Rimpy Dhingra

Striking a Balance: Autophagy, Apoptosis, and Necrosis in a Normal and Failing Heart

Despite the progress that has been made over the past two decades in cardiovascular research, heart failure remains a major cause of morbidity and mortality worldwide. Insight into the cellular and molecular mechanisms that underlie the heart failure in individuals with ischemic heart disease have identified defects in cellular processes that govern autophagy, apoptosis and necrosis as a prevailing underlying cause. Indeed, programmed cell death of cardiac cells by apoptosis or necrosis is believed to involve the intrinsic mitochondrial pathway and/or extrinsic death receptor pathway by certain Bcl-2 family members as well as components of the TNFα signaling pathway. In this review, we discuss recent advances in the molecular signaling factors that govern cardiac cell fate under normal and disease conditions.

Mitochondrial dynamics: Orchestrating the journey to advanced age.

Aging is a degenerative process that unfortunately is an inevitable part of life and risk factor for cardiovascular disease including heart failure. Among the several theories purported to explain the effects of age on cardiac dysfunction, the mitochondrion has emerged a central regulator of this process. Hence, it is not surprising that abnormalities in mitochondrial quality control including biogenesis and turnover have such detrimental effects on cardiac function. In fact mitochondria serve as a conduit for biological signals for apoptosis, necrosis and autophagy respectively. The removal of damaged mitochondria by autophagy/mitophagy is essential for mitochondrial quality control and cardiac homeostasis. Defects in mitochondrial dynamism fission/fusion events have been linked to cardiac senescence and heart failure. In this review we discuss the impact of aging on mitochondrial dynamics and senescence on cardiovascular health. This article is part of a Special Issue entitled: CV Aging.

Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling

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. Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3−/− mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy.

A Novel Hypoxia-Inducible Spliced Variant of Mitochondrial Death Gene Bnip3 Promotes Survival of Ventricular Myocytes

Rationale: Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. Previously, we established hypoxia-inducible death factor Bnip3 as a critical component of the intrinsic death pathway. Objective: To investigate alternative splicing of Bnip3 pre-mRNA in postnatal ventricular myocytes during hypoxia. Methods and Results: We identify a novel previously unrecognized spliced variant of Bnip3 (Bnip3&Dgr;ex3) generated by alternative splicing of exon3 exclusively in cardiac myocytes subjected to hypoxia. Sequencing of Bnip3&Dgr;ex3 revealed a frame shift mutation that terminated transcription up-stream of exon5 and exon6 ablating translation of the BH3-like domain and critical carboxyl-terminal transmembrane domain crucial for mitochondrial localization and cell death. Notably, although the 26-kDa Bnip3 protein (Bnip3FL) encoded by full-length mRNA was localized to mitochondria and provoked cell death, the 8.2-kDa Bnip3&Dgr;ex3 protein encoded by the truncated spliced mRNA was defective for mitochondrial targeting but interacted with Bnip3FL resulting in less association of Bnip3FL with mitochondria and diminished apoptotic and necrotic cell death. Forced expression of Bnip3FL in cardiac myocytes or Bnip3−/− mouse embryonic fibroblasts triggered widespread cell death that was inhibited by coexpression of Bnip3&Dgr;ex3. Conversely, RNA interference targeted against sequences encompassing the unique exon2-exon4 junction of the Bnip3&Dgr;ex3 sensitized cardiac myocytes to mitochondrial perturbations and cell death induced by Bnip3FL. Conclusions: Given the otherwise lethal consequences of deregulated Bnip3FL expression in postmitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the mitochondrial defects and cell death of ventricular myocytes that is obligatorily linked and mutually dependent on alternative splicing of Bnip3FL during hypoxia or ischemic stress.

