RESUMO
By uncoupling oxidative phosphorylation, 2,4-dinitrophenol (DNP) attenuates reactive oxygen species (ROS) biosynthesis, which are known to aggravate infectious myocarditis in Chagas disease. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. C56BL/6 mice uninfected and infected untreated and treated daily with 100 mg/kg benznidazole (Bz, reference drug), 5 and 10 mg/kg DNP by gavage for 11 days after confirmation of T. cruzi infection were investigated. Twenty-four hours âafter the last treatment, the animals were euthanized and the heart was collected for microstructural, immunological and biochemical analyses. T. cruzi inoculation induced systemic inflammation (e.g., cytokines and anti-T. cruzi IgG upregulation), cardiac infection (T. cruzi DNA), oxidative stress, inflammatory infiltrate and microstructural myocardial damage in untreated mice. DNP treatment aggravated heart infection and microstructural damage, which were markedly attenuated by Bz. DNP (10 mg/kg) was also effective in attenuating ROS (total ROS, H2O2, and O2-), nitric oxide (NO), lipid (malondialdehyde - MDA) and protein (protein carbonyl - PCn) oxidation, TNF, IFN-γ, IL-10, and MCP-1/CCL2, anti-T. cruzi IgG, cardiac troponin I levels, as well as inflammatory infiltrate and cardiac damage in T. cruzi-infected mice. Our findings indicate that DNP aggravated heart infection and microstructural cardiomyocytes damage in infected mice. These responses were related to the antioxidant and anti-inflammatory properties of DNP, which favors infection by weakening the pro-oxidant and pro-inflammatory protective mechanisms of the infected host. Conversely, Bz-induced cardioprotective effects combined effective anti-inflammatory and antiparasitic responses, which protect against heart infection, oxidative stress, and microstructural damage in Chagas disease.
Assuntos
2,4-Dinitrofenol , Cardiomiopatia Chagásica , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Trypanosoma cruzi , Animais , 2,4-Dinitrofenol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Cardiomiopatia Chagásica/tratamento farmacológico , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Trypanosoma cruzi/efeitos dos fármacos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Desacopladores/farmacologia , Desacopladores/toxicidade , Camundongos , Miocárdio/patologia , Miocárdio/metabolismo , Nitroimidazóis/farmacologia , Doença Aguda , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Miocardite/parasitologia , Miocardite/metabolismo , Miocardite/tratamento farmacológico , Miocardite/patologia , Miocardite/induzido quimicamente , Doença de Chagas/tratamento farmacológico , Doença de Chagas/metabolismo , Doença de Chagas/patologia , Doença de Chagas/parasitologiaRESUMO
Diabetes mellitus-induced heart disease, including diabetic cardiomyopathy, is an important medical problem and is difficult to treat. Diabetes mellitus increases the risk for heart failure and decreases cardiac myocyte function, which are linked to changes in cardiac mitochondrial energy metabolism. The free mitochondrial calcium concentration ([Ca2+]m) is fundamental in activating the mitochondrial respiratory chain complexes and ATP production and is also known to regulate the activity of key mitochondrial dehydrogenases. The mitochondrial calcium uniporter complex (MCUC) plays a major role in mediating mitochondrial Ca2+ import, and its expression and function therefore may have a marked impact on cardiac myocyte metabolism and function. Here, we summarize the pathophysiological role of [Ca2+]m handling and MCUC in the diabetic heart. In addition, we evaluate potential therapeutic targets, directed to the machinery that regulates mitochondrial calcium handling, to alleviate diabetes-related cardiac disease.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cardiomiopatias Diabéticas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Cardiomiopatias Diabéticas/patologia , Humanos , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologiaRESUMO
RATIONALE: Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. OBJECTIVE: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. METHODS AND RESULTS: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. CONCLUSIONS: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Gatos , Células Cultivadas , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Humanos , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Cinética , Masculino , Proteínas de Membrana/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Proteínas Musculares/genética , Mutação , Miócitos Cardíacos/patologia , Biogênese de Organelas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de RianodinaRESUMO
The mitochondrial permeability transition pore (mPTP) opening is involved in the pathophysiology of multiple cardiac diseases, such as ischemia/reperfusion injury and heart failure. A growing number of evidence provided by proteomic screening techniques has demonstrated the role of post-translational modifications (PTMs) in several key components of the pore in response to changes in the extra/intracellular environment and bioenergetic demand. This could lead to a fine, complex regulatory mechanism that, under pathological conditions, can shift the state of mitochondrial functions and, thus, the cell's fate. Understanding the complex relationship between these PTMs is still under investigation and can provide new, promising therapeutic targets and treatment approaches. This review, using a systematic review of the literature, presents the current knowledge on PTMs of the mPTP and their role in health and cardiac disease.
