RESUMO
Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.
Assuntos
Gasderminas , Piroptose , Proteínas de Neoplasias/metabolismo , Cardiolipinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inflamassomos/metabolismoRESUMO
Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.
RESUMO
Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.
Assuntos
Morte Celular , Citotoxicidade Imunológica , Cardiopatias/patologia , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Cardiopatias/imunologia , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Humanos , Mitocôndrias Cardíacas/imunologia , Mitocôndrias Cardíacas/metabolismo , Miocárdio/imunologia , Miocárdio/metabolismo , Necrose , Piroptose , Transdução de SinaisRESUMO
The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.
Assuntos
Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras , Camundongos , Animais , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Peptidil-Prolil Isomerase F , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Cálcio/metabolismoRESUMO
At least seven cell death programs are activated during myocardial infarction (MI), but which are most important in causing heart damage is not understood. Two of these programs are mitochondrial-dependent necrosis and apoptosis. The canonical function of the pro-cell death BCL-2 family proteins BAX and BAK is to mediate permeabilization of the outer mitochondrial membrane during apoptosis allowing apoptogen release. BAX has also been shown to sensitize cells to mitochondrial-dependent necrosis, although the underlying mechanisms remain ill-defined. Genetic deletion of Bax or both Bax and Bak in mice reduces infarct size following reperfused myocardial infarction (MI/R), but the contribution of BAK itself to cardiomyocyte apoptosis and necrosis and infarction has not been investigated. In this study, we use Bak-deficient mice and isolated adult cardiomyocytes to delineate the role of BAK in the pathogenesis of infarct generation and post-infarct remodeling during MI/R and non-reperfused MI. Generalized homozygous deletion of Bak reduced infarct size â¼50% in MI/R in vivo, which was attributable primarily to decreases in necrosis. Protection from necrosis was also observed in BAK-deficient isolated cardiomyocytes suggesting that the cardioprotection from BAK loss in vivo is at least partially cardiomyocyte-autonomous. Interestingly, heterozygous Bak deletion, in which the heart still retains â¼28% of wild type BAK levels, reduced infarct size to a similar extent as complete BAK absence. In contrast to MI/R, homozygous Bak deletion did not attenuate acute infarct size or long-term scar size, post-infarct remodeling, cardiac dysfunction, or mortality in non-reperfused MI. We conclude that BAK contributes significantly to cardiomyocyte necrosis and infarct generation during MI/R, while its absence does not appear to impact the pathogenesis of non-reperfused MI. These observations suggest BAK may be a therapeutic target for MI/R and that even partial pharmacological antagonism may provide benefit.
Assuntos
Infarto do Miocárdio , Proteína Killer-Antagonista Homóloga a bcl-2 , Animais , Camundongos , Apoptose/fisiologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Homozigoto , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Necrose/genética , Deleção de Sequência , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism typified by stable proliferative arrest, a persistent DNA damage response, and the senescence-associated secretory phenotype (SASP), which helps to maintain the senescent state and triggers bystander senescence in a paracrine fashion. Here, we demonstrate that the tumor suppressive histone variant macroH2A1 is a critical component of the positive feedback loop that maintains SASP gene expression and triggers the induction of paracrine senescence. MacroH2A1 undergoes dramatic genome-wide relocalization during OIS, including its removal from SASP gene chromatin. The removal of macroH2A1 from SASP genes results from a negative feedback loop activated by SASP-mediated endoplasmic reticulum (ER) stress. ER stress leads to increased reactive oxygen species and persistent DNA damage response including activation of ATM, which mediates removal macroH2A1 from SASP genes. Together, our findings indicate that macroH2A1 is a critical control point for the regulation of SASP gene expression during senescence.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Senescência Celular/genética , Senescência Celular/fisiologia , Histonas/genética , Histonas/metabolismo , Linhagem Celular , Dano ao DNA , Estresse do Retículo Endoplasmático , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Oncogenes , Comunicação Parácrina , FenótipoRESUMO
Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/química , Permeabilidade , Conformação Proteica/efeitos dos fármacosRESUMO
RATIONALE: Thioredoxin-interacting protein (Txnip) is a novel molecular target with translational potential in diverse human diseases. Txnip has several established cellular actions including binding to thioredoxin, a scavenger of reactive oxygen species (ROS). It has been long recognized from in vitro evidence that Txnip forms a disulfide bridge through cysteine 247 (C247) with reduced thioredoxin to inhibit the anti-oxidative properties of thioredoxin. However, the physiological significance of the Txnip-thioredoxin interaction remains largely undefined in vivo. OBJECTIVE: A single mutation of Txnip, C247S, abolishes the binding of Txnip with thioredoxin. Using a conditional and inducible approach with a mouse model of a mutant Txnip that does not bind thioredoxin, we tested whether the interaction of thioredoxin with Txnip is required for Txnip's pro-oxidative or cytotoxic effects in the heart. METHODS AND RESULTS: Overexpression of Txnip C247S in cells resulted in a reduction in ROS, due to an inability to inhibit thioredoxin. Hypoxia (1% O2, 24 h)-induced killing effects of Txnip were decreased by lower levels of cellular ROS in Txnip C247S-expressing cells compared with wild-type Txnip-expressing cells. Then, myocardial ischemic injuries were assessed in the animal model. Cardiomyocyte-specific Txnip C247S knock-in mice had better survival with smaller infarct size following myocardial infarction (MI) compared to control animals. The absence of Txnip's inhibition of thioredoxin promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage induced by MI. Furthermore, an unbiased RNA sequencing screen identified that hypoxia-inducible factor 1 signaling pathway was involved in Txnip C247S-mediated cardioprotective mechanisms. CONCLUSION: Txnip is a cysteine-containing redox protein that robustly regulates the thioredoxin system via a disulfide bond-switching mechanism in adult cardiomyocytes. Our results provide the direct in vivo evidence that regulation of redox state by Txnip is a crucial component for myocardial homeostasis under ischemic stress.
Assuntos
Alelos , Substituição de Aminoácidos , Proteínas de Transporte/genética , Resistência à Doença/genética , Mutação , Infarto do Miocárdio/etiologia , Tiorredoxinas/genética , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores , Proteínas de Transporte/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eletrocardiografia , Expressão Gênica , Glucose/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/metabolismo , Especificidade de Órgãos/genética , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Previously we reported that adipocyte SNAP23 (synaptosome-associated protein of 23 kDa) deficiency blocks the activation of macroautophagy, leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion, BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, whereas in the nutrient-depleted state, BAX binds to SNAP23 to form a ternary BAX-SNAP23-ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program.
Assuntos
Apoptose/fisiologia , Proteínas Relacionadas à Autofagia/fisiologia , Autofagia/fisiologia , Proteína X Associada a bcl-2/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Camundongos , Células NIH 3T3 , Ligação Proteica , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Frações Subcelulares/metabolismoRESUMO
BAX is a critical effector of the mitochondrial cell death pathway in response to a diverse range of stimuli in physiological and disease contexts. Upon binding by BH3-only proteins, cytosolic BAX undergoes conformational activation and translocation, resulting in mitochondrial outer-membrane permeabilization. Efforts to rationally target BAX and develop inhibitors have been elusive, despite the clear therapeutic potential of inhibiting BAX-mediated cell death in a host of diseases. Here, we describe a class of small-molecule BAX inhibitors, termed BAIs, that bind directly to a previously unrecognized pocket and allosterically inhibit BAX activation. BAI binding around the hydrophobic helix α5 using hydrophobic and hydrogen bonding interactions stabilizes key areas of the hydrophobic core. BAIs inhibit conformational events in BAX activation that prevent BAX mitochondrial translocation and oligomerization. Our data highlight a novel paradigm for effective and selective pharmacological targeting of BAX to enable rational development of inhibitors of BAX-mediated cell death.
Assuntos
Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Sítios de Ligação/fisiologia , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Mitocôndrias/fisiologia , Modelos Moleculares , Fragmentos de Peptídeos/fisiologia , Permeabilidade , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches.