p53 Mediates Autophagy and Cell Death by a Mechanism Contingent On Bnip3

Myocardial ischemia and angiotensin II activate the tumor suppressor p53 protein, which promotes cell death. Previously, we showed that the Bcl-2 death gene Bnip3 is highly induced during ischemia, where it triggers mitochondrial perturbations resulting in autophagy and cell death. However, whether p53 regulates Bnip3 and autophagy is unknown. Herein, we provide new compelling evidence for a novel signaling axis that commonly links p53 and Bnip3 for autophagy and cell death. p53 overexpression increased endogenous Bnip3 mRNA and protein levels resulting in mitochondrial defects leading to loss of mitochondrial &Dgr;&PSgr;m. This was accompanied by an increase in autophagic flux and cell death. Notably, genetic loss of function studies, such as Atg7 knock-down or pharmacological inhibition of autophagy with 3-methyl adenine, suppressed cell death induced by p53—indicating that p53 induces maladaptive autophagy. Our previous work demonstrated that Bnip3 induces mitochondrial defects and autophagic cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3−/− mouse embryonic fibroblasts prevented mitochondrial targeting of p53, autophagy, and cell death. To our knowledge, these data provide the first evidence for the dual regulation of autophagy and cell death of cardiac myocytes by p53 that is mutually dependent on and obligatorily linked to Bnip3 gene activation. Hence, our findings may explain more fundamentally, how, autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

Bi-Directional Regulation of NF-κB and mTOR Signaling Functionally Links Bnip3 Gene Repression and Cell Survival of Ventricular Myocytes

Background—Tumor necrosis factor-&agr; and other proinflammatory cytokines activate the canonical Nuclear Factor (NF)-&kgr;B pathway through the kinase IKK&bgr;. Previously, we established that IKK&bgr; is also critical for Akt-mediated NF-&kgr;B activation in ventricular myocytes. Akt activates the kinase mammalian target of rapamycin (mTOR), which mediates important processes such as cardiac hypertrophy. However, whether mTOR regulates cardiac myocyte cell survival is unknown. Methods and Results—Herein, we demonstrate bidirectional regulation between NF-&kgr;B signaling and mTOR, the balance which determines ventricular myocyte survival. Overexpression of IKK&bgr; resulted in mTOR activation and conversely overexpression of mTOR lead to NF-&kgr;B activation. Loss of function approaches demonstrated that endogenous levels of IKK&bgr; and mTOR also signal through this pathway. NF-&kgr;B activation by mTOR was mediated by phosphorylation of the NF-&kgr;B p65 subunit increasing p65 nuclear translocation and activation of gene transcription. This circuit was also important for NF-&kgr;B activation by the canonical tumor necrosis factor-&agr; pathway. Our previous work has shown that NF-&kgr;B signaling suppresses transcription of the death gene Bnip3 resulting in ventricular myocyte survival. Inhibition of mTOR with rapamycin decreased NF-&kgr;B activation resulting in increased Bnip3 expression and cell death. Conversely, mTOR overexpression suppressed Bnip3 levels and cell death of ventricular myocytes in response to hypoxia. Conclusions—To our knowledge, these data provide the first evidence for a bidirectional link between NF-&kgr;B signaling and mTOR that is critical in the regulation of Bnip3 expression and cardiac myocyte death. Hence, modulation of this axis may be cardioprotective during ischemia.

Bidirectional Regulation of Nuclear Factor-κB and Mammalian Target of Rapamycin Signaling Functionally Links Bnip3 Gene Repression and Cell Survival of Ventricular Myocytes

Background—Tumor necrosis factor-&agr; and other proinflammatory cytokines activate the canonical Nuclear Factor (NF)-&kgr;B pathway through the kinase IKK&bgr;. Previously, we established that IKK&bgr; is also critical for Akt-mediated NF-&kgr;B activation in ventricular myocytes. Akt activates the kinase mammalian target of rapamycin (mTOR), which mediates important processes such as cardiac hypertrophy. However, whether mTOR regulates cardiac myocyte cell survival is unknown. Methods and Results—Herein, we demonstrate bidirectional regulation between NF-&kgr;B signaling and mTOR, the balance which determines ventricular myocyte survival. Overexpression of IKK&bgr; resulted in mTOR activation and conversely overexpression of mTOR lead to NF-&kgr;B activation. Loss of function approaches demonstrated that endogenous levels of IKK&bgr; and mTOR also signal through this pathway. NF-&kgr;B activation by mTOR was mediated by phosphorylation of the NF-&kgr;B p65 subunit increasing p65 nuclear translocation and activation of gene transcription. This circuit was also important for NF-&kgr;B activation by the canonical tumor necrosis factor-&agr; pathway. Our previous work has shown that NF-&kgr;B signaling suppresses transcription of the death gene Bnip3 resulting in ventricular myocyte survival. Inhibition of mTOR with rapamycin decreased NF-&kgr;B activation resulting in increased Bnip3 expression and cell death. Conversely, mTOR overexpression suppressed Bnip3 levels and cell death of ventricular myocytes in response to hypoxia. Conclusions—To our knowledge, these data provide the first evidence for a bidirectional link between NF-&kgr;B signaling and mTOR that is critical in the regulation of Bnip3 expression and cardiac myocyte death. Hence, modulation of this axis may be cardioprotective during ischemia.