Assuntos
Insuficiência Cardíaca/metabolismo , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Processamento de Proteína Pós-Traducional , Insuficiência Cardíaca/patologia , Humanos , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/patologia , ProteômicaRESUMO
Cardiac fibrosis is mediated by the activation of resident cardiac fibroblasts, which differentiate into myofibroblasts in response to injury or stress. Although myofibroblast formation is a physiological response to acute injury, such as myocardial infarction, myofibroblast persistence, as occurs in heart failure, contributes to maladaptive remodeling and progressive functional decline. Although traditional pathways of activation, such as TGFß (transforming growth factor ß) and AngII (angiotensin II), have been well characterized, less understood are the alterations in mitochondrial function and cellular metabolism that are necessary to initiate and sustain myofibroblast formation and function. In this review, we highlight recent reports detailing the mitochondrial and metabolic mechanisms that contribute to myofibroblast differentiation, persistence, and function with the hope of identifying novel therapeutic targets to treat, and potentially reverse, tissue organ fibrosis.
Assuntos
Diferenciação Celular , Metabolismo Energético , Cardiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miofibroblastos/metabolismo , Animais , Sinalização do Cálcio , Fibrose , Cardiopatias/patologia , Humanos , Mitocôndrias Cardíacas/patologia , Miofibroblastos/patologiaRESUMO
Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05-5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients.
Assuntos
Maleatos/metabolismo , Mitocôndrias Cardíacas/patologia , Mioblastos Cardíacos/patologia , Propionatos/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Fracionamento Celular , Linhagem Celular , Metabolismo Energético , Humanos , Masculino , Mitocôndrias Cardíacas/metabolismo , Dilatação Mitocondrial , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/metabolismo , Oxigênio/análise , Oxigênio/metabolismo , Acidemia Propiônica/complicações , Acidemia Propiônica/metabolismo , Acidemia Propiônica/patologia , RatosRESUMO
Hypothyroidism is considered a cardiac risk factor, but there is controversial evidence about its effects on coronary disease. The aim of this work was to evaluate the influence of hypothyroidism in rat hearts exposed to 2 degrees of stunning due to ischemia and reperfusion (I/R) as well as the underlying mechanisms. Hypothyroid (HypoT) rats were obtained by drinking 0.02% methimazole during 15 days. Isolated hearts were perfused and introduced in a flow calorimeter to measure contractile performance (P), total heat rate (Ht), and muscle economy (P/Ht). Hearts were exposed to 2 models of I/R, moderate and severe (respectively 20 or 30 minutes I/45 minutes R). Moreover, free cytosolic and mitochondrial calcium changes were measured by confocal fluorometry on cardiomyocytes. Comparison to euthyroid (EuT) hearts was done. Hypothyroidism was cardioprotective, but HypoT hearts were more sensitive than EuT hearts to the preischemic blockade of mitochondrial transporters mNCX and mKATP channels. Moreover, the postischemic recovery of P and P/Ht in HypoT hearts was strongly reduced by inhibition of the cellular pathways of PI3K/Akt and protein kinase C (PKC), and it was increased by nitric oxide synthase (NOS) inhibition. However, physiological concentrations of adrenaline reduced the cardioprotection of HypoT, but oral treatment with 20 mg/kg/day carvedilol prevented it. Results show that hypothyroidism reduces the mitochondrial Ca2+ overload during I/R by mKATP channel activation and Ca2+ extrusion through mNCX, while the PI3K/Akt and PKC pathways are involved in that cardioprotection. Contrarily, NOS activation and adrenaline blunt such cardioprotection, but carvedilol prevented the adrenergic dysfunction. These results would explain why hypothyroidism is a clinical risk factor in angor patients under adrenergic exacerbation but reduced the incidence of acute episodes of coronary syndrome in hospitalized patients. Results suggest that a treatment with carvedilol could be a potential therapeutic agent to prevent cardiac postischemic dysfunction in hypothyroid patients.