Assuntos
Sistema Cardiovascular , Educação/métodos , Insuficiência Cardíaca/terapia , Mitocôndrias/fisiologia , National Heart, Lung, and Blood Institute (U.S.) , Relatório de Pesquisa , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Sistema Cardiovascular/patologia , Educação/tendências , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/epidemiologia , Humanos , National Heart, Lung, and Blood Institute (U.S.)/tendências , Relatório de Pesquisa/tendências , Pesquisa Translacional Biomédica/métodos , Pesquisa Translacional Biomédica/tendências , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Advances in congestive heart failure (CHF) management depend on biomarkers for monitoring disease progression and therapeutic response. During systole, intracellular Ca2+ is released from the sarcoplasmic reticulum into the cytoplasm through type-2 ryanodine receptor/Ca2+ release channels. In CHF, chronically elevated circulating catecholamine levels cause pathological remodeling of type-2 ryanodine receptor/Ca2+ release channels resulting in diastolic sarcoplasmic reticulum Ca2+ leak and decreased myocardial contractility. Similarly, skeletal muscle contraction requires sarcoplasmic reticulum Ca2+ release through type-1 ryanodine receptors (RyR1), and chronically elevated catecholamine levels in CHF cause RyR1-mediated sarcoplasmic reticulum Ca2+ leak, contributing to myopathy and weakness. Circulating B-lymphocytes express RyR1 and catecholamine-responsive signaling cascades, making them a potential surrogate for defects in intracellular Ca2+ handling because of leaky RyR channels in CHF. METHODS: Whole blood was collected from patients with CHF, CHF following left-ventricular assist device implant, and controls. Blood was also collected from mice with ischemic CHF, ischemic CHF+S107 (a drug that specifically reduces RyR channel Ca2+ leak), and wild-type controls. Channel macromolecular complex was assessed by immunostaining RyR1 immunoprecipitated from lymphocyte-enriched preparations. RyR1 Ca2+ leak was assessed using flow cytometry to measure Ca2+ fluorescence in B-lymphocytes in the absence and presence of RyR1 agonists that empty RyR1 Ca2+ stores within the endoplasmic reticulum. RESULTS: Circulating B-lymphocytes from humans and mice with CHF exhibited remodeled RyR1 and decreased endoplasmic reticulum Ca2+ stores, consistent with chronic intracellular Ca2+ leak. This Ca2+ leak correlated with circulating catecholamine levels. The intracellular Ca2+ leak was significantly reduced in mice treated with the Rycal S107. Patients with CHF treated with left-ventricular assist devices exhibited a heterogeneous response. CONCLUSIONS: In CHF, B-lymphocytes exhibit remodeled leaky RyR1 channels and decreased endoplasmic reticulum Ca2+ stores consistent with chronic intracellular Ca2+ leak. RyR1-mediated Ca2+ leak in B-lymphocytes assessed using flow cytometry provides a surrogate measure of intracellular Ca2+ handling and systemic sympathetic burden, presenting a novel biomarker for monitoring response to pharmacological and mechanical CHF therapy.
Assuntos
Linfócitos B/metabolismo , Sinalização do Cálcio , Cálcio/sangue , Retículo Endoplasmático/metabolismo , Insuficiência Cardíaca/sangue , Canal de Liberação de Cálcio do Receptor de Rianodina/sangue , Idoso , Animais , Linfócitos B/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Estudos de Casos e Controles , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Coração Auxiliar , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Norepinefrina/sangue , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Tiazepinas/farmacologia , Função Ventricular EsquerdaAssuntos
Coração , Cadeias Pesadas de Miosina , Animais , Camundongos , Miocárdio , Cadeias Pesadas de Miosina/genética , OrganogêneseRESUMO
Cardiovascular disease is a major leading cause of morbidity and mortality in the United States and elsewhere. Alterations in mitochondrial function are increasingly being recognized as a contributing factor in myocardial infarction and in patients presenting with cardiomyopathy. Recent understanding of the complex interaction of the mitochondria in regulating metabolism and cell death can provide novel insight and therapeutic targets. The purpose of this statement is to better define the potential role of mitochondria in the genesis of cardiovascular disease such as ischemia and heart failure. To accomplish this, we will define the key mitochondrial processes that play a role in cardiovascular disease that are potential targets for novel therapeutic interventions. This is an exciting time in mitochondrial research. The past decade has provided novel insight into the role of mitochondria function and their importance in complex diseases. This statement will define the key roles that mitochondria play in cardiovascular physiology and disease and provide insight into how mitochondrial defects can contribute to cardiovascular disease; it will also discuss potential biomarkers of mitochondrial disease and suggest potential novel therapeutic approaches.