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.

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.

Immunomodulatory Cell Therapy Using αGalCer-Pulsed Dendritic Cells Ameliorates Heart Failure in a Murine Dilated Cardiomyopathy Model.

BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening disease, resulting in refractory heart failure. An immune disorder underlies the pathophysiology associated with heart failure progression. Invariant natural killer T (iNKT) cell activation is a prospective therapeutic strategy for ischemic heart disease. However, its efficacy in nonischemic cardiomyopathy, such as DCM, remains to be elucidated, and the feasible modality for iNKT cell activation in humans is yet to be validated. METHODS: Dendritic cells isolated from human volunteers were pulsed with α-galactosylceramide ex vivo, which were used as α-galactosylceramide-pulsed dendritic cells (αGCDCs). We treated DCM mice harboring mutated troponin TΔK210/ΔK210 with αGCDCs and evaluated the efficacy of iNKT cell activation on heart failure in DCM mice. Furthermore, we investigated the molecular basis underlying its therapeutic effects in these mice and analyzed primary cardiac cells under iNKT cell-secreted cytokines. RESULTS: The number of iNKT cells in the spleens of DCM mice was reduced compared with that in wild-type mice, whereas αGCDC treatment activated iNKT cells, prolonged survival of DCM mice, and prevented decline in the left ventricular ejection fraction for 4 weeks, accompanied by suppressed interstitial fibrosis. Mechanistically, αGCDC treatment suppressed TGF (transforming growth factor)-ß signaling and expression of fibrotic genes and restored vasculature that was impaired in DCM hearts by upregulating angiopoietin 1 (Angpt1) expression. Consistently, IFNγ (interferon gamma) suppressed TGF-ß-induced Smad2/3 signaling and the expression of fibrotic genes in cardiac fibroblasts and upregulated Angpt1 expression in cardiomyocytes via Stat1. CONCLUSIONS: Immunomodulatory cell therapy with αGCDCs is a novel therapeutic strategy for heart failure in DCM.

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.

GFAT2 mediates cardiac hypertrophy through HBP-O-GlcNAcylation-Akt pathway.

Molecular mechanisms mediating cardiac hypertrophy by glucose metabolism are incompletely understood. Hexosamine biosynthesis pathway (HBP), an accessory pathway of glycolysis, is known to be involved in the attachment of O-linked N-acetylglucosamine motif (O-GlcNAcylation) to proteins, a post-translational modification. We here demonstrate that glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2), a critical HBP enzyme, is a major isoform of GFAT in the heart and is increased in response to several hypertrophic stimuli, including isoproterenol (ISO). Knockdown of GFAT2 suppresses ISO-induced cardiomyocyte hypertrophy, accompanied by suppression of Akt O-GlcNAcylation and activation. Knockdown of GFAT2 does not affect anti-hypertrophic effect by Akt inhibition. Administration of glucosamine, a substrate of HBP, induces protein O-GlcNAcylation, Akt activation, and cardiomyocyte hypertrophy. In mice, 6-diazo-5-oxo-L-norleucine, an inhibitor of GFAT, attenuates ISO-induced protein O-GlcNAcylation, Akt activation, and cardiac hypertrophy. Our results demonstrate that GFAT2 mediates cardiomyocyte hypertrophy by HBP-O-GlcNAcylation-Akt pathway and could be a critical therapeutic target of cardiac hypertrophy.

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.

Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity.

