RESUMO
Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.
Assuntos
Smegmamorpha , Animais , Smegmamorpha/metabolismo , Mitocôndrias/metabolismo , Metabolismo Energético , Glicólise , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Mamíferos/metabolismoRESUMO
Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.
Assuntos
Criopreservação , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Masculino , CamundongosRESUMO
The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Ensaios Enzimáticos , Técnicas de Introdução de Genes , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Estresse Oxidativo , Fosfoproteínas Fosfatases/genética , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/genética , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Normal contractile function of the heart depends on a constant and reliable production of ATP by cardiomyocytes. Dysregulation of cardiac energy metabolism can result in immature heart development and disrupt the ability of the adult myocardium to adapt to stress, potentially leading to heart failure. Further, restoration of abnormal mitochondrial function can have beneficial effects on cardiac dysfunction. Previously, we identified a novel protein termed Perm1 (PGC-1 and estrogen-related receptor (ERR)-induced regulator, muscle 1) that is enriched in skeletal and cardiac-muscle mitochondria and transcriptionally regulated by PGC-1 (peroxisome proliferator-activated receptor gamma coactivator 1) and ERR. The role of Perm1 in the heart is poorly understood and is studied here. We utilized cell culture, mouse models, and human tissue, to study its expression and transcriptional control, as well as its role in transcription of other factors. Critically, we tested Perm1's role in cardiomyocyte mitochondrial function and its ability to protect myocytes from stress-induced damage. Our studies show that Perm1 expression increases throughout mouse cardiogenesis, demonstrate that Perm1 interacts with PGC-1α and enhances activation of PGC-1 and ERR, increases mitochondrial DNA copy number, and augments oxidative capacity in cultured neonatal mouse cardiomyocytes. Moreover, we found that Perm1 reduced cellular damage produced as a result of hypoxia and reoxygenation-induced stress and mitigated cell death of cardiomyocytes. Taken together, our results show that Perm1 promotes mitochondrial biogenesis in mouse cardiomyocytes. Future studies can assess the potential of Perm1 to be used as a novel therapeutic to restore cardiac dysfunction induced by ischemic injury.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Biogênese de Organelas , Oxigênio/metabolismo , Animais , Hipóxia Celular , DNA Mitocondrial/genética , Regulação para Baixo/genética , Coração/embriologia , Insuficiência Cardíaca/genética , Ventrículos do Coração/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Oxirredução , Fosforilação Oxidativa , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied (13)C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.
Assuntos
Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismo , Ácido Pirúvico/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciclo do Ácido Cítrico , Ácidos Graxos/metabolismo , Glutamina/metabolismo , Humanos , Lipogênese , Análise do Fluxo Metabólico , Camundongos , Fibras Musculares Esqueléticas/metabolismo , OxirreduçãoRESUMO
The neuropathogenesis of HIV associated neurocognitive disorders (HAND) involves disruption of mitochondrial homeostasis and increased neuroinflammation. However, it is unknown if alterations in mitochondrial biogenesis in the brain underlie the neuropathogenesis of HAND. In this study, neuropathological and molecular analyses of mitochondrial biogenesis and inflammatory pathways were performed in brain specimens from a well-characterized cohort of HIV+ cases that were on antiretroviral regimens. In vitro investigations using primary human astroglia and neurons were used to probe the underlying mechanisms of mitochondrial alterations. In frontal cortices from HAND brains compared to cognitive normal brains, total levels of transcription factors that regulate mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were decreased. Immunohistochemical analyses revealed that TFAM was decreased in neurons and increased in astroglia. These changes were accompanied by decreased total mitochondrial DNA per cell and increased levels of messenger RNA for the proinflammatory cytokine interleukin (IL)-1ß. To determine how IL-1ß affects astroglial bioenergetic processes and mitochondrial activity, human astroglial cultures were exposed to recombinant IL-1ß. IL-1ß induced mitochondrial activity within 30â¯min of treatment, altered mitochondrial related gene expression, altered mitochondrial morphology, enhanced adenoside triphosphate (ATP) utilization and increased the expression of inflammatory cytokines. WIN55,212-2 (WIN), an aminoalkylindole derivative and cannabinoid receptor agonist, blocked IL-1ß-induced bioenergetic fluctuations and inflammatory gene expression in astroglia independent of cannabinoid receptor (CB)1 and peroxisome proliferator-activated receptor (PPAR) γ. A PPARα antagonist reversed the anti-inflammatory effects of WIN in human astroglia. These results show that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HAND brains and that targeting astroglial bioenergetic processes may be a strategy to modulate neuroinflammation.