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1.
Proc Natl Acad Sci U S A ; 117(26): 15363-15373, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554501

RESUMO

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.


Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Oligopeptídeos/farmacologia , Envelhecimento , Animais , Masculino , Camundongos , Modelos Químicos , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Ligação Proteica
2.
PLoS One ; 15(6): e0234872, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559203

RESUMO

The leading cause of death in Pulmonary Arterial Hypertension (PAH) is right ventricular (RV) failure. The tumor suppressor p53 has been associated with left ventricular hypertrophy (LVH) and remodeling but its role in RV hypertrophy (RVH) is unclear. The purpose of this study was to determine whether pharmacological activation of p53 by Quinacrine affects RV remodeling and function in the pulmonary artery banding (PAB) model of compensated RVH in mice. The effects of p53 activation on cellular functions were studied in isolated cardiomyocytes, cardiac fibroblasts and endothelial cells (ECs). The expression of p53 was examined both on human RV tissues from patients with compensated and decompensated RVH and in mouse RV tissues early and late after the PAB. As compared to control human RVs, there was no change in p53 expression in compensated RVH, while a marked upregulation was found in decompensated RVH. Similarly, in comparison to SHAM-operated mice, unaltered RV p53 expression 7 days after PAB, was markedly induced 21 days after the PAB. Quinacrine induced p53 accumulation did not further deteriorate RV function at day 7 after PAB. Quinacrine administration did not increase EC death, neither diminished EC number and capillary density in RV tissues. No major impact on the expression of markers of sarcomere organization, fatty acid and mitochondrial metabolism and respiration was noted in Quinacrine-treated PAB mice. p53 accumulation modulated the expression of Heme Oxygenase 1 (HO-1) and Glucose Transporter (Glut1) in mouse RVs and in adult cardiomyocytes. We conclude that early p53 activation in PAB-induced RVH does not cause substantial detrimental effects on right ventricular remodeling and function.


Assuntos
Hipertrofia Ventricular Direita/metabolismo , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Animais , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ácidos Graxos/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Hipertrofia Ventricular Direita/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Quinacrina/farmacologia , Sarcômeros/metabolismo , Proteína Supressora de Tumor p53/metabolismo
3.
Am J Physiol Heart Circ Physiol ; 318(6): H1516-H1524, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32383992

RESUMO

Engineered heart tissues (EHTs) have emerged as a robust in vitro model to study cardiac physiology. Although biomimetic culture environments have been developed to better approximate in vivo conditions, currently available methods do not permit full recapitulation of the four phases of the cardiac cycle. We have developed a bioreactor which allows EHTs to undergo cyclic loading sequences that mimic in vivo work loops. EHTs cultured under these working conditions exhibited enhanced concentric contractions but similar isometric contractions compared with EHTs cultured isometrically. EHTs that were allowed to shorten cyclically in culture had increased capacity for contractile work when tested acutely. Increased work production was correlated with higher levels of mitochondrial proteins and mitochondrial biogenesis; this effect was eliminated when tissues were cyclically shortened in the presence of a myosin ATPase inhibitor. Leveraging our novel in vitro method to precisely apply mechanical loads in culture, we grew EHTs under two loading regimes prescribing the same work output but with different associated afterloads. These groups showed no difference in mitochondrial protein expression. In loading regimes with the same afterload but different work output, tissues subjected to higher work demand exhibited elevated levels of mitochondrial protein. Our findings suggest that regulation of mitochondrial mass in cultured human EHTs is potently modulated by the mechanical work the tissue is permitted to perform in culture, presumably communicated through ATP demand. Precise application of mechanical loads to engineered heart tissues in culture represents a novel in vitro method for studying physiological and pathological cardiac adaptation.NEW & NOTEWORTHY In this work, we present a novel bioreactor that allows for active length control of engineered heart tissues during extended tissue culture. Specific length transients were designed so that engineered heart tissues generated complete cardiac work loops. Chronic culture with various work loops suggests that mitochondrial mass and biogenesis are directly regulated by work output.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Humanos , Engenharia Tecidual
4.
Am J Physiol Regul Integr Comp Physiol ; 318(5): R972-R980, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233925

