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.

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.

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.

Mitochondrial quality control: The role of mitophagy in aging.

Autophagy is a catabolic process for eliminating macromolecules and damaged organelles by a highly regulated lysosomal pathway. Importantly, autophagy serves as an integral quality control mechanism by recycling cellular constituents for energy consumption and cellular rejuvenation under basal and stress conditions. Nevertheless, there is growing evidence that under certain conditions autophagy can switch from an adaptive survival mechanism to maladaptive process that promotes cell death. Furthermore, defects in autophagy have been linked to mitochondria injury and cell death associated with aging. In this review, we describe the role of autophagy as a physiological mechanism for maintaining homeostasis with its specific involvement in mitochondrial quality control and cardiac aging.

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.