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
Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.
Assuntos
Cálcio , Mitocôndrias Cardíacas , Mitocôndrias Hepáticas , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Ratos Wistar , Animais , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Masculino , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ratos , Consumo de Oxigênio , Fígado/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Ciclosporina/farmacologiaRESUMO
The myocardium is a highly oxidative tissue in which mitochondria are essential to supply the energy required to maintain pump function. When pathological hypertrophy develops, energy consumption augments and jeopardizes mitochondrial capacity. We explored the cardiac consequences of chronic swimming training, focusing on the mitochondrial network, in spontaneously hypertensive rats (SHR). Male adult SHR were randomized to sedentary or trained (T: 8-week swimming protocol). Blood pressure and echocardiograms were recorded, and hearts were removed at the end of the training period to perform molecular, imaging, or isolated mitochondria studies. Swimming improved cardiac midventricular shortening and decreased the pathological hypertrophic marker atrial natriuretic peptide. Oxidative stress was reduced, and even more interesting, mitochondrial spatial distribution, dynamics, function, and ATP were significantly improved in the myocardium of T rats. In the signaling pathway triggered by training, we detected an increase in the phosphorylation level of both AKT and glycogen synthase kinase-3 ß, key downstream targets of insulin-like growth factor 1 signaling that are crucially involved in mitochondria biogenesis and integrity. Aerobic exercise training emerges as an effective approach to improve pathological cardiac hypertrophy and bioenergetics in hypertension-induced cardiac hypertrophy.
Assuntos
Mitocôndrias Cardíacas , Condicionamento Físico Animal , Ratos Endogâmicos SHR , Animais , Masculino , Ratos , Mitocôndrias Cardíacas/metabolismo , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/fisiologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Natação/fisiologia , Estresse Oxidativo , Transdução de Sinais/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Pressão Sanguínea/fisiologia , Fator Natriurético Atrial/metabolismoRESUMO
Calorie restriction is a nutritional intervention that reproducibly protects against the maladaptive consequences of cardiovascular diseases. Pathological cardiac hypertrophy leads to cellular growth, dysfunction (with mitochondrial dysregulation), and oxidative stress. The mechanisms behind the cardiovascular protective effects of calorie restriction are still under investigation. In this study, we show that this dietetic intervention prevents cardiac protein elevation, avoids fetal gene reprogramming (atrial natriuretic peptide), and blocks the increase in heart weight per tibia length index (HW/TL) seen in isoproterenol-induced cardiac hypertrophy. Our findings suggest that calorie restriction inhibits cardiac pathological growth while also lowering mitochondrial reverse electron transport-induced hydrogen peroxide formation and improving mitochondrial content. Calorie restriction also attenuated the opening of the Ca2+-induced mitochondrial permeability transition pore. We also found that calorie restriction blocked the negative correlation of antioxidant enzymes (superoxide dimutase and glutatione peroxidase activity) and HW/TL, leading to the maintenance of protein sulphydryls and glutathione levels. Given the nature of isoproterenol-induced cardiac hypertrophy, we investigated whether calorie restriction could alter cardiac beta-adrenergic sensitivity. Using isolated rat hearts in a Langendorff system, we found that calorie restricted hearts have preserved beta-adrenergic signaling. In contrast, hypertrophic hearts (treated for seven days with isoproterenol) were insensitive to beta-adrenergic activation using isoproterenol (50 nM). Despite protecting against cardiac hypertrophy, calorie restriction did not alter the lack of responsiveness to isoproterenol in isolated hearts harvested from isoproterenol-treated rats. These results suggest (through a series of mitochondrial, oxidative stress, and cardiac hemodynamic studies) that calorie restriction possesses beneficial effects against hypertrophic cardiomyopathy.
Assuntos
Cálcio , Restrição Calórica , Estresse Oxidativo , Animais , Ratos , Cálcio/metabolismo , Masculino , Cardiomegalia/metabolismo , Cardiomegalia/prevenção & controle , Transporte de Elétrons , Isoproterenol , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Ratos Sprague-DawleyRESUMO
AIMS: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. METHODS: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. RESULTS: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. CONCLUSION: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.