RESUMO

Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS) but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis, and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (Jo2) from tissue mitochondrial preparations is an attractive investigative approach, but it introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluorespirometry to evaluate Jo2 and hydrogen peroxide release (Jh2o2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in Jh2o2 across tissues and sample preparations during nonphosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2] but a linear decline in Jh2o2 of 5-15% per 50-µM decrease in chamber [O2] in all samples. Jo2 was generally stable in Mt and Hm across [O2]s above 50 µM but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of Jh2o2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2 sensitive, emphasizing relevant calibration considerations. This study highlights the importance of monitoring and reporting the chamber [O2] at which Jo2 and Jh2o2 are recorded during fluorespirometry experiments and provides a basis for selecting sample preparations for studies addressing the role of mtROS in physiology and disease.


Assuntos
Peróxido de Hidrogênio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Consumo de Oxigênio , Oxigênio/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Respiração Celular , Fluorometria , Cinética , Masculino , Camundongos , Modelos Biológicos , Fosforilação Oxidativa
5.
Circ Heart Fail ; 13(3): e006298, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32160771

RESUMO

BACKGROUND: MicroRNAs are small, noncoding RNAs that play a key role in gene expression. Accumulating evidence suggests that aberrant microRNA expression contributes to the heart failure (HF) phenotype; however, the underlying molecular mechanisms are not well understood. A better understanding of the mechanisms of action of microRNAs could potentially lead to targeted therapies that could halt the progression or even reverse HF. METHODS AND RESULTS: We found that microRNA-152 (miR-152) expression was upregulated in the failing human heart and experimental animal models of HF. Transgenic mice with cardiomyocyte-specific miR-152 overexpression developed systolic dysfunction (mean difference, -38.74% [95% CI, -45.73% to -31.74%]; P<0.001) and dilated cardiomyopathy. At the cellular level, miR-152 overexpression perturbed mitochondrial ultrastructure and dysregulated key genes involved in cardiomyocyte metabolism and inflammation. Mechanistically, we identified Glrx5 (glutaredoxin 5), a critical regulator of mitochondrial iron homeostasis and iron-sulfur cluster synthesis, as a direct miR-152 target. Finally, a proof-of-concept of the therapeutic efficacy of targeting miR-152 in vivo was obtained by utilizing a locked nucleic acid-based inhibitor of miR-152 (LNA 152) in a murine model of HF subjected to transverse aortic constriction. We demonstrated that animals treated with LNA-152 (n=10) showed preservation of systolic function when compared with locked nucleic acid-control treated animals (n=9; mean difference, 18.25% [95% CI, 25.10% to 11.39%]; P<0.001). CONCLUSIONS: The upregulation of miR-152 expression in the failing myocardium contributes to HF pathophysiology. Preclinical evidence suggests that miR-152 inhibition preserves cardiac function in a model of pressure overload-induced HF. These findings offer new insights into the pathophysiology of HF and point to miR-152-Glrx5 axis as a potential novel therapeutic target.


Assuntos
Antagomirs/administração & dosagem , Inativação Gênica , Insuficiência Cardíaca/prevenção & controle , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Aorta/fisiopatologia , Aorta/cirurgia , Estudos de Casos e Controles , Modelos Animais de Doenças , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Ligadura , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Estudo de Prova de Conceito , Volume Sistólico , Função Ventricular Esquerda
6.
Am J Physiol Heart Circ Physiol ; 318(4): H778-H786, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142354