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Astrócitos/metabolismo , Encéfalo/metabolismo , Soropositividade para HIV/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Fármacos Anti-HIV/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Soropositividade para HIV/tratamento farmacológico , Soropositividade para HIV/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Neuroscience is a technology-driven discipline and brain energy metabolism is no exception. Once satisfied with mapping metabolic pathways at organ level, we are now looking to learn what it is exactly that metabolic enzymes and transporters do and when, where do they reside, how are they regulated, and how do they relate to the specific functions of neurons, glial cells, and their subcellular domains and organelles, in different areas of the brain. Moreover, we aim to quantify the fluxes of metabolites within and between cells. Energy metabolism is not just a necessity for proper cell function and viability but plays specific roles in higher brain functions such as memory processing and behavior, whose mechanisms need to be understood at all hierarchical levels, from isolated proteins to whole subjects, in both health and disease. To this aim, the field takes advantage of diverse disciplines including anatomy, histology, physiology, biochemistry, bioenergetics, cellular biology, molecular biology, developmental biology, neurology, and mathematical modeling. This article presents a well-referenced synopsis of the technical side of brain energy metabolism research. Detail and jargon are avoided whenever possible and emphasis is given to comparative strengths, limitations, and weaknesses, information that is often not available in regular articles.
Assuntos
Encéfalo/metabolismo , Metabolismo Energético , Neurociências/métodos , Animais , Humanos , Neurociências/instrumentaçãoRESUMO
OBJECTIVES: Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage. METHODS: HK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells. RESULTS: HK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage. CONCLUSION: HK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.
Assuntos
Artrite Reumatoide/enzimologia , Hexoquinase/metabolismo , Animais , Artrite Experimental/enzimologia , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Movimento Celular/fisiologia , Regulação da Expressão Gênica , Hexoquinase/genética , Humanos , Mediadores da Inflamação/metabolismo , Camundongos Transgênicos , Osteoartrite/enzimologia , Osteoartrite/genética , Osteoartrite/patologia , RNA Interferente Pequeno/genética , Membrana Sinovial/enzimologia , Sinoviócitos/enzimologia , Sinoviócitos/fisiologia , Sinovite/enzimologia , Sinovite/patologiaRESUMO
Activities of enzymes localized to the mitochondrial matrix of mammalian cells are often critical regulatory steps in cellular metabolism. As such, measurement of matrix enzyme activities in response to genetic modifications or drug interventions is often desired. However, measurements in intact cells are often hampered by the presence of other isozymes in the cytoplasm as well as the inability to deliver enzyme substrates across cellular membranes. Classic approaches to liberate matrix enzymes utilize harsh treatments that disrupt intracellular architecture or require significant starting material to allow mitochondrial isolation prior to sample extraction. We describe a method using permeabilization reagents for both the plasma and mitochondrial membranes to allow in situ measurement of matrix enzyme activities. It is applied to adherent cell monolayers in 96-well plates treated with perfringolysin O to permeabilize the plasma membrane and alamethicin to permeabilize the mitochondrial inner membrane. We present three examples validated with inhibitor sensitivity: (i) Complex I-mediated oxygen consumption driven by NADH, (ii) ATP hydrolysis by the F1FO complex measuring pH changes in an Agilent Seahorse XF Analyzer, and (iii) Mitochondrial glutaminase (GLS1) activity in a coupled reaction monitoring NADH fluorescence in a plate reader.
