Abstract 25: Polyphenolic - Ellagic Acid Suppresses Mitophagy- Induced Necrotic Cell Death During Doxorubicin Cardiotoxicity

Reactive oxygen species (ROS) play a major role in cardiac dysfunction during myocardial ischemia. ROS production has been linked to oxidative stress injury and mitochondrial perturbations including permeability transition pore opening (mPTP), loss of mitochondrial membrane potential ([[Unable to Display Character: ∆]]Ψm) and necrotic cell death. Previously we identified the inducible Bcl-2 protein, Bnip3 as critical regulator of mitochondrial function and cell death of ventricular myocytes. Polyphenolic compounds including ellagic acid from pomegranate, have strong anti-oxidant properties. The effects of ellagic acid on oxidative stress injury in the heart has not been explored. In this report, we provide new compelling evidence that ellagic acid suppressed mitochondrial ROS production, loss of [[Unable to Display Character: ∆]]Ψm and necrotic cell death of cardiac myocytes induced by doxorubicin (DOX) or hypoxia. We further show mechanistically that the cytoprotective effects of ellagic acid were related to the transcriptional repression of Bnip3. In contrast to vehicle treated cells, cells treated with DOX or hypoxia displayed a marked increase in Bnip3 expression and mitochondrial association, concordant with increased ROS, mPTP, and loss of [[Unable to Display Character: ∆]]Ψm. Consistent with these mitochondrial defects there was a marked increase in mitophagy as confirmed by the dual emission Mitokeima probe that detects autophagic degradation by labelling mitochondria containing autophagosomes fused with lysosome. Mitophagy was accompanied by a marked increase in LDH release, loss of nuclear HMGB1 immunostaining and cell death. Interestingly, cells treated with ellagic acid were resistant to mitochondrial and the cytotoxic effects of DOX displaying reduced ROS production, mitophagy and were indistinguishable from vehicle treated control cells with respect to cell viability. Notably, Dox-induced Bnip3 expression was dramatically reduced in cells treated with ellagic acid. Hence, the findings of the present study demonstrate that ellagic acid suppresses mitochondrial perturbations and cell death of cardiac myocytes by mechanism that links to the repression of mitochondrial Bnip3.

Download Full-text

Abstract 24: Bnip3 Provokes ROS Production and Maladaptive Autophagy by Displacing Uncoupling Protein3 (UCP3) From Cytochrome c Oxidase of Respiration Chain Complex in Cardiotoxicity

Circulation Research ◽

10.1161/res.117.suppl_1.24 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Rimpy Dhingra ◽

Victoria Margulets ◽

Davinder Jassal ◽

Gerald Dorn II ◽

Lorrie A. Kirshenbaum

Keyword(s):

Cell Death ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Cardiac Myocytes ◽

Mitochondrial Function ◽

Uncoupling Protein ◽

Mitochondrial Morphology ◽

Necrotic Cell Death ◽

Ros Production ◽

Necrotic Cell

Doxorubicin is known for its cardiotoxic effects and inducing cardiac failure, however, the underlying mechanisms remain cryptic. Earlier we established the inducible - death protein, Bcl-2-like Nineteen- Kilodalton- Interacting - Protein 3 (Bnip3) to be crucial for disrupting mitochondrial function and inducing cell death of cardiac myocytes. Whether Bnip3 underlies cardiotoxic effects of doxorubicin toxicity is unknown. Herein we demonstrate a novel signaling pathway that functionally links activation and preferential mitochondrial targeting of Bnip3 to the cardiotoxic properties of doxorubicin. Perturbations to mitochondria including increased calcium loading, ROS, loss of αΨm and mPTP opening were observed in cardiac myocytes treated with doxorubicin. In mitochondria, Bnip3 forms strong association with Cytochrome c oxidase subunit1 (COX1) of respiratory chain and displaces uncoupling protein 3 (UCP3) resulting in increased ROS production, decline in maximal and reserved respiration capacity and cell viability. Impaired mitochondrial function was accompanied by an accumulated increase in autophagosomes and necrosis demonstrated by increase release of LDH, cTnT and loss of nuclear High Mobility Group Protein 1 (HMGB-1) immunoreactivity. Interestingly, pharmacological or genetic inhibition of autophagy with 3-methyl adenine (3-MA), or Atg7 knock-down suppressed necrotic cell death induced by doxorubicin. Loss of function of Bnip3 restored UCP3-COX complexes, mitochondrial respiratory integrity and abrogated necrotic cell death induced by doxorubicin. Mice germ-line deficient for Bnip3 were resistant to doxorubicin cardiotoxicity displaying normal mitochondrial morphology, cardiac function and survival rates comparable to vehicle treated mice. The findings of the present study demonstrate that doxorubicin provokes maladaptive autophagy and necrotic cell death of ventricular myocytes that is mutually dependent and obligatorily linked to Bnip3.