RESUMO

Sepsis-induced cardiomyopathy (SIC) is associated with increased patient mortality. At present, there are no specific therapies for SIC. Previous studies have reported increased reactive oxygen species (ROS) and mitochondrial dysfunction during SIC. However, a unifying mechanism remains to be defined. We hypothesized that PKCδ is required for abnormal calcium handling and cardiac mitochondrial dysfunction during sepsis and that genetic deletion of PKCδ would be protective. Polymicrobial sepsis induced by cecal ligation and puncture (CLP) surgery decreased the ejection fraction of wild-type (WT) mice but not PKCδ knockout (KO) mice. Similarly, WT cardiomyocytes exposed to lipopolysaccharide (LPS) demonstrated decreases in contractility and calcium transient amplitude that were not observed in PKCδ KO cardiomyocytes. LPS treatment decreased sarcoplasmic reticulum calcium stores in WT cardiomyocytes, which correlated with increased ryanodine receptor-2 oxidation in WT hearts but not PKCδ KO hearts after sepsis. LPS exposure increased mitochondrial ROS and decreased mitochondrial inner membrane potential in WT cardiomyocytes. This corresponded to morphologic changes consistent with mitochondrial dysfunction such as decreased overall size and cristae disorganization. Increased cellular ROS and changes in mitochondrial morphology were not observed in PKCδ KO cardiomyocytes. These data show that PKCδ is required in the pathophysiology of SIC by generating ROS and promoting mitochondrial dysfunction. Thus, PKCδ is a potential target for cardiac protection during sepsis.NEW & NOTEWORTHY Sepsis is often complicated by cardiac dysfunction, which is associated with a high mortality rate. Our work shows that the protein PKCδ is required for decreased cardiac contractility during sepsis. Mice with deletion of PKCδ are protected from cardiac dysfunction after sepsis. PKCδ causes mitochondrial dysfunction in cardiac myocytes, and reducing mitochondrial oxidative stress improves contractility in wild-type cardiomyocytes. Thus, PKCδ is a potential target for cardiac protection during sepsis.


Assuntos
Cardiomiopatias/genética , Mitocôndrias Cardíacas/metabolismo , Proteína Quinase C-delta/genética , Sepse/complicações , Animais , Sinalização do Cálcio , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Células Cultivadas , Feminino , Deleção de Genes , Lipopolissacarídeos/toxicidade , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Estresse Oxidativo , Proteína Quinase C-delta/metabolismo
7.
Phytomedicine ; 68: 153171, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32018211

RESUMO

BACKGROUND: Cardiac hypertrophy is a prominent feature of heart remodeling, which may eventually lead to heart failure. Tongmaiyangxin (TMYX) pills are a clinically used botanical drug for treating multiple cardiovascular diseases including chronic heart failure. The aim of the current study was to identify the bioactive compounds in Tongmaiyangxin pills that attenuate cardiomyocytes hypertrophy, and to investigate the underlying mechanism of action. METHODS AND RESULTS: The anti-hypertrophy effect of TMYX was validated in isoproterenol-induced cardiac hypertrophy model in C57BL/6 mice. After TMYX treatment for 2 weeks, the heart ejection fraction and fractional shortening of the mice model was increased by approximately 20% and 15%, respectively, (p < 0.05). Besides, TMYX dose-dependently reduced the cross section area of cardiomyocytes in the angiotensin-II induced hypertrophy H9c2 model (p < 0.01). Combining high content screening and liquid chromatography mass spectrometry, four compounds with anti-cardiac hypertrophy effects were identified from TMYX, which includes emodin, licoisoflavone A, licoricone and glyasperin A. Licoisoflavone A is one of the compounds with most significant protective effect and we continued to investigate the mechanism. Primary cultures of neonatal rat cardiomyocytes were treated with a hypertrophic agonist phenylephrine (PE) in the presence or absence of licoisoflavone A. After 48 h of treatment, cells were harvested and mitochondrial acetylation was analyzed by western blotting and Image analysis. Interestingly, the results suggested that the anti-hypertrophic effects of licoisoflavone A depend on the activation of the deacetylase Sirt3 (p < 0.01). Finally, we showed that licoisoflavone A-treatment was able to decrease relative ANF and BNP levels in the hypertrophic cardiac cells (p < 0.01), but not in cells co-treated with Sirt3 inhibitors (3-TYP) (p > 0.05). CONCLUSION: TMYX exerts its anti-hypertrophy effect possibly through upregulating Sirt3 expression. Four compounds were identified from TMYX which may be responsible for the anti-hypertrophy effect. Among these compounds, licoisoflavone A was demonstrated to block the hypertrophic response of cardiomyocytes, which required its positive regulation on the expression of Sirt3. These results suggested that licoisoflavone A is a potential Sirt3 activator with therapeutic effect on cardiac hypertrophy.