Assuntos
Toxinas Bacterianas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proteínas Hemolisinas/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Células A549 , Glutaminase/metabolismo , Células Hep G2 , Humanos , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , NAD/metabolismo , Consumo de OxigênioRESUMO
Adipose tissue plays important roles in regulating carbohydrate and lipid homeostasis, but less is known about the regulation of amino acid metabolism in adipocytes. Here we applied isotope tracing to pre-adipocytes and differentiated adipocytes to quantify the contributions of different substrates to tricarboxylic acid (TCA) metabolism and lipogenesis. In contrast to proliferating cells, which use glucose and glutamine for acetyl-coenzyme A (AcCoA) generation, differentiated adipocytes showed increased branched-chain amino acid (BCAA) catabolic flux such that leucine and isoleucine from medium and/or from protein catabolism accounted for as much as 30% of lipogenic AcCoA pools. Medium cobalamin deficiency caused methylmalonic acid accumulation and odd-chain fatty acid synthesis. Vitamin B12 supplementation reduced these metabolites and altered the balance of substrates entering mitochondria. Finally, inhibition of BCAA catabolism compromised adipogenesis. These results quantitatively highlight the contribution of BCAAs to adipocyte metabolism and suggest that BCAA catabolism has a functional role in adipocyte differentiation.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Lipogênese , Obesidade/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Células 3T3-L1/efeitos dos fármacos , Acetilcoenzima A/metabolismo , Adipócitos/efeitos dos fármacos , Adipogenia/fisiologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Obesidade/cirurgia , Ácidos Tricarboxílicos/metabolismo , Vitamina B 12/farmacologiaRESUMO
RATIONALE: Lysosomal associated membrane protein type-2 (LAMP-2) is a highly conserved, ubiquitous protein that is critical for autophagic flux. Loss of function mutations in the LAMP-2 gene cause Danon disease, a rare X-linked disorder characterized by developmental delay, skeletal muscle weakness, and severe cardiomyopathy. We previously found that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from Danon patients exhibited significant mitochondrial oxidative stress and apoptosis. Understanding how loss of LAMP-2 expression leads to cardiomyocyte dysfunction and heart failure has important implications for the treatment of Danon disease as well as a variety of other cardiac disorders associated with impaired autophagy. OBJECTIVE: Elucidate the pathophysiology of cardiac dysfunction in Danon disease. METHODS AND RESULTS: We created hiPSCs from two patients with Danon disease and differentiated those cells into hiPSC-CMs using well-established protocols. Danon hiPSC-CMs demonstrated an accumulation of damaged mitochondria, disrupted mitophagic flux, depressed mitochondrial respiratory capacity, and abnormal gene expression of key mitochondrial pathways. Restoring the expression of LAMP-2B, the most abundant LAMP-2 isoform in the heart, rescued mitophagic flux as well as mitochondrial health and bioenergetics. To confirm our findings in vivo, we evaluated Lamp-2 knockout (KO) mice. Impaired autophagic flux was noted in the Lamp-2 KO mice compared to WT reporter mice, as well as an increased number of abnormal mitochondria, evidence of incomplete mitophagy, and impaired mitochondrial respiration. Physiologically, Lamp-2 KO mice demonstrated early features of contractile dysfunction without overt heart failure, indicating that the metabolic abnormalities associated with Danon disease precede the development of end-stage disease and are not merely part of the secondary changes associated with heart failure. CONCLUSIONS: Incomplete mitophagic flux and mitochondrial dysfunction are noted in both in vitro and in vivo models of Danon disease, and proceed overt cardiac contractile dysfunction. This suggests that impaired mitochondrial clearance may be central to the pathogenesis of disease and a potential target for therapeutic intervention.
Assuntos
Doença de Depósito de Glicogênio Tipo IIb/genética , Doença de Depósito de Glicogênio Tipo IIb/metabolismo , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitofagia/genética , Animais , Técnicas de Inativação de Genes , Doença de Depósito de Glicogênio Tipo IIb/diagnóstico , Hemodinâmica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Imageamento por Ressonância Magnética , Camundongos Knockout , Mitocôndrias Cardíacas/ultraestrutura , Modelos Biológicos , Miócitos Cardíacos/metabolismoRESUMO
Metabolic reprogramming is emerging as a hallmark of the innate immune response, and the dynamic control of metabolites such as succinate serves to facilitate the execution of inflammatory responses in macrophages and other immune cells. Immunoresponsive gene 1 (Irg1) expression is induced by inflammatory stimuli, and its enzyme product cis-aconitate decarboxylase catalyzes the production of itaconate from the tricarboxylic acid cycle. Here we identify an immunometabolic regulatory pathway that links Irg1 and itaconate production to the succinate accumulation that occurs in the context of innate immune responses. Itaconate levels and Irg1 expression correlate strongly with succinate during LPS exposure in macrophages and non-immune cells. We demonstrate that itaconate acts as an endogenous succinate dehydrogenase inhibitor to cause succinate accumulation. Loss of itaconate production in activated macrophages from Irg1(-/-) mice decreases the accumulation of succinate in response to LPS exposure. This metabolic network links the innate immune response and tricarboxylic acid metabolism to function of the electron transport chain.
