. We biochemically analyzed the myocardium on day 5, additionally observed the mice for 10 days, and analyzed the rates of cardiac rupture and non-cardiac rupture death, and survival after MI. RESULTS: HR was significantly lower in the IVA-treated mice, whereas blood pressure was comparable between the two groups. Compared to the Veh-treated mice, apoptosis was significantly reduced in the MI border zone in the IVA-treated mice. Although there were no differences in the infarct size of the surviving MI mice between the two groups, HR reduction with IVA significantly reduced cardiac rupture (rupture rate 26 and 8% in the Veh-treated and IVA-treated groups, respectively) and improved survival after MI. CONCLUSION: Our findings suggest that HR reduction with IVA prevents cardiac rupture after MI. This may be particularly effective in MI patients with a high HR who are either unable to adequately tolerate ß-blockers or whose HR remains high despite receiving ß-blockers.

Excessive Hypoxia-Inducible Factor-1α Expression Induces Cardiac Rupture via p53-Dependent Apoptosis After Myocardial Infarction.

Background Apoptosis plays a pivotal role in cardiac rupture after myocardial infarction (MI), and p53 is a key molecule in apoptosis during cardiac rupture. Hif-1α (hypoxia-inducible factor-1α), upregulated under hypoxia, is a known p53 inducer. However, the role of Hif-1α in the regulatory mechanisms underlying p53 upregulation, apoptosis, and cardiac rupture after MI is unclear. Methods and Results We induced MI in mice by ligating the left anterior descending artery. Hif-1α and p53 expressions were upregulated in the border zone at day 5 after MI, accompanied by apoptosis. In rat neonatal cardiomyocytes, treatment with cobalt chloride (500 µmol/L), which mimics severe hypoxia by inhibiting PHD (prolyl hydroxylase domain-containing protein), increased Hif-1α and p53, accompanied by myocyte death with caspase-3 cleavage. Silencing Hif-1α or p53 inhibited caspase-3 cleavage, and completely prevented myocyte death under PHD inhibition. In cardiac-specific Hif-1α hetero-knockout mice, expression of p53 and cleavage of caspase-3 and poly (ADP-ribose) polymerase were reduced, and apoptosis was suppressed on day 5. Furthermore, the cleavage of caspase-8 and IL-1ß (interleukin-1ß) was also suppressed in hetero knockout mice, accompanied by reduced macrophage infiltration and matrix metalloproteinase/tissue inhibitor of metalloproteinase activation. Although there was no intergroup difference in infarct size, the cardiac rupture and survival rates were significantly improved in the hetero knockout mice until day 10 after MI. Conclusions Hif-1α plays a pivotal role in apoptosis, inflammation, and cardiac rupture after MI, in which p53 is a critical mediator, and may be a prospective therapeutic target for preventing cardiac rupture.