Assuntos
Cardiomegalia/tratamento farmacológico , Medicamentos de Ervas Chinesas/química , Isoflavonas/farmacologia , Sirtuína 3/metabolismo , Acetilação , Angiotensina II/efeitos adversos , Animais , Cardiomegalia/induzido quimicamente , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/farmacologia , Isoproterenol/efeitos adversos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenilefrina/efeitos adversos , Ratos
8.
Clin Chim Acta ; 505: 92-97, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32070725

RESUMO

INTRODUCTION: Carnitine is essential for long-chain fatty acid oxidation in muscle and heart. Tissue stores are regulated by organic cation/Cn transporter plasmalemmal Octn2. We previously demonstrated low carnitine in quadriceps/gluteus and heart of adult mdx mice. METHODS: We studied protein and mRNA expression of Octn2, mitochondrial Octn1 and peroxisomal Octn3 in adult male C57BL/10ScSn-DMD mdx/J quadriceps, heart, and diaphragm compared to C57BL/10SnJ mice. RESULTS: We demonstrated reduction in mOctn2 expression on Western blot and similar expression of mOctn1 and mOctn3 in mdx quadriceps, heart and diaphragm. There was a significant upregulation of mOctn1 and mOctn2 mRNA by qRT-PCR in mdx quadriceps and of mOctn2 and mOctn3 mRNA in mdx heart. We showed upregulation of mdx mOctn1 and mOctn3 mRNA but no increase in protein expression. DISCUSSION: Dystrophin deficiency likely disrupts Octn2 expression decreasing muscle carnitine uptake thus contributing to membranotoxic long-chain acyl-CoAs with sarcolemmal and organellar membrane oxidative injury providing a treatment rationale for early L-carnitine in DMD.


Assuntos
Carnitina/química , Carnitina/uso terapêutico , Músculo Esquelético/química , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Miocárdio/química , Proteínas de Transporte de Cátions Orgânicos/biossíntese , Proteínas de Transporte de Cátions Orgânicos/genética , Membro 5 da Família 22 de Carreadores de Soluto/biossíntese , Membro 5 da Família 22 de Carreadores de Soluto/genética , Simportadores/biossíntese , Simportadores/genética , Animais , Carnitina/metabolismo , Diafragma/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
9.
PLoS One ; 15(2): e0228710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084168

RESUMO

Our study revisits the role of cardiac mitochondrial adjustments during the progression of type 2 diabetes mellitus (T2DM), while considering age and sex as potential confounding factors. We used the Nile Grass rats (NRs) as the animal model. After weaning, animals were fed either a Standard Rodent Chow Diet (SRCD group) or a Mazuri Chinchilla Diet (MCD group) consisting of high-fiber and low-fat content. Both males and females in the SRCD group, exhibited increased body mass, body mass index, and plasma insulin compared to the MCD group animals. However, the females were able to preserve their fasting blood glucose throughout the age range on both diets, while the males showed significant hyperglycemia starting at 6 months in the SRCD group. In the males, a higher citrate synthase activity-a marker of mitochondrial content-was measured at 2 months in the SRCD compared to the MCD group, and this was followed by a decline with age in the SRCD group only. In contrast, females preserved their mitochondrial content throughout the age range. In the males exclusively, the complex IV capacity expressed independently of mitochondrial content varied with age in a diet-specific pattern; the capacity was elevated at 2 months in the SRCD group, and at 6 months in the MCD group. In addition, females, but not males, were able to adjust their capacity to oxidize long-chain fatty acid in accordance with the fat content of the diet. Our results show clear sexual dimorphism in the variation of mitochondrial content and oxidative phosphorylation capacity with diet and age. The SRCD not only leads to T2DM but also exacerbates age-related cardiac mitochondrial defects. These observations, specific to male NRs, might reflect deleterious dietary-induced changes on their metabolism making them more prone to the cardiovascular consequences of aging and T2DM.