Assuntos
Hidroliases/fisiologia , Succinato Desidrogenase/antagonistas & inibidores , Succinatos/farmacologia , Ácido Succínico/metabolismo , Animais , Linhagem Celular , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , CamundongosRESUMO
The Liver Kinase B1 (LKB1) tumor suppressor acts as a metabolic energy sensor to regulate AMP-activated protein kinase (AMPK) signaling and is commonly mutated in various cancers, including non-small cell lung cancer (NSCLC). Tumor cells deficient in LKB1 may be uniquely sensitized to metabolic stresses, which may offer a therapeutic window in oncology. To address this question we have explored how functional LKB1 impacts the metabolism of NSCLC cells using 13C metabolic flux analysis. Isogenic NSCLC cells expressing functional LKB1 exhibited higher flux through oxidative mitochondrial pathways compared to those deficient in LKB1. Re-expression of LKB1 also increased the capacity of cells to oxidize major mitochondrial substrates, including pyruvate, fatty acids, and glutamine. Furthermore, LKB1 expression promoted an adaptive response to energy stress induced by anchorage-independent growth. Finally, this diminished adaptability sensitized LKB1-deficient cells to combinatorial inhibition of mitochondrial complex I and glutaminase. Together, our data implicate LKB1 as a major regulator of adaptive metabolic reprogramming and suggest synergistic pharmacological strategies for mitigating LKB1-deficient NSCLC tumor growth.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metabolismo Energético , Neoplasias Pulmonares/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Células A549 , Quinases Proteína-Quinases Ativadas por AMP , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
RATIONALE: Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation. OBJECTIVE: To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq. METHODS AND RESULTS: Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion. CONCLUSIONS: Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cardiomegalia/enzimologia , Cardiomiopatia Dilatada/etiologia , Insuficiência Cardíaca/etiologia , Mitocôndrias Cardíacas/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cardiomegalia/fisiopatologia , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Dilatada/prevenção & controle , Células Cultivadas , Progressão da Doença , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Perfilação da Expressão Gênica , Insuficiência Cardíaca/fisiopatologia , Canais Iônicos/biossíntese , Canais Iônicos/genética , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , PPAR alfa/biossíntese , PPAR alfa/genética , Mutação Puntual , Pressão , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Ratos , Espécies Reativas de Oxigênio , Análise de Sequência de RNA , Sulfonamidas/farmacologia , Transfecção , Proteína Desacopladora 3RESUMO
Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.
Assuntos
Proliferação de Células/genética , DNA Mitocondrial/genética , Mutação , Células-Tronco/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Miocárdio/citologia , Miocárdio/metabolismo , Biogênese de Organelas , Fosforilação Oxidativa , Consumo de Oxigênio/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses, but the role of mitochondria in such responses is poorly understood. To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wild-type (WT) and kin(-) (PKA-null) murine S49 lymphoma cells. Basally, 75 proteins significantly differed in abundance between WT and kin(-) S49 cells. WT, but not kin(-), S49 cells incubated with the cAMP analog 8-(4-chlorophenylthio)adenosine cAMP (CPT-cAMP) for 16 h have (a) increased expression of mitochondria-related genes and proteins, including ones in pathways of branched-chain amino acid and fatty acid metabolism and (b) increased maximal capacity of respiration on branched-chain keto acids and fatty acids. CPT-cAMP also regulates the cellular rate of ATP-utilization, as the rates of both ATP-linked respiration and proton efflux are decreased in WT but not kin(-) cells. CPT-cAMP protected WT S49 cells from glucose or glutamine deprivation, In contrast, CPT-cAMP did not protect kin(-) cells or WT cells treated with the PKA inhibitor H89 from glutamine deprivation. Under basal conditions, the mitochondrial structure of WT and kin(-) S49 cells is similar. Treatment with CPT-cAMP produced apoptotic changes (i.e. decreased mitochondrial density and size and loss of cristae) in WT, but not kin(-) cells. Together, these findings show that cAMP acts via PKA to regulate multiple aspects of mitochondrial function and structure. Mitochondrial perturbation thus likely contributes to cAMP/PKA-mediated cellular responses.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteômica/métodos , Aminoácidos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , AMP Cíclico/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Immunoblotting , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Microscopia Eletrônica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tionucleotídeos/farmacologiaRESUMO
HIV-associated neurocognitive disorders (HAND) still occur in approximately 50% of HIV patients, and therapies to combat HAND progression are urgently needed. HIV proteins are released from infected cells and cause neuronal damage, possibly through mitochondrial abnormalities. Altered mitochondrial fission and fusion is implicated in several neurodegenerative disorders. Here, we hypothesized that mitochondrial fission/fusion may be dysregulated in neurons during HAND. We have identified decreased mitochondrial fission protein (dynamin 1-like; DNM1L) in frontal cortex tissues of HAND donors, along with enlarged and elongated mitochondria localized to the soma of damaged neurons. Similar pathology was observed in the brains of GFAP-gp120 tg mice. In vitro, recombinant gp120 decreased total and active DNM1L levels, reduced the level of Mitotracker staining, and increased extracellular acidification rate (ECAR) in primary neurons. DNM1L knockdown enhanced the effects of gp120 as measured by reduced Mitotracker signal in the treated cells. Interestingly, overexpression of DNM1L increased the level of Mitotracker staining in primary rat neurons and reduced neuroinflammation and neurodegeneration in the GFAP-gp120-tg mice. These data suggest that mitochondrial biogenesis dynamics are shifted towards mitochondrial fusion in brains of HAND patients and this may be due to gp120-induced reduction in DNM1L activity. Promoting mitochondrial fission during HIV infection of the CNS may restore mitochondrial biogenesis and prevent neurodegeneration.
