Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.

BACKGROUND: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9 (Ras-related protein Rab-9A)-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial. We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are. METHODS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy was evaluated using cardiac-specific Mito-Keima mice. The role of DRP1 was evaluated using tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mice. RESULTS: Mitophagy was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in Drp1 MCM mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3 (microtubule-associated protein 1 light chain 3)-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 (fission protein 1) only during the chronic, but not acute, phase of HFD consumption. CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.

HTLV-1 viral oncoprotein HBZ contributes to the enhancement of HAX-1 stability by impairing the ubiquitination pathway.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia (ATL). The viral protein HTLV-1 basic leucine-zipper factor (HBZ), which is constitutively expressed in all ATL patient cells, contributes toward the development of ATL; however, the underlying mechanism has not been elucidated yet. Here, we identified HS-1-associated protein X-1 (HAX-1) as a novel binding partner of HBZ. Interestingly, HAX-1 specifically associated with HBZ-US, but not HBZ-SI, in the cytoplasm. HBZ suppressed the polyubiquitination levels of HAX-1 protein by inhibiting the association HAX-1 with F-box protein 25 (FBXO25), which is a member of the SCF E3 ubiquitin ligase complex, and promoted the stabilization of HAX-1 levels. In fact, the protein levels of HAX-1 were significantly increased in HTLV-1 infected and the overexpressing HBZ in uninfected T-cell lines. Enhanced HAX-1 correlated well to suppression of caspase 9 processing, suggesting that HBZ may contribute to the enhancement of antiapoptotic function for HAX-1. Our results revealed a role for HBZ on HAX-1 stabilization by abrogating the ubiquitination-mediated degradation pathway, which may play an important role in understanding the potential mechanisms of HTLV-1 related pathogenesis.

Hippo Deficiency Leads to Cardiac Dysfunction Accompanied by Cardiomyocyte Dedifferentiation During Pressure Overload.

RATIONALE: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. OBJECTIVE: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). METHODS AND RESULTS: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. CONCLUSIONS: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Overexpression of Transmembrane Protein BST2 is Associated with Poor Survival of Patients with Esophageal, Gastric, or Colorectal Cancer.

BACKGROUND: Gastrointestinal (GI) cancer, including gastric cancer (GC), colorectal cancer (CRC), and esophageal squamous cell carcinoma (ESCC), is the most common malignancy worldwide. To identify genes that encode transmembrane proteins present in GI cancer, Escherichia coli ampicillin secretion trap libraries were generated from MKN-74 GC cells, and BST2 was identified as overexpressed in GC. This study analyzed the expression and function of the BST2 gene in human GI cancers and examined the relationship between bone marrow stromal antigen-2 (BST-2) expression and GI patient clinicopathologic characteristics. METHODS: Expression and distribution of BST-2 protein was analyzed by immunohistochemistry in 180 GC cases, 140 CRC cases, and 132 ESCC cases. Cell growth was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Immunohistochemical analysis of BST-2 in GC tissues showed that 65 (36 %) of 180 GC cases were positive for BST-2. Uni- and multivariate analyses demonstrated that BST-2 expression is an independent prognostic classifier of GC patients. Immunohistochemical analysis showed that 46 % of 140 CRC cases and 27 % of 132 ESCC cases were positive for BST-2. In ESCC, BST-2 expression was an independent prognostic predictor for survival. The growth of BST2 small interfering RNA (siRNA)-transfected GC cells was significantly slower than the growth of negative control siRNA-transfected GC cells. The levels of phosphorylated epidermal growth factor receptor, extracellular signal-regulated kinase, and Akt were lower in BST2 siRNA-transfected GC cells than in control cells. CONCLUSIONS: The results suggest that BST-2 is involved in tumor progression and serves as an independent prognostic classifier for patients with GC. Because BST-2 is expressed on the cell membrane, BST-2 could be a therapeutic target for GC, CRC, and ESCC.

HTLV-1 bZIP factor suppresses the centromere protein B (CENP-B)-mediated trimethylation of histone H3K9 through the abrogation of DNA-binding ability of CENP-B.

Human T-cell leukaemia virus type-1 (HTLV-1) infection causes adult T-cell leukaemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in the pathogenesis of HTLV-1-associated disease. Here, we identified centromere protein B (CENP-B) as a novel interacting partner of HBZ. HBZ and CENP-B associate with their central regions in cells. Furthermore, overexpression of HBZ abrogated the DNA-binding activity of CENP-B to the α-satellite DNA region containing the CENP-B box motif, which in turn inhibited the CENP-B-mediated trimethylation of histone H3K9 in T-cells.