Assuntos
Envelhecimento/patologia , Diabetes Mellitus Tipo 2/patologia , Mitocôndrias Cardíacas/patologia , Caracteres Sexuais , Animais , Glicemia/metabolismo , Citrato (si)-Sintase/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Jejum/sangue , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Masculino , Mitocôndrias Cardíacas/metabolismo , Murinae , NAD/metabolismo , Oxirredução , Fenótipo
10.
FASEB J ; 34(1): 663-675, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914595

RESUMO

Mitochondrial fusion and fission are essential for heart function. Abrogating mitochondrial dynamism leads to cardiomyopathy. Excessive mitochondrial fragmentation is involved in most heart diseases, thus enhancing mitochondrial fusion will be a potential therapeutic strategy. To understand the effects of promoting mitochondrial fusion in adult cardiac, we investigated mice hearts, and cultured murine embryonic fibroblasts (MEFs), in which mitofusin 2 (Mfn2) overexpressed or dynamin-related protein 1 (Drp1) was abrogated concomitantly forcing mitochondrial fusion. Parallel studies revealed that fission-defective Drp1 knockout hearts and MEFs evoked stronger mitochondrial enlargement, enhanced mitophagy with mitochondrial volume decrease and increased mitochondrial calcium uptake, superoxide production, and permeability transition pore opening, contributed to cardiomyocyte apoptosis and dilated cardiomyopathy. Mfn2 overexpression in the adult heart is comparable with the control except for slight mitochondrial enlargement and mitochondrial volume increase, but without mitophagy induction. Moreover, Mfn2 overexpression increases mitochondrial biogenesis and fusion could protect against mitochondrial fragmentation and Drp1 deletion evoking mitophagy in MEFs. Our findings indicate that mitochondrial fusion provoked by fusion promotion and fission inhibition direct the different fate of heart, Mfn2 upregulation other than Drp1 downregulation well maintains heart mitochondrial function is a more safe strategy for correcting excessive mitochondrial fragmentation in hearts.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Dinâmica Mitocondrial/fisiologia , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Dinaminas/metabolismo , Fibroblastos/metabolismo , Camundongos Transgênicos , Mitofagia/fisiologia
11.
Circ Res ; 126(2): 280-293, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31944918

RESUMO

Adult cardiomyocytes are postmitotic cells that undergo very limited cell division. Thus, cardiomyocyte death as occurs during myocardial infarction has very detrimental consequences for the heart. Mitochondria have emerged as an important regulator of cardiovascular health and disease. Mitochondria are well established as bioenergetic hubs for generating ATP but have also been shown to regulate cell death pathways. Indeed many of the same signals used to regulate metabolism and ATP production, such as calcium and reactive oxygen species, are also key regulators of mitochondrial cell death pathways. It is widely hypothesized that an increase in calcium and reactive oxygen species activate a large conductance channel in the inner mitochondrial membrane known as the PTP (permeability transition pore) and that opening of this pore leads to necroptosis, a regulated form of necrotic cell death. Strategies to reduce PTP opening either by inhibition of PTP or inhibiting the rise in mitochondrial calcium or reactive oxygen species that activate PTP have been proposed. A major limitation of inhibiting the PTP is the lack of knowledge about the identity of the protein(s) that form the PTP and how they are activated by calcium and reactive oxygen species. This review will critically evaluate the candidates for the pore-forming unit of the PTP and discuss recent data suggesting that assumption that the PTP is formed by a single molecular identity may need to be reconsidered.