Assuntos
Encéfalo/metabolismo , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/virologia , Infecções por HIV/complicações , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Neurônios/metabolismo , Adulto , Animais , Encéfalo/ultraestrutura , Encéfalo/virologia , Dinaminas , Encefalite/metabolismo , Encefalite/virologia , Feminino , Lobo Frontal/metabolismo , Lobo Frontal/ultraestrutura , Lobo Frontal/virologia , GTP Fosfo-Hidrolases/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Mitocôndrias/virologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/ultraestrutura , Neurônios/virologia , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Facilitated pyruvate transport across the mitochondrial inner membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. We report that clinically relevant concentrations of thiazolidinediones (TZDs), a widely used class of insulin sensitizers, acutely and specifically inhibit mitochondrial pyruvate carrier (MPC) activity in a variety of cell types. Respiratory inhibition was overcome with methyl pyruvate, localizing the effect to facilitated pyruvate transport, and knockdown of either paralog, MPC1 or MPC2, decreased the EC50 for respiratory inhibition by TZDs. Acute MPC inhibition significantly enhanced glucose uptake in human skeletal muscle myocytes after 2 h. These data (i) report that clinically used TZDs inhibit the MPC, (ii) validate that MPC1 and MPC2 are obligatory components of facilitated pyruvate transport in mammalian cells, (iii) indicate that the acute effect of TZDs may be related to insulin sensitization, and (iv) establish mitochondrial pyruvate uptake as a potential therapeutic target for diseases rooted in metabolic dysfunction.
Assuntos
Respiração Celular/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Redes e Vias Metabólicas/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Acrilatos/farmacologia , Análise de Variância , Animais , Proteínas de Transporte de Ânions , Western Blotting , Linhagem Celular , Citocromos c/metabolismo , Glucose/metabolismo , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos , Músculo Esquelético/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Carreadoras de Solutos , Tiazolidinedionas/metabolismoRESUMO
It is known that loss-of-function mutations in the gene encoding Parkin lead to development of Parkinson disease. Recently, Parkin was found to play an important role in the removal of dysfunctional mitochondria via autophagy in neurons. Although Parkin is expressed in the heart, its functional role in this tissue is largely unexplored. In this study, we have investigated the role of Parkin in the myocardium under normal physiological conditions and in response to myocardial infarction. We found that Parkin-deficient (Parkin(-/-)) mice had normal cardiac function for up to 12 months of age as determined by echocardiographic analysis. Although ultrastructural analysis revealed that Parkin-deficient hearts had disorganized mitochondrial networks and significantly smaller mitochondria, mitochondrial function was unaffected. However, Parkin(-/-) mice were much more sensitive to myocardial infarction when compared with wild type mice. Parkin(-/-) mice had reduced survival and developed larger infarcts when compared with wild type mice after the infarction. Interestingly, Parkin protein levels and mitochondrial autophagy (mitophagy) were rapidly increased in the border zone of the infarct in wild type mice. In contrast, Parkin(-/-) myocytes had reduced mitophagy and accumulated swollen, dysfunctional mitochondria after the infarction. Overexpression of Parkin in isolated cardiac myocytes also protected against hypoxia-mediated cell death, whereas nonfunctional Parkinson disease-associated mutants ParkinR42P and ParkinG430D had no effect. Our results suggest that Parkin plays a critical role in adapting to stress in the myocardium by promoting removal of damaged mitochondria.