IKKß stabilizes Mitofusin 2 and suppresses doxorubicin cardiomyopathy.

AIMS: The mitochondrial dynamics protein Mitofusin 2 (MFN2) coordinates critical cellular processes including mitochondrial bioenergetics, quality control, and cell viability. The NF-κB kinase IKKß suppresses mitochondrial injury in doxorubicin cardiomyopathy, but the underlying mechanism is undefined. METHODS AND RESULTS: Herein, we identify a novel signalling axis that functionally connects IKKß and doxorubicin cardiomyopathy to a mechanism that impinges upon the proteasomal stabilization of MFN2. In contrast to vehicle-treated cells, MFN2 was highly ubiquitinated and rapidly degraded by the proteasomal-regulated pathway in cardiac myocytes treated with doxorubicin. The loss of MFN2 activity resulted in mitochondrial perturbations, including increased reactive oxygen species (ROS) production, impaired respiration, and necrotic cell death. Interestingly, doxorubicin-induced degradation of MFN2 and mitochondrial-regulated cell death were contingent upon IKKß kinase activity. Notably, immunoprecipitation and proximity ligation assays revealed that IKKß interacted with MFN2 suggesting that MFN2 may be a phosphorylation target of IKKß. To explore this possibility, mass spectrometry analysis identified a novel MFN2 phospho-acceptor site at serine 53 that was phosphorylated by wild-type IKKß but not by a kinase-inactive mutant IKKßK-M. Based on these findings, we reasoned that IKKß-mediated phosphorylation of serine 53 may influence MFN2 protein stability. Consistent with this view, an IKKß-phosphomimetic MFN2 (MFN2S53D) was resistant to proteasomal degradation induced by doxorubicin whereas wild-type MFN2 and IKKß-phosphorylation defective MFN2 mutant (MFNS53A) were readily degraded in cardiac myocytes treated with doxorubicin. Concordantly, gain of function of IKKß or MFN2S53D suppressed doxorubicin-induced mitochondrial injury and cell death. CONCLUSIONS: The findings of this study reveal a novel survival pathway for IKKß that is mutually dependent upon and obligatory linked to the phosphorylation and stabilization of the mitochondrial dynamics protein MFN2.

Ellagic acid inhibits mitochondrial fission protein Drp-1 and cell proliferation in cancer.

Anthracyclines such as doxorubicin (Dox) are widely used to treat a variety of adult and childhood cancers, however, a major limitation to many of these compounds is their propensity for inducing heart failure. A naturally occurring polyphenolic compound such as Ellagic acid (EA) has been shown by our laboratory to mitigate the cardiotoxic effects of Dox, however, the effects of EA on cancer cell viability have not been established. In this study, we explored the effects of EA alone and in combination with Dox on cancer cell viability and tumorigenesis. Herein, we show that EA induces cell cycle exit and reduces proliferation in colorectal cancer (HCT116) and breast adenocarcinoma cells (MCF7). We show that EA promotes cell cycle exit by a mechanism that inhibits mitochondrial dynamics protein Drp-1. EA treatment of HCT116 and MCF7 cells resulted in a hyperfused mitochondrial morphology that coincided with mitochondrial perturbations including loss of mitochondrial membrane potential, impaired respiratory capacity. Moreover, impaired mitochondrial function was accompanied by a reduction in cell cycle and proliferation markers, CDK1, Ki67, and Cyclin B. This resulted in a reduction in proliferation and widespread death of cancer cells. Furthermore, while Dox treatment alone promoted cell death in both HCT116 and MCF7 cancer cell lines, EA treatment lowered the effective dose of Dox to promote cell death. Hence, the findings of the present study reveal a previously unreported anti-tumor property of EA that impinges on mitochondrial dynamics protein, Drp-1 which is crucial for cell division and tumorigenesis. The ability of EA to lower the therapeutic threshold of Dox for inhibiting cancer cell growth may prove beneficial in reducing cardiotoxicity in cancer patients undergoing anthracycline therapy.

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

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

NF-κB p65 Attenuates Cardiomyocyte PGC-1α Expression in Hypoxia.