Doxorubicin (DOX), a chemotherapeutic agent, induces a cardiotoxicity referred to as doxorubicin-induced cardiomyopathy (DIC). This cardiotoxicity often limits chemotherapy for malignancies and is associated with poor prognosis. However, the molecular mechanism underlying this cardiotoxicity is yet to be fully elucidated. Here, we show that DOX downregulated glutathione peroxidase 4 (GPx4) and induced excessive lipid peroxidation through DOX-Fe2+ complex in mitochondria, leading to mitochondria-dependent ferroptosis; we also show that mitochondria-dependent ferroptosis is a major cause of DOX cardiotoxicity. In DIC mice, the left ventricular ejection fraction was significantly impaired, and fibrosis and TUNEL+ cells were induced at day 14. Additionally, GPx4, an endogenous regulator of ferroptosis, was downregulated, accompanied by the accumulation of lipid peroxides, especially in mitochondria. These cardiac impairments were ameliorated in GPx4 Tg mice and exacerbated in GPx4 heterodeletion mice. In cultured cardiomyocytes, GPx4 overexpression or iron chelation targeting Fe2+ in mitochondria prevented DOX-induced ferroptosis, demonstrating that DOX triggered ferroptosis in mitochondria. Furthermore, concomitant inhibition of ferroptosis and apoptosis with ferrostatin-1 and zVAD-FMK fully prevented DOX-induced cardiomyocyte death. Our findings suggest that mitochondria-dependent ferroptosis plays a key role in progression of DIC and that ferroptosis is the major form of regulated cell death in DOX cardiotoxicity.

Roxadustat Markedly Reduces Myocardial Ischemia Reperfusion Injury in Mice.

BACKGROUND: Ischemic preconditioning (IPC) is an effective procedure to protect against ischemia/reperfusion (I/R) injury. Hypoxia-inducible factor-1α (Hif-1α) is a key molecule in IPC, and roxadustat (RXD), a first-in-class prolyl hydroxylase domain-containing protein inhibitor, has been recently developed to treat anemia in patients with chronic kidney disease. Thus, we investigated whether RXD pretreatment protects against I/R injury.Methods and Results:RXD pretreatment markedly reduced the infarct size and suppressed plasma creatinine kinase activity in a murine I/R model. Analysis of oxygen metabolism showed that RXD could produce ischemic tolerance by shifting metabolism from aerobic to anaerobic respiration. CONCLUSIONS: RXD pretreatment may be a novel strategy against I/R injury.

DPP (Dipeptidyl Peptidase)-4 Inhibitor Attenuates Ang II (Angiotensin II)-Induced Cardiac Hypertrophy via GLP (Glucagon-Like Peptide)-1-Dependent Suppression of Nox (Nicotinamide Adenine Dinucleotide Phosphate Oxidase) 4-HDAC (Histone Deacetylase) 4 Pathway.

Nox4 (NADPH [Nicotinamide adenine dinucleotide phosphate] oxidase 4) is a major source of oxidative stress and is intimately involved in cardiac hypertrophy. DPP (Dipeptidyl peptidase)-4 inhibitor has been reported to regulate Nox4 expression in adipose tissues. However, its effects on Nox4 in cardiac hypertrophy are still unclear. We investigated whether DPP-4 inhibitor could ameliorate cardiac hypertrophy by regulating Nox4 and its downstream targets. Ang II (Angiotensin II; 1.44 mg/kg per day) or saline was continuously infused into C57BL/6J mice with or without teneligliptin (a DPP-4 inhibitor, 30 mg/kg per day) in the drinking water for 1 week. Teneligliptin significantly suppressed plasma DPP-4 activity without any significant changing aortic blood pressure or metabolic parameters such as blood glucose and insulin levels. It attenuated Ang II-induced increases in left ventricular wall thickness and the ratio of heart weight to body weight. It also significantly suppressed Ang II-induced increases in Nox4 mRNA, 4-hydroxy-2-nonenal, and phosphorylation of HDAC4 (histone deacetylase 4), a downstream target of Nox4 and a crucial suppressor of cardiac hypertrophy, in the heart. Exendin-3 (150 pmol/kg per minute), a GLP-1 (glucagon-like peptide 1) receptor antagonist, abrogated these inhibitory effects of teneligliptin on Nox4, 4-hydroxy-2-nonenal, phosphorylation of HDAC4, and cardiac hypertrophy. In cultured neonatal cardiomyocytes, exendin-4 (100 nmol/L, 24 hours), a GLP-1 receptor agonist, ameliorated Ang II-induced cardiomyocyte hypertrophy and decreased in Nox4, 4-hydroxy-2-nonenal, and phosphorylation of HDAC4. Furthermore, exendin-4 prevented Ang II-induced decrease in nuclear HDAC4 in cardiomyocytes. In conclusion, GLP-1 receptor stimulation by DPP-4 inhibitor can attenuate Ang II-induced cardiac hypertrophy by suppressing of the Nox4-HDAC4 axis in cardiomyocytes.