Assuntos
Metabolismo Energético , Hipotireoidismo/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Animais , Sinalização do Cálcio , Modelos Animais de Doenças , Feminino , Frequência Cardíaca , Hipotireoidismo/patologia , Hipotireoidismo/fisiopatologia , Preparação de Coração Isolado , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Canais de Potássio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Trocador de Sódio e Cálcio/metabolismoRESUMO
BACKGROUND: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (mCa2+) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. METHODS: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. RESULTS: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing mCa2+ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa2+ overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa2+ uptake impair mitochondrial energetics and contractile function. CONCLUSIONS: We detail a new regulatory mechanism to modulate mtCU function and mCa2+ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa2+ uptake and cellular physiology.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Sistemas CRISPR-Cas , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Técnicas de Inativação de Genes , Células HeLa , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Função Ventricular EsquerdaRESUMO
There is current awareness about the central role of mitochondrial dysfunction in the development of cardiac dysfunction in systemic inflammatory syndromes, especially in sepsis and endotoxemia. The aim of this work was to elucidate the mechanism that governs the link between the severity of the systemic inflammatory insult and mitochondrial function, analysing the consequences on heart function, particularly in cardiac contractile state. Female Sprague-Dawley rats were subjected to low-grade endotoxemia (i.p. injection LPS 0.5 mg kg-1 body weight) and severe endotoxemia (i.p. injection LPS 8 mg kg-1 body weight) for 6 h. Blood NO, as well as cardiac TNF-α and IL-1ß mRNA, were found increased as the severity of the endotoxemia increases. Cardiac relaxation was altered only in severe endotoxemia, although contractile and lusitropic reserves were found impaired in both treatments in response to work-overload. Cardiac ultrastructure showed disorientation of myofibrillar structure in both endotoxemia degrees, but mitochondrial swelling and cristae disruption were only observed in severe endotoxemia. Mitochondrial ATP production, O2 consumption and mitochondrial inner membrane potential decreases were related to blood NO levels and mitochondrial protein nitration, leading to diminished ATP availability and impairment of contractile state. Co-treatment with the NOS inhibitor L-NAME or the administration of the NO scavenger c-PTIO leads to the observation that mitochondrial bioenergetics status depends on the degree of the inflammatory insult mainly determined by blood NO levels. Unravelling the mechanisms involved in the onset of sepsis and endotoxemia improves the interpretation of the pathology, and provides new horizons for novel therapeutic targets.
Assuntos
Endotoxemia/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Inflamação/fisiopatologia , Mitocôndrias Cardíacas/fisiologia , Contração Miocárdica/fisiologia , Animais , Endotoxemia/complicações , Metabolismo Energético , Feminino , Insuficiência Cardíaca/etiologia , Mitocôndrias Cardíacas/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Myocardial infarction triggers cellular events that starts with the activation of inflammatory response and fibrogenic pathways involved in cardiac tissue remodeling. Angiotensin-(1-7) (Ang-(1-7)) is an endogenous heptapeptide from the renin-angiotensin system with a cardioprotective role due to its anti-inflammatory and anti-fibrotic activities in cardiac cells. Although the beneficial aspects of Ang-(1-7) in animal models of cardiac ischemia have been reported, the molecular events underlying Ang-(1-7) cardioprotective effect remains elusive. This study investigated the impact of oral treatment with Ang-(1-7) included in hydroxypropyl ß-cyclodextrin (HPßCD/Ang-(1-7)) on the cardiac proteome dysregulation due to experimental myocardial infarction. Wistar male rats were submitted to experimental myocardial infarction and treated daily with HPßCD/Ang-(1-7) during 7â¯days or 60â¯days by gavage. Our results showed that HPßCD/Ang-(1-7) treatment ameliorates the post-infarction condition due to the modulation of proteins that initially favor the resolution of inflammation and mitochondrial dysfunction. Moreover, this study reported for the first time that Ang-(1-7) treatment after experimental myocardial infarction leads to the downregulation of the C-X-C chemokine receptor type 4 (CXCR4). SIGNIFICANCE: Myocardial infarction triggers a sequence of cellular and molecular events that starts with an intense inflammatory response that is resolved in the proliferative phase. Prolonged inflammatory phase can lead to adverse cardiac repair and heart failure. In this context, we proposed a post-MI treatment using Ang-(1-7) included in HPßCD and administrated orally. We observed that HPßCD/Ang-(1-7) treatment led to CXCR4 downregulation, highlighting this C-X-C chemokine receptor as a potential therapeutic target for ischemic heart diseases.