Download Full-text

Abstract 23: Disruption of TRAF2-TAK1-NF-κB Signaling Axis Triggers K-48 Linked Poly-Ubiquitylation of RIP1 and Necrotic Cell Death in Doxorubicin Cardiotoxicity

Circulation Research ◽

10.1161/res.117.suppl_1.23 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Rimpy Dhingra ◽

Victoria Margulets ◽

Floribeth Aguilar ◽

Lorrie A. Kirshenbaum

Keyword(s):

Cell Death ◽

Cardiac Myocytes ◽

Cell Injury ◽

Ventricular Myocytes ◽

Necrotic Cell Death ◽

Ros Production ◽

Necrotic Cell ◽

Interacting Protein

The anthracycline doxorubicin (Dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -mTOR-NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, Dox impairs IKKβ-mTOR- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of mitochondrial membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63- ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complexes and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1, in vivo and in vitro. This was accompanied by a decline in K63- and concomitant increase in K-48 linked polyubiquitination on RIP1, impaired NF-kB activation and necrotic cell death of cardiac myocytes. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 linked poly ubiquitylation of RIP1 provides a functional switch that impairs NF-kB activation and signals necrosis in cells treated with dox. Interventions that modulate NF-kB activity may prove beneficial in mitigating the cardiotoxic effects of dox.

Download Full-text

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

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2017.08.010 ◽

2017 ◽

Vol 112 ◽

pp. 411-422 ◽

Cited By ~ 20

Author(s):

Abhinav Dhingra ◽

Rahul Jayas ◽

Pegah Afshar ◽

Matthew Guberman ◽

Graham Maddaford ◽

...

Keyword(s):

Cell Death ◽

Cardiac Myocytes ◽

Ellagic Acid ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Mitochondrial Injury

Download Full-text

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

Cardiovascular Research ◽

10.1093/cvr/cvz240 ◽

2019 ◽

Vol 116 (6) ◽

pp. 1161-1174 ◽

Cited By ~ 7

Author(s):

Rimpy Dhingra ◽

Matthew Guberman ◽

Inna Rabinovich-Nikitin ◽

Jonathon Gerstein ◽

Victoria Margulets ◽

...

Keyword(s):

Heart Failure ◽

Cell Death ◽

Cardiac Myocytes ◽

Gene Activation ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Necrotic Cell Death ◽

Cyclophilin D ◽

Necrotic Cell ◽

Mptp Opening

Abstract 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 IKKβ–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.

Download Full-text

Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death

Pharmacological Research ◽

10.1016/j.phrs.2013.03.002 ◽

2013 ◽

Vol 72 ◽

pp. 25-34 ◽

Cited By ~ 5

Author(s):

Csaba Hegedűs ◽

Petra Lakatos ◽

Attila Kiss-Szikszai ◽

Tamás Patonay ◽

Szabolcs Gergely ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Necrotic Cell Death ◽

Necrotic Cell

Download Full-text

Targeting of the c-Abl Tyrosine Kinase to Mitochondria in the Necrotic Cell Death Response to Oxidative Stress

Journal of Biological Chemistry ◽

10.1074/jbc.m101414200 ◽

2001 ◽

Vol 276 (20) ◽

pp. 17281-17285 ◽

Cited By ~ 74

Author(s):

Shailendra Kumar ◽

Ajit Bharti ◽

Neerad C. Mishra ◽

Deepak Raina ◽

Surender Kharbanda ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Tyrosine Kinase ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Abl Tyrosine Kinase ◽

Cell Death Response

Download Full-text

The TGFβ1 Receptor Antagonist GW788388 Reduces JNK Activation and Protects Against Acetaminophen Hepatotoxicity in Mice

Toxicological Sciences ◽

10.1093/toxsci/kfz122 ◽

2019 ◽

Vol 170 (2) ◽

pp. 549-561 ◽

Cited By ~ 3

Author(s):

Matthew McMillin ◽

Stephanie Grant ◽

Gabriel Frampton ◽

Anca D Petrescu ◽

Elaina Williams ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Growth Factor ◽

Liver Injury ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Growth Factor Beta ◽

And Oxidative Stress

Abstract Acute liver failure is a serious consequence of acetaminophen (APAP)-induced hepatotoxic liver injury with high rates of morbidity and mortality. Transforming growth factor beta 1 (TGFβ1) is elevated during liver injury and influences hepatocyte senescence during APAP-induced hepatotoxicity. This study investigated TGFβ1 signaling in the context of inflammation, necrotic cell death, and oxidative stress during APAP-induced liver injury. Male C57Bl/6 mice were injected with 600 mg/kg APAP to generate liver injury in the presence or absence of the TGFβ receptor 1 inhibitor, GW788388, 1 h prior to APAP administration. Acetaminophen-induced liver injury was characterized using histological and biochemical measures. Transforming growth factor beta 1 expression and signal transduction were assessed using immunohistochemistry, Western blotting and ELISA assays. Hepatic necrosis, liver injury, cell proliferation, hepatic inflammation, and oxidative stress were assessed in all mice. Acetaminophen administration significantly induced necrosis and elevated serum transaminases compared with control mice. Transforming growth factor beta 1 staining was observed in and around areas of necrosis with phosphorylation of SMAD3 observed in hepatocytes neighboring necrotic areas in APAP-treated mice. Pretreatment with GW788388 prior to APAP administration in mice reduced hepatocyte cell death and stimulated regeneration. Phosphorylation of SMAD3 was reduced in APAP mice pretreated with GW788388 and this correlated with reduced hepatic cytokine production and oxidative stress. These results support that TGFβ1 signaling plays a significant role in APAP-induced liver injury by influencing necrotic cell death, inflammation, oxidative stress, and hepatocyte regeneration. In conclusion, targeting TGFβ1 or downstream signaling may be a possible therapeutic target for the management of APAP-induced liver injury.

Download Full-text