Dichotomous Actions of NF-κB Signaling Pathways in Heart

Despite the substantial progress in heart research over the past two decades heart failure still remains a major cause of morbidity and mortality in North America and is reaching pandemic proportions worldwide. Though the underlying causes are varied, the functional loss of contractile myocytes through apoptosis, necrosis, and autophagy has emerged a central unifying theme to explain diminished cardiac performance in individuals with heart failure. At the molecular level, there has been considerable interest in understanding the signaling pathways that regulate cell death in the heart with specific interest in the extrinsic and intrinsic cell death pathways. The cellular factor nuclear factor-κB (NF-κB) is a key transcription factor involved in the regulation of a wide range of genes involved in cellular process including inflammation, immune cell maturation, cell proliferation, and, most recently, cell survival. NF-κB signaling is important for the normal cellular growth and is a major target of inflammatory cytokines. Several studies have highlighted a protective role of NF-κB in the heart under certain circumstances including hypoxic or ischemic myocardial injury. The diverse nature and involvement of NF-κB in regulation of vital cellular processes including cell survival notably in the post-mitotic heart has sparked considerable interest in understanding the signaling pathways involved in regulating NF-κB in the heart under normal and pathological conditions. However, whether NF-κB is adaptive, maladaptive or is a homeostatic response to cardiac injury may simply depend on the context and timing of its activation. In this forum we discuss NF-κB signaling pathways and therapeutic opportunities to modulate NF-κB activity in heart failure.

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

In hypoxia, the survival property of cancer cells mediated by the glycolytic enzyme PDK2 is obligatorily linked to alternative splicing and generation of a novel isoform of death gene Bnip3, which suppresses mitochondrial injury and promotes survival.

Epigenetic regulation of canonical TNFα pathway by HDAC1 determines survival of cardiac myocytes

Gene transcription is regulated by post-translation modifications. Histone deacetylases (HDACs) remove acetyl groups from histone and non-histone factors inhibiting transcription. Proinflammatory cytokines such as TNFα activate the canonical nuclear factor-κB (NF-κB) pathway. Earlier we established a cytoprotective role for NF-κB in the heart. Though a causal relationship for HDAC1 and NF-κB has been established, the impact of HDAC1 on TNFα signaling is unknown. Herein, we demonstrate that HDAC1 provides a molecular switch for determining cell survival in the TNFα pathway. In contrast to vehicle-treated control cells, TNFα-treated cells displayed a marked increase in NF-κB gene transcription. Notably, cells treated with TNFα were indistinguishable from vehicle controls cells with respect to viability. Interestingly, HDAC activity was reduced in cells treated with TNFα. Conversely, in the presence of HDAC1, NF-κB gene transcription by TNFα was repressed, resulting in mitochondrial perturbations and widespread cell death. Heterologous fusion proteins comprised of yeast Gal4 DNA binding domain fused in frame to the NF-κB p65 transactivation domain were preferentially repressed by HDAC1. Moreover, transcription mediated by Gal4VP16 protein from herpes virus was unaffected by HDAC1 in cardiac myocytes. Mutations that abrogate known catalytic activities of HDAC1, small interference RNA, or pharmacological inhibition of HDAC1 restored NF-κB signaling and suppressed cell death induced by TNFα. These data provide the first evidence for an obligate link between HDAC1 and canonical TNFα pathway for cell survival of cardiac myocytes.