Hypoxia exerts broad effects on cardiomyocyte function and viability, ranging from altered metabolism and mitochondrial physiology to apoptotic or necrotic cell death. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of cardiomyocyte metabolism and mitochondrial function and is down-regulated in hypoxia; however, the underlying mechanism is incompletely resolved. Using primary rat cardiomyocytes coupled with electrophoretic mobility shift and luciferase assays, we report that hypoxia impaired mitochondrial energetics and resulted in an increase in nuclear localization of the Nuclear Factor-κB (NF-κB) p65 subunit, and the association of p65 with the PGC-1α proximal promoter. Tumor necrosis factor α (TNFα), an activator of NF-κB signaling, similarly reduced PGC-1α expression and p65 binding to the PGC-1α promoter in a dose-dependent manner, and TNFα-mediated down-regulation of PGC-1α expression could be reversed by the NF-κB inhibitor parthenolide. RNA-seq analysis revealed that cardiomyocytes isolated from p65 knockout mice exhibited alterations in genes associated with chromatin remodeling. Decreased PGC-1α promoter transactivation by p65 could be partially reversed by the histone deacetylase inhibitor trichostatin A. These results implicate NF-κB signaling, and specifically p65, as a potent inhibitor of PGC-1α expression in cardiac myocyte hypoxia.

Mitochondrial autophagy and cell survival is regulated by the circadian Clock gene in cardiac myocytes during ischemic stress.

Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK.Abbreviations: ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATG14: autophagy related 14; ATG7: autophagy related 7; ATP: adenosine triphosphate; BCA: bovine serum albumin; BECN1: beclin 1, autophagy related; bHLH: basic helix- loop-helix; CLOCK: circadian locomotor output cycles kaput; CMV: cytomegalovirus; COQ5: coenzyme Q5 methyltransferase; CQ: chloroquine; CRY1: cryptochrome 1 (photolyase-like); DNM1L/DRP1: dynamin 1-like; EF: ejection fraction; EM: electron microscopy; FS: fractional shortening; GFP: green fluorescent protein; HPX: hypoxia; i.p.: intraperitoneal; I-R: ischemia-reperfusion; LAD: left anterior descending; LVIDd: left ventricular internal diameter diastolic; LVIDs: left ventricular internal diameter systolic; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; MI: myocardial infarction; mPTP: mitochondrial permeability transition pore; NDUFA4: Ndufa4, mitochondrial complex associated; NDUFA8: NADH: ubiquinone oxidoreductase subunit A8; NMX: normoxia; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PER1: period circadian clock 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; qPCR: quantitative real-time PCR; RAB7A: RAB7, member RAS oncogene family; ROS: reactive oxygen species; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TMRM: tetra-methylrhodamine methyl ester perchlorate; WT: wild -type; ZT: zeitgeber time.

Hypoxia-induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.

Ageing-associated increase in SGLT2 disrupts mitochondrial/sarcoplasmic reticulum Ca2+ homeostasis and promotes cardiac dysfunction.

The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age-associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co-transporter gene (SGLT2) in disrupted cellular Ca2+ -homeostasis, and mitochondrial dysfunction in age-associated cardiac dysfunction. In contrast to younger rats (6-month of age), older rats (24-month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca2+ ([Ca2+ ]i ) overload, indicative of disrupted cellular Ca2+ -homeostasis. Interestingly, increased [Ca2+ ]i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged-cardiomyocytes also displayed high Na+ /Ca2+ -exchanger (NCX) activity and blood glucose levels compared with young-controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age-associated defects in [Ca2+ ]i -homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca2+ -loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca2+ ]i -homeostasis. Our studies support the notion that interventions that modulate SGLT2-activity can provide benefits in maintaining [Ca2+ ]i and cardiac function with advanced age.

Impaired NF-κB signalling underlies cyclophilin D-mediated mitochondrial permeability transition pore opening in doxorubicin cardiomyopathy.

AIMS: The chemotherapy drug doxorubicin (Dox) is commonly used for treating a variety of human cancers; however, it is highly cardiotoxic and induces heart failure. We previously reported that the Bcl-2 mitochondrial death protein Bcl-2/19kDa interaction protein 3 (Bnip3), is critical for provoking mitochondrial perturbations and necrotic cell death in response to Dox; however, the underlying mechanisms had not been elucidated. Herein, we investigated mechanism that drives Bnip3 gene activation and downstream effectors of Bnip3-mediated mitochondrial perturbations and cell death in cardiac myocytes treated with Dox. METHODS AND RESULTS: Nuclear factor-κB (NF-κB) signalling, which transcriptionally silences Bnip3 activation under basal states in cardiac myocytes was dramatically reduced following Dox treatment. This was accompanied by Bnip3 gene activation, mitochondrial injury including calcium influx, permeability transition pore (mPTP) opening, loss of nuclear high mobility group protein 1, reactive oxygen species production, and cell death. Interestingly, impaired NF-κB signalling in cells treated with Dox was accompanied by protein complexes between Bnip3 and cyclophilin D (CypD). Notably, Bnip3-mediated mPTP opening was suppressed by inhibition of CypD-demonstrating that CypD functionally operates downstream of Bnip3. Moreover, restoring IKKß-NF-κB activity in cardiac myocytes treated with Dox suppressed Bnip3 expression, mitochondrial perturbations, and necrotic cell death. CONCLUSIONS: The findings of the present study reveal a novel signalling pathway that functionally couples NF-κB and Dox cardiomyopathy to a mechanism that is mutually dependent upon and obligatorily linked to the transcriptional control of Bnip3. Our findings further demonstrate that mitochondrial injury and necrotic cell death induced by Bnip3 is contingent upon CypD. Hence, maintaining NF-κB signalling may prove beneficial in reducing mitochondrial dysfunction and heart failure in cancer patients undergoing Dox chemotherapy.