A lipidomics approach reveals novel phospholipid changes in palmitate-treated C2C12 myotubes.

Type 2 diabetes mellitus (T2DM) is a highly prevalent metabolic disorder. Insulin resistance and oxidative stress are associated with T2DM development. The hypothesis that patients with T2DM show excess accumulation of lipids, such as ceramides (Cers) and diacylglycerols (DAGs), in their skeletal muscles has been widely supported; however, detailed lipidomic data at the molecular species level are limited. Therefore, in this study, we aimed to investigate the in vitro dynamics of total lipids, including phospholipids (PLs), sphingolipids, and neutral lipids, in palmitic acid-induced insulin-resistant C2C12 skeletal muscle cells. Our data demonstrated that the profiles of not only Cers and DAGs but also those of PLs showed considerably differences after palmitate treatment. We found that PL synthesis reduced and PL degradation increased after palmitate treatment. These findings may aid in the development of treatments to ameliorate muscle dysfunction caused by lipid accumulation in muscles.

Improved mitochondrial function in the hearts of sarcolipin-deficient dystrophin and utrophin double-knockout mice.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin double-knockout (mdx:utrn-/-) mice in a sarcolipin (SLN) heterozygous-knockout (sln+/-) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn-/- mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes showed that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiomyocytes. These findings indicate that SLN upregulation plays a substantial role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD cardiomyopathy.

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia.

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload.

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including l-serine, l-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1α in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect.

Ulk1-dependent alternative mitophagy plays a protective role during pressure overload in the heart.

AIMS: Well-controlled mitochondrial homeostasis, including a mitochondria-specific form of autophagy (hereafter referred to as mitophagy), is essential for maintaining cardiac function. The molecular mechanism mediating mitophagy during pressure overload (PO) is poorly understood. We have shown previously that mitophagy in the heart is mediated primarily by Atg5/Atg7-independent mechanisms, including Unc-51-like kinase 1 (Ulk1)-dependent alternative mitophagy, during myocardial ischaemia. Here, we investigated the role of alternative mitophagy in the heart during PO-induced hypertrophy. METHODS AND RESULTS: Mitophagy was observed in the heart in response to transverse aortic constriction (TAC), peaking at 3-5 days. Whereas mitophagy is transiently up-regulated by TAC through an Atg7-dependent mechanism in the heart, peaking at 1 day, it is also activated more strongly and with a delayed time course through an Ulk1-dependent mechanism. TAC induced more severe cardiac dysfunction, hypertrophy, and fibrosis in ulk1 cardiac-specific knock-out (cKO) mice than in wild-type mice. Delayed activation of mitophagy was characterized by the co-localization of Rab9 dots and mitochondria and phosphorylation of Rab9 at Ser179, major features of alternative mitophagy. Furthermore, TAC-induced decreases in the mitochondrial aspect ratio were abolished and the irregularity of mitochondrial cristae was exacerbated, suggesting that mitochondrial quality control mechanisms are impaired in ulk1 cKO mice in response to TAC. TAT-Beclin 1 activates mitophagy even in Ulk1-deficient conditions. TAT-Beclin 1 treatment rescued mitochondrial dysfunction and cardiac dysfunction in ulk1 cKO mice during PO. CONCLUSION: Ulk1-mediated alternative mitophagy is a major mechanism mediating mitophagy in response to PO and plays an important role in mediating mitochondrial quality control mechanisms and protecting the heart against cardiac dysfunction.

Dual effects of HTLV-1 bZIP factor in suppression of interferon regulatory factor 1.

Human T-cell leukemia virus type-1 (HTLV-1) causes ATL in 2.5% of carriers after a long period of latent infection. Moreover, half of adult T-cell leukemia (ATL) patients succumb to this disease within 1year of onset. HTLV-1 bZIP factor (HBZ) is constitutively expressed in all the ATL cells. Thus, suggesting that HBZ may play a key role in cellular leukemogenesis. Herein we present evidence that interferon regulatory factor IRF-1, which is a member of IRF transcription family, interacts with HBZ. The N-terminal of HBZ interacted with IRF-1. HBZ reduced both IRF-1 DNA-binding activity and stability via a proteasome-dependent pathway. In addition, IRF-1-mediated apoptosis is significantly reduced by ectopic production of the HBZ. These results suggested that HBZ has dual suppressive effects on IRF-1 function, which may contribute to HTLV-1 related pathogenesis.