Blockade of L-type Ca2+ channel attenuates doxorubicin-induced cardiomyopathy via suppression of CaMKII-NF-κB pathway.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor-kappa B (NF-κB) play crucial roles in pathogenesis of doxorubicin (DOX)-induced cardiomyopathy. Their activities are regulated by intracellular Ca2+. We hypothesized that blockade of L-type Ca2+ channel (LTCC) could attenuate DOX-induced cardiomyopathy by regulating CaMKII and NF-κB. DOX activated CaMKII and NF-κB through their phosphorylation and increased cleaved caspase 3 in cardiomyocytes. Pharmacological blockade or gene knockdown of LTCC by nifedipine or small interfering RNA, respectively, suppressed DOX-induced phosphorylation of CaMKII and NF-κB and apoptosis in cardiomyocytes, accompanied by decreasing intracellular Ca2+ concentration. Autocamtide 2-related inhibitory peptide (AIP), a selective CaMKII inhibitor, inhibited DOX-induced phosphorylation of NF-κB and cardiomyocyte apoptosis. Inhibition of NF-κB activity by ammonium pyrrolidinedithiocarbamate (PDTC) suppressed DOX-induced cardiomyocyte apoptosis. DOX-treatment (18 mg/kg via intravenous 3 injections over 1 week) increased phosphorylation of CaMKII and NF-κB in mouse hearts. Nifedipine (10 mg/kg/day) significantly suppressed DOX-induced phosphorylation of CaMKII and NF-κB and cardiomyocyte injury and apoptosis in mouse hearts. Moreover, it attenuated DOX-induced left ventricular dysfunction and dilatation. Our findings suggest that blockade of LTCC attenuates DOX-induced cardiomyocyte apoptosis via suppressing intracellular Ca2+ elevation and activation of CaMKII-NF-κB pathway. LTCC blockers might be potential therapeutic agents against DOX-induced cardiomyopathy.

[An usual case of vasculo- and neuro-Behçet's disease with MEFV mutations].

  A 35-year-old man had a 2-year history of uveitis and arterial wall thickening of an aortic branching artery detected by magnetic resonance imaging. He was admitted to our hospital because of mental exaltation. He had been treated with a moderate dose of prednisolone (30 mg/ml) plus methotrexate (6 mg/week) under the diagnosis of Takayasu arteritis for 2 years. Neurological examinations and brain magnetic resonance imaging taken on admission showed no abnormal findings. Although cerebrospinal fluid showed four cells/mm3 and 45 mg/dl of protein, cerebrospinal fluid interleukin-6 levels were markedly elevated (65.4 pg/mL), suggesting the neuro-Behçet's disease. The diagnosis of coexistence of vasculo- and neuro-Behçet's disease was made because vascular lesions are considered as a complication of Behçet's disease. We performed DNA testing using direct sequencing of all exons of the MEFV gene because of the patient's history of periodic fever since 30 year-old. The E148Q/L110P compound heterozygous mutation was found. His neurological symptoms and periodic fever were well controlled after the administration of infliximab (5 mg/kg every 2-month interval). Although occurrence by chance cannot be ruled out, the unusual findings of this patient suggest that MEFV mutations have some etiopathogenic mechanisms of an atypical phenotype of Behçet's disease.