Assuntos
Cálcio , Mitocôndrias Cardíacas , Animais , Camundongos , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Masculino , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Hipóxia/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Oxigênio/metabolismoRESUMO
Introduction: Clinical studies have shown that low levels of endogenous testosterone are associated with cardiovascular diseases. Considering the intimate connection between oxidative metabolism and myocardial contractility, we determined the effects of testosterone deficiency on the two spatially distinct subpopulations of cardiac mitochondria, subsarcolemmal (SSM) and interfibrillar (IFM). Methods: We assessed cardiac function and cardiac mitochondria structure of SSM and IFM after 12 weeks of testosterone deficiency in male Wistar rats. Results and Discussion: Results show that low testosterone reduced myocardial contractility. Orchidectomy increased total left ventricular mitochondrial protein in the SSM, but not in IFM. The membrane potential, size and internal complexity in the IFM after orchidectomy were higher compared to the SHAM group. However, the rate of oxidative phosphorylation with all substrates in the IFM after orchidectomy was lower compared to the SHAM group. Testosterone replacement restored these changes. In the testosterone-deficient SSM group, oxidative phosphorylation was decreased with palmitoyl-L-carnitine as substrate; however, the mitochondrial calcium retention capacity in IFM was increased. There was no difference in swelling of the mitochondria in either group. These changes in IFM were followed by a reduction in phosphorylated form of AMP-activated protein kinase (p-AMPK-α), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) translocation to mitochondria and decreased mitochondrial transcription factor A (TFAM). Testosterone deficiency increased NADPH oxidase (NOX), angiotensin converting enzyme (ACE) protein expression and reduced mitochondrial antioxidant proteins such as manganese superoxide dismutase (Mn-SOD) and catalase in the IFM. Treatment with apocynin (1.5 mM in drinking water) normalized myocardial contractility and interfibrillar mitochondrial function in the testosterone depleted animals. In conclusion, our findings demonstrate that testosterone deficiency leads to reduced myocardial contractility and impaired cardiac interfibrillar mitochondrial function. Our data suggest the involvement of reactive oxygen species, with a possibility of NOX as an enzymatic source.
Assuntos
Mitocôndrias Cardíacas , Miocárdio , Ratos , Animais , Masculino , Ratos Wistar , Miocárdio/metabolismo , Estresse Oxidativo , Testosterona/farmacologia , Testosterona/metabolismoRESUMO
Direct analysis of isolated mitochondria from old mice enables a better understanding of heart senescence dysfunction. Despite a well-defined senescent phenotype in cardiomyocytes, the mitochondrial state in aged cardiomyocytes is still unclear. Here, we report data about mitochondrial function in old mice. Isolated cardiomyocytes' mitochondria were obtained by differential centrifugation from old and young mice hearts to perform functional analyses of mitochondrial O2 consumption, transmembrane potential, ROS formation, ATP production, and swelling. Our results show that mitochondria from old mouse hearts have reduced oxygen consumption during the phosphorylative states of complexes I and II. Additionally, these mitochondria produced more ROS and less ATP than those of young hearts. Mitochondria from old hearts also showed a depolarized membrane potential than mitochondria from young hearts and, as expected, a greater electron leak. Our results indicate that mitochondria from senescent cardiomyocytes are less efficient in O2 consumption, generating more ROS and producing less ATP. Furthermore, the phosphorylative state of complexes I and II presents a functional defect, contributing to greater leakage of protons and ROS production that can be harmful to the cell.
Assuntos
Envelhecimento , Mitocôndrias Cardíacas , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos , Trifosfato de Adenosina/metabolismo , Potencial da Membrana MitocondrialRESUMO
BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.
Assuntos
Sinalização do Cálcio , Retículo Sarcoplasmático , Feminino , Camundongos , Animais , Retículo Sarcoplasmático/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Cálcio/metabolismoRESUMO
Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 µM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 µM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.
Assuntos
Cardiomiopatias , Doença de Refsum , Ratos , Animais , Doença de Refsum/metabolismo , Ácido Fitânico/farmacologia , Ácido Fitânico/metabolismo , Cálcio/metabolismo , Ratos Wistar , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/metabolismo , Metabolismo Energético , Mitocôndrias Cardíacas/metabolismo , Ácidos Graxos/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , HomeostaseRESUMO
Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2-at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2-, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2- and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2-, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H202 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2- and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2-with k3 values of 0.1 to 1.4 M-1 s-1 and H2O2 with k4 values of 0.009 to 4.3 m-1·s-1.