Lats2 promotes heart failure by stimulating p53-mediated apoptosis during pressure overload.

The Hippo pathway plays a wide variety of roles in response to stress in the heart. Lats2, a component of the Hippo pathway, is phosphorylated by Mst1/2 and, in turn, phosphorylates YAP, causing inactivation of YAP. Lats2 stimulates apoptosis and negatively affects hypertrophy in cardiomyocytes. However, the role of Lats2 during cardiac stress is poorly understood in vivo. Lats2 is activated in the mouse heart in response to transverse aortic constriction (TAC). We used systemic Lats2 +/- mice to elucidate the role of endogenous Lats2. Cardiac hypertrophy and dysfunction induced by 4 weeks of TAC were attenuated in Lats2 +/- mice, and interstitial fibrosis and apoptosis were suppressed. Although TAC upregulated the Bcl-2 family proapoptotic (Bax and Bak) and anti-apoptotic (Bcl-2 and Bcl-xL) molecules in non-transgenic mice, TAC-induced upregulation of Bax and Bak was alleviated and that of Bcl-2 was enhanced in Lats2 +/- mice. TAC upregulated p53, but this upregulation was abolished in Lats2 +/- mice. Lats2-induced increases in apoptosis and decreases in survival in cardiomyocytes were inhibited by Pifithrin-α, a p53 inhibitor, suggesting that Lats2 stimulates apoptosis via a p53-dependent mechanism. In summary, Lats2 is activated by pressure overload, thereby promoting heart failure by stimulating p53-dependent mechanisms of cell death.

HTLV-1 basic leucine-zipper factor, HBZ, interacts with MafB and suppresses transcription through a Maf recognition element.

HTLV-1 infection causes adult T-cell leukemia (ATL). The development of ATL is thought to be associated with disruption of transcriptional control of cellular genes. HTLV-1 basic leucine-zipper (bZIP) factor, HBZ, is encoded by the complementary strand of the provirus. We previously reported that HBZ interacts with c-Jun and suppresses its transcriptional activity. To identify the cellular factor(s) that interact with HBZ, we conducted a yeast two-hybrid screen using full-length HBZ as bait and identified MafB. HBZ heterodimerizes with MafB via each bZIP domain. Luciferase analysis revealed a significant decrease in transcription through Maf recognition element (MARE) in a manner dependent on the bZIP domain of HBZ. Indeed, production of full-length HBZ in cells decreased the MARE-bound MafB protein, indicating that HBZ abrogates the DNA-binding activity of MafB. In addition, HBZ reduced the steady-state levels of MafB, and the levels were restored by treatment with a proteasome inhibitor. These results suggest a suppressive effect of HBZ on Maf function, which may have a significant role in HTLV-1 related pathogenesis.

Comprehensive autophagy evaluation in cardiac disease models.

Autophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system.

Perm1 regulates cardiac energetics as a downstream target of the histone methyltransferase Smyd1.

The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.

Intermittent Fasting Reverses an Advanced Form of Cardiomyopathy.

See Article by Ma et al.

Yes-Associated Protein (YAP) Facilitates Pressure Overload-Induced Dysfunction in the Diabetic Heart.

Patients with diabetes are more prone to developing heart failure in the presence of high blood pressure than those without diabetes. Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is persistently activated in diabetic hearts, and YAP plays an essential role in mediating the exacerbation of heart failure in response to pressure overload in the hearts of mice fed a high-fat diet. YAP induced dedifferentiation of cardiomyocytes through activation of transcriptional enhancer factor 1 (TEAD1), a transcription factor. Thus, YAP and TEAD1 are promising therapeutic targets for diabetic patients with high blood pressure to prevent the development of heart failure.

An alternative mitophagy pathway mediated by Rab9 protects the heart against ischemia.

Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of unc-51 like kinase 1 (Ulk1), Rab9, receptor-interacting serine/thronine protein kinase 1 (Rip1), and dynamin-related protein 1 (Drp1). This complex allowed the recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through S179 phosphorylation of Rab9 by Ulk1 and S616 phosphorylation of Drp1 by Rip1. Knockin of Rab9 (S179A) abolished mitophagy and exacerbated the injury in response to myocardial ischemia, without affecting conventional autophagy. Mitophagy mediated through the Ulk1/Rab9/Rip1/Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.