Assuntos
Cálcio/metabolismo , Morte Celular , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismo
12.
PLoS One ; 15(1): e0226142, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940313

RESUMO

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 µM cytochrome c, 10 mM succinate, 2.5 µM oligomycin, 1.5 µM FCCP, 3.5 µM rotenone, 4 µM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Adulto , Idoso , Citrato (si)-Sintase/metabolismo , Criopreservação , Metabolismo Energético , Ácidos Graxos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Membranas Mitocondriais/metabolismo , Oxirredução , Oxigênio/metabolismo
13.
Arch Biochem Biophys ; 681: 108258, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31917961

RESUMO

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.


Assuntos
Glioxilatos/metabolismo , Ácidos Mandélicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Animais , Cálcio/metabolismo , Magnésio/metabolismo , Suínos
14.
Eur J Clin Invest ; 50(3): e13204, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31990365

RESUMO

BACKGROUND: The heart is a highly oxidative tissue, thus mitochondria play a major role in maintaining optimal cardiac function. Our previous study established a dietary-induced obese minipig with cardiac fibrosis. The aim of this study was to elucidate the role of mitochondrial dynamics in cardiac fibrosis of obese minipigs. DESIGN: Four-month-old Lee-Sung minipigs were randomly divided into two groups: a control group (C) and an obese group (O) by feeding a control diet or a high-fat diet (HFD) for 6 months. Exposure of H9c2 cardiomyoblasts to palmitate was used to explore the effects of high-fat on induction of myocardial injury in vitro. RESULTS: The O pigs displayed greater heart weight and cardiac collagen accumulation. Obese pigs exhibited a lower antioxidant capacity, ATP concentration, and higher oxidative stress in the left ventricle (LV). The HFD caused downregulation in protein expression of PGC-1α and OPA1, and upregulation of DRP1, FIS1, and PINK1 in the LV of O compared to C pigs. Furthermore, palmitate induced apoptosis and decreased ATP content in H9c2 cells. Palmitate elevated the protein expression of DRP1 and PINK1 in these cells. Inhibition of DRP1 protein expression by siDRP1 in H9c2 cells resulted in enhanced ATP and decreased palmitate-induced apoptosis. CONCLUSIONS: These results suggest that mitochondrial dynamics were linked to the progression of obesity-related cardiac injury. Inhibition of DRP1 after palmitate exposure in H9c2 cells resulted in improved ATP level and decreased apoptosis in vitro suggesting that mitochondrial fission serves a key role in progression of obesity-induced cardiac fibrosis.


Assuntos
Dinaminas/metabolismo , Cardiopatias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Dinaminas/genética , Fibrose/metabolismo , Mitocôndrias Cardíacas/metabolismo , Obesidade , Ratos , Respiração , Suínos , Porco Miniatura
15.
Am J Physiol Heart Circ Physiol ; 318(2): H295-H300, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31922888

RESUMO

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.


Assuntos
Inibidores Enzimáticos/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Consumo de Oxigênio/efeitos dos fármacos , Amidinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ornitina/análogos & derivados , Ornitina/farmacologia
16.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R227-R233, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774307