Ulk1/Rab9-mediated alternative mitophagy confers cardioprotection during energy stress.

The heart relies on mitochondria-derived energy production for continuous contraction and relaxation; therefore, the maintenance of a pool of healthy mitochondria is essential for sustaining normal cardiac performance. Mitophagy serves as a critical process for maintaining mitochondrial quality control and involves the PTEN-induced kinase 1/Parkin (Pink1/Parkin) pathway and autophagosomes labeled with the autophagy proteins autophagy-related 7 (ATG) and light chain 3 (LC3). In this issue of the JCI, Saito and colleagues identify an alternative pathway for mitophagy that utilizes the serine/threonine protein kinase Unc-51-like kinase 1 (Ulk1) and the small GTPase Rab9 to clear damaged mitochondria independently of conventional autophagy proteins. Together, the results of this study reveal that Ulk1 phosphorylation of Rab9 at serine 179 is critical for alternative mitophagy and cardioprotection under energy stress conditions.

Ellagic acid antagonizes Bnip3-mediated mitochondrial injury and necrotic cell death of cardiac myocytes.

The Bcl-2 protein Bnip3 is crucial for provoking oxidative injury to mitochondria following anthracycline treatment or ischemia-reperfusion injury. Herein, we investigate the effects of the polyphenolic compound ellagic acid (EA) on Bnip3 mediated mitochondrial injury and necrotic cell death in cardiac myocytes. In contrast to vehicle treated cardiomyocytes, Bnip3 was highly enriched in mitochondrial fractions of cardiac myocytes treated with the anthracycline doxorubicin or in cells subjected to hypoxia (HPX). Mitochondrial associated Bnip3 was accompanied by mPTP opening and loss of ∆Ψm. The dynamin related fission protein Drp-1 was phosphorylated (Drp1616) and coincided with excessive mitochondrial fragmentation, mitophagy and necrosis in cardiac myocytes treated with doxorubicin or subjected to hypoxia. Moreover, knock-down of Bnip3 was sufficient to prevent mitochondrial fission and doxorubicin-induced cell death supporting the involvement of Bnip3 in doxorubicin cardiotoxity. Interestingly, mitochondrial associated Bnip3 in cells treated with doxorubicin was markedly reduced by EA. This resulted in significantly less mitochondrial fission and cell death. Notably, EA similarly suppressed mitochondrial injury and cell death induced by hypoxia or Bnip3 over-expression. Herein, we identify a novel signaling axis that operationally links EA and Bnip3 for suppression of cardiac cell death. We provide compelling new evidence that EA suppresses mitochondrial injury and necrotic cell death of cardiac myocytes by functionally abrogating Bnip3 activity. Hence, by suppressing mitochondrial injury induced by Bnip3, EA may provide a therapeutic advantage in reducing oxidative injury and cardiac dysfunction in cancer patients undergoing anthracycline treatment or individuals with ischemic cardiac stress.

PDK2-mediated alternative splicing switches Bnip3 from cell death to cell survival.

Herein we describe a novel survival pathway that operationally links alternative pre-mRNA splicing of the hypoxia-inducible death protein Bcl-2 19-kD interacting protein 3 (Bnip3) to the unique glycolytic phenotype in cancer cells. While a full-length Bnip3 protein (Bnip3FL) encoded by exons 1-6 was expressed as an isoform in normal cells and promoted cell death, a truncated spliced variant of Bnip3 mRNA deleted for exon 3 (Bnip3Δex3) was preferentially expressed in several human adenocarcinomas and promoted survival. Reciprocal inhibition of the Bnip3Δex3/Bnip3FL isoform ratio by inhibiting pyruvate dehydrogenase kinase isoform 2 (PDK2) in Panc-1 cells rapidly induced mitochondrial perturbations and cell death. The findings of the present study reveal a novel survival pathway that functionally couples the unique glycolytic phenotype in cancer cells to hypoxia resistance via a PDK2-dependent mechanism that switches Bnip3 from cell death to survival. Discovery of the survival Bnip3Δex3 isoform may fundamentally explain how certain cells resist Bnip3 and avert death during hypoxia.