The progression from cardiac injury to symptomatic heart failure has been intensely studied over the last decade, and is largely attributable to a loss of functional cardiac myocytes through necrosis, intrinsic and extrinsic apoptosis pathways and autophagy. Therefore, the molecular regulation of these cellular programs has been rigorously investigated in the hopes of identifying a potential cell target that could promote cell survival and/or inhibit cell death to avert, or at least prolong, the degeneration toward symptomatic heart failure. The nuclear factor (NF)-κB super family of transcription factors has been implicated in the regulation of immune cell maturation, cell survival, and inflammation in many cell types, including cardiac myocytes. Recent studies have shown that NF-κB is cardioprotective during acute hypoxia and reperfusion injury. However, prolonged activation of NF-κB appears to be detrimental and promotes heart failure by eliciting signals that trigger chronic inflammation through enhanced elaboration of cytokines including tumor necrosis factor α, interleukin-1, and interleukin-6, leading to endoplasmic reticulum stress responses and cell death. The underlying mechanisms that account for the multifaceted and differential outcomes of NF-κB on cardiac cell fate are presently unknown. Herein, we posit a novel paradigm in which the timing, duration of activation, and cellular context may explain mechanistically the differential outcomes of NF-κB signaling in the heart that may be essential for future development of novel therapeutic interventions designed to target NF-κB responses and heart failure following myocardial injury.

Significance: We provide new, exciting evidence for a previously unidentified signaling pathway that mechanistically links mitochondrial respiratory chain defects to necrosis and heart failure induced by the chemotherapy agent doxorubicin (DOX). We specifically show that DOX disrupts protein complexes between the key respiratory chain proteins, including uncoupling protein 3 and cytochrome c oxidase, resulting in abnormal mitochondrial respiration and necrosis through a mechanism contingent on Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Perhaps most compelling is our finding that inhibiting Bnip3 completely abrogated the cardiotoxic effects of DOX. These exciting findings have important clinical implications not only for preventing heart failure by targeting Bnip3 in cancer patients undergoing chemotherapy, but also for understanding the pathogenesis of other diseases in which mitochondrial function is compromised.

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

The transcription factor E2F-1 drives proliferation and death, but the mechanisms that differentially regulate these divergent actions are poorly understood. The hypoxia-inducible death factor Bnip3 is an E2F-1 target gene and integral component of the intrinsic mitochondrial death pathway. The mechanisms that govern Bnip3 gene activity remain cryptic. Herein we show that the transcription factor NF-κB provides a molecular switch that determines whether E2F-1 signals proliferation or death under physiological conditions. We show under basal nonapoptotic conditions that NF-κB constitutively occupies and transcriptionally silences Bnip3 gene transcription by competing with E2F-1 for Bnip3 promoter binding. Conversely, in the absence of NF-κB, or during hypoxia when NF-κB abundance is reduced, basal Bnip3 gene transcription is activated by the unrestricted binding of E2F-1 to the Bnip3 promoter. Genetic knock-down of E2F-1 or retinoblastoma gene product over-expression in cardiac and human pancreatic cancer cells deficient for NF-κB signaling abrogated basal and hypoxia-inducible Bnip3 transcription. The survival kinase PI3K/Akt inhibited Bnip3 expression levels in cells in a manner dependent upon NF-κB activation. Hence, by way of example, we show that the transcriptional inhibition of E2F-1-dependent Bnip3 expression by NF-κB highlights a survival pathway that overrides the E2F-1 tumor suppressor program. Our data may explain more fundamentally how cells, by selectively inhibiting E2F-1-dependent death gene transcription, avert apoptosis down-stream of the retinoblastoma/E2F-1 cell cycle pathway.

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.