RESUMO

Doxorubicin (DOX) is a highly effective antineoplastic agent used in cancer treatment. Unfortunately, clinical use of DOX is limited due to the development of dose-dependent toxicity to cardiac and respiratory (i.e., diaphragm) muscles. After administration, DOX preferentially localizes to the inner mitochondrial membrane, where it promotes cellular toxicity via enhanced mitochondrial reactive oxygen species (ROS) production. Although recent evidence suggests that amelioration of mitochondrial ROS emission preserves cardiorespiratory muscle function following DOX treatment, the mechanisms responsible for this protection remain unknown. Therefore, we tested the hypothesis that DOX-induced mitochondrial ROS production is required to stimulate pathological signaling by the autophagy/lysosomal system (ALS), the ubiquitin-proteasome pathway (UPP), and the unfolded protein response (UPR). Cause and effect were determined by administration of the mitochondria-targeted peptide SS-31 to DOX-treated animals. Interestingly, while SS-31 abrogated aberrant ROS emission in cardiorespiratory muscles of DOX-treated animals, our results revealed muscle-specific regulation of effector pathways. In the heart, SS-31 prevented DOX-induced proteolytic signaling through the ALS and UPP. In contrast, ALS signaling was inhibited by SS-31 in the diaphragm, but the UPP was not affected. UPR signaling was activated in both muscles at eukaryotic translation initiation factor 2α (eIF2α) S51 in the heart and diaphragm of DOX-treated animals and was attenuated with SS-31 treatment in both tissues. However, downstream signaling of eIF2α (activating transcription factor 4 and CCAAT/enhancer-binding protein homologous protein) was diminished in the heart but upregulated in the diaphragm with DOX. Collectively, these results show that DOX-induced ROS production plays distinct roles in the regulation of cardiac and diaphragm muscle proteolysis.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Diafragma/efeitos dos fármacos , Doxorrubicina/toxicidade , Cardiopatias/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cardiotoxicidade , Diafragma/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Cardiopatias/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas/efeitos dos fármacos
17.
Life Sci ; 247: 116942, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31715185

RESUMO

AIMS: The cardioprotective effects of preconditioning against ischemia-reperfusion (I/R) injury depend on the structural integrity of membrane caveolae and signaling through G protein-coupled receptors (GPCRs). However, the mechanisms underlying opioid preconditioning are not fully understood. Here, we examined whether caveolins transmitted opioid-GPCR signals to the mitochondria to mediate cardioprotection. MAIN METHODS: Mice were treated with pertussis toxin (PTX) or saline. Thirty-six hours later, mice from each group were randomly assigned to receive the δ-opioid receptor agonist SNC-121 or saline intraperitoneally 15 min before in vivo I/R. Infarct sizes in each group were compared, and immunoblot analysis was used to detect caveolin expression. The structures of caveolae and mitochondria were determined by electron microscopy (EM). The opening degree of the mitochondrial permeability transition pore (mPTP) was assessed by colorimetry, and mitochondrial respiratory function was assessed by Oxygraph-2k. KEY FINDINGS: Treatment with an opioid receptor agonist reduced the myocardial infarct size after I/R injury, increased caveolin expression, decreased mitochondrial mPTP opening, and improved mitochondrial respiratory function. EM analysis revealed that opioids induced caveolae formation in myocytes and tended to promote translocation to mitochondria. However, these protective effects were blocked by PTX. SIGNIFICANCE: Opioid-induced preconditioning depended on Gi signaling, which promoted caveolin translocation to mitochondria, supported their functional integrity, and enhanced cardiac stress adaption. Verification of this pathway will establish new targets for opioid agents in the field of cardiac protection.


Assuntos
Benzamidas/farmacologia , Cardiotônicos/farmacologia , Caveolinas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Piperazinas/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Animais , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Masculino , Camundongos , Mitocôndrias Cardíacas/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/ultraestrutura , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Receptores Acoplados a Proteínas-G/metabolismo
18.
Life Sci ; 247: 116560, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31200000

RESUMO

Mitochondrial dysfunction caused by calcium overload is a vital factor for mediating cardiomyocyte death following ischemia/reperfusion (I/R) injury. The stromal interactive molecule 2 (STIM2) is a calcium sensor protein that regulates the store-operated calcium entry (SOCE). Whereas, whether STIM2 is associated with I/R injury remains largely unclear. We report here that STIM2, but not its homologue STIM1, is upregulated in cultured H9c2 cells, a cell model for cardiomyocytes, following I/R injury. In addition, the knockdown of STIM2, but not STIM1, reduces H9c2 cell apoptosis following I/R injury, and similar results were obtained in primary neonatal cardiomyocytes. This anti-apoptotic effect could be attributed to the inhibited activation of mitochondrial apoptosis pathway. Moreover, STIM2 knockdown reduces ER calcium release and simultaneously alleviates mitochondrial calcium overload in H9c2 cells following I/R injury. Furthermore, STIM2 knockdown decreases mitochondrial injury and preserves mitochondrial function following I/R injury. Collectively, these results suggest that the protective role of STIM2 knockdown against I/R injury in cardiomyocytes is associated with the reduced mitochondrial calcium overload and preserved mitochondrial function. Hence, our study may provide a novel insight into the regulation of mitochondrial-mediated cardiomyocyte apoptosis following I/R injury.


Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/metabolismo , Animais , Apoptose , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Transporte de Íons/fisiologia , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno , Ratos , Transfecção
19.
Artigo em Inglês | MEDLINE | ID: mdl-31678269

RESUMO

Naked mole-rats (Heterocephalus glaber; NMRs) are among the most hypoxia-tolerant mammals described to date and exhibit plastic responses during hypoxia exposure. The goal of the present study was to determine if heart mitochondria from NMRs functionally differ from those of hypoxia-intolerant common laboratory mice (Mus musculus). We assessed heart mitochondrial respiratory flux, proton leak kinetics, responses to in vitro anoxia-recovery, and maximal complex enzyme activities. When investigated at their respective body temperatures (28 °C for NMR and 37 °C for mice), NMR heart mitochondria had lower respiratory fluxes relative to mice, particularly for state 2 and oligomycin-induced state 4 leak respiration rates. When leak respiration rates were standardized to the same membrane potential, NMR mitochondria had lower complex II-stimulated state 2 respiration rates than mice. Both mice and NMRs responded similarly to an in vitro anoxia-recovery challenge and decreased state 3 respiration rate post-anoxia. Finally, NMRs had overall lower maximal complex enzyme activities compared with mice, but the magnitude of the difference did not correspond with observed differences in respiratory fluxes. Overall, heart mitochondria from NMRs appear more coupled than those of mice, but in both species the heart appears equally susceptible to ischemic-reperfusion injury.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Consumo de Oxigênio , Estresse Fisiológico , Animais , Camundongos , Mitocôndrias Cardíacas/patologia , Ratos-Toupeira , Traumatismo por Reperfusão Miocárdica/patologia , Especificidade da Espécie
20.
Circ Res ; 126(4): 439-452, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31852393

RESUMO

RATIONALE: Hypertension represents a major risk factor for stroke, myocardial infarction, and heart failure and affects 30% of the adult population. Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear. The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level. OBJECTIVE: We hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in hypertension, but increased Sirt3 protects vascular function and decreases hypertension. METHODS AND RESULTS: To test the therapeutic potential of targeting Sirt3 expression, we developed new transgenic mice with global Sirt3OX (Sirt3 overexpression), which protects from endothelial dysfunction, vascular oxidative stress, and hypertrophy and attenuates Ang II (angiotensin II) and deoxycorticosterone acetate-salt induced hypertension. Global Sirt3 depletion in Sirt3-/- mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM [vascular cell adhesion molecule-1], ICAM [intercellular adhesion molecule-1], and MCP1 [monocyte chemoattractant protein 1]), increases inflammatory cell infiltration in the kidney, reduces telomerase expression, and accelerates vascular senescence and age-dependent hypertension; conversely, increased Sirt3 expression in Sirt3OX mice prevents these deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with normotensive subjects. CONCLUSIONS: We suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of targeting Sirt3 expression in vascular dysfunction and hypertension.


Assuntos
Hipertensão Essencial/metabolismo , Coração/fisiopatologia , Inflamação/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Sirtuína 3/metabolismo , Angiotensina II , Animais , Acetato de Desoxicorticosterona , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hipertensão Essencial/induzido quimicamente , Hipertensão Essencial/genética , Feminino , Inflamação/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/genética , Miocárdio/metabolismo , Miocárdio/patologia , Sirtuína 3/genética
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