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INTRODUCTION: Rattlesnake (Crotalus spp., Sistrurus spp.) bites in the southwestern United States are associated with significant morbidity. This study aims to describe 25 years of rattlesnake encounters reported to the Arizona Poison and Drug Information Center to identify vulnerable populations and circumstances where encounters occur to create public education to reduce future bites. METHODS: Cases of suspected rattlesnake encounters in Arizona reported to the Arizona Poison and Drug Information Center between 1999 and 2023 were analyzed to identify populations and circumstances associated with encounters. RESULTS: A total of 3,808 cases were analyzed overall and by age subgroups. Most encounters occurred in men (69.9%), during the evening (16:00-21:59; 49.2%), in summer (41.9%), and close to home (38.2%). Most bites occurred to the lower extremity (51%). Children 0 to 12-years-old have more encounters than those 13-years-old and older in rural zip codes (27.7% versus 14.8%; P = 0.005), during spring (31.8% versus 22.3%; P = 0.0005), and during the evening (64.4% versus 48.1%; P < 0.001). DISCUSSION: Rattlesnakes are encountered when rattlesnake and human behavior patterns overlap. Many people spend time outside during evening hours in the summer, and valuable resources like food, water, and shelter can be found near houses where humans spend much of their time. Most age groups have similar encounter circumstances but encounters among children 0 to 12-years-old differ in time of day, season, and urbanization level than encounters of those 13-years-old and older. Limitations of this study include underreporting of encounters, incomplete case details, potential reporting bias, potential snake misidentification, and geographic coverage of the poison center. CONCLUSION: Prevention of rattlesnake bites by reducing encounters is the most effective way to reduce suffering and healthcare costs. Future steps include creating and disseminating targeted public health education using the data collected.
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Crotalus , Centros de Controle de Intoxicações , Mordeduras de Serpentes , Humanos , Arizona/epidemiologia , Criança , Masculino , Pré-Escolar , Adolescente , Lactente , Feminino , Adulto , Adulto Jovem , Pessoa de Meia-Idade , Centros de Controle de Intoxicações/estatística & dados numéricos , Animais , Mordeduras de Serpentes/epidemiologia , Mordeduras de Serpentes/terapia , Recém-Nascido , Idoso , Estações do AnoRESUMO
Gliding is only present in six extant groups of mammals-interestingly, despite divergent evolutionary histories, all mammalian gliders are strictly nocturnal. Gliding mammals also seem to have relatively high rates of ultrasound use and ultraviolet-induced photoluminescence (UVP) in contrast with their close relatives. Therefore, we hypothesized that, despite diverging lineages, gliding mammals use similar modes of cryptic communication compared to their non-gliding counterparts. We developed two datasets containing the vocal range (minimum-maximum of the dominant harmonic; kHz) and UVP of 73 and 82 species, respectively; we report four novel vocal repertoires and 57 novel observations of the presence or absence of UVP. We complemented these datasets with information about body size, diel activity patterns, habitat openness, and sociality to explore possible covariates related to vocal production and UVP. We found that the maximum of the dominant harmonic was significant higher in gliding mammals when vocalizing than their non-gliding relatives. Additionally, we found that nocturnality was the only significant predictor of UVP, consistent with the previous hypothesis that luminophores primarily drive UVP in mammal fur. In contrast, however, we did not find UVP ubiquitous in nocturnal mammals, suggesting that some unknown process may contribute to variation in this trait.
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Mamíferos , Esportes , Animais , Evolução Biológica , EcossistemaRESUMO
The One Health framework links animal, human, and environmental health, and focuses on emerging zoonotic pathogens. Understanding the interface between wildlife and human activity is critical due to the unpredictable nature of spillover of zoonotic pathogens from animals to humans. Zoos are important partners in One Health because of their contributions to education, conservation, and animal health monitoring. In addition, the housing of wildlife in captive and semi-natural settings makes zoos, especially relevant for detecting animal-related pathogens. A first step to determine the utility of zoos in contributing to pathogen surveillance is to survey the peer-reviewed literature. We, therefore, retrieved data from the previous 20 years and performed a meta-analysis to determine global patterns of viral seroprevalence in mammals housed in zoo collections from peer-reviewed literature. We analysed 50 articles, representing a total of 11,300 terrestrial mammals. Increased prevalence was found in viruses strictly targeting specific host taxonomy, especially in viruses transmitted through direct contact. Potentially complex patterns with geography were also identified, despite uneven sampling. This research highlights the role zoos could play in public health and encourages future standardized epidemiological surveillance of zoological collections.
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Animais de Zoológico , Vírus , Animais , Humanos , Estudos Soroepidemiológicos , Animais Selvagens , MamíferosRESUMO
BACKGROUND: California has experienced an increase in reported cases of disseminated gonococcal infection (DGI). Given significant morbidity associated with DGI and the ability of Neisseria gonorrhoeae to rapidly develop antibiotic resistance, characterization of these cases can inform diagnosis, management, and prevention of DGI. METHODS: As part of the public health response to increased reports of DGI, we used gonorrhea surveillance data reported to the California Department of Public Health to identify all DGI cases in a geographically-bound region. Standardized case report forms were used to collect epidemiologic risk factors and clinical information obtained from provider/laboratory reports, medical records, and patient interviews. RESULTS: From 1 July 2020 to 31 July 2021, we identified 149 DGI patients among 63 338 total gonorrhea infections, representing 0.24% of gonorrhea cases. Estimated incidence was 0.47 DGI cases per 100 000 person-years. Mean age of DGI patients was 40 years, and 75 (50%) were cisgender men, of whom only 13 were known to have male partners. Where reported, more than one-third (36%) used methamphetamine and nearly one-quarter (23%) experienced homelessness. Clinically, 61% lacked urogenital, pharyngeal, or rectal symptoms; 2 patients died in the hospital. Among 47 isolates from patients with antimicrobial susceptibility testing (AST) results available, all were susceptible to ceftriaxone and cefixime. CONCLUSIONS: Most DGI patients lacked urogenital symptoms and were not among populations for which routine gonorrhea screening is currently recommended. Expanding gonorrhea screening might prevent DGI. Cefixime is likely the best option if transitioning from parenteral to oral therapy when AST results are unavailable.
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Gonorreia , Humanos , Masculino , Adulto , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Cefixima/uso terapêutico , Neisseria gonorrhoeae , Ceftriaxona/uso terapêutico , California/epidemiologia , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência BacterianaRESUMO
While an array of taxa are capable of producing fluorescent pigments, fluorescence in mammals is a novel and poorly understood phenomenon. A first step towards understanding the potential adaptive functions of fluorescence in mammals is to develop an understanding of fluorescent compounds, or fluorophores, that are present in fluorescent tissue. Here we use Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) of flying squirrel fur known to fluoresce under ultraviolet (UV) light to identify potentially fluorescent compounds in squirrel fur. All of the potentially fluorescent compounds we identified were either present in non-fluorescent fur or were not present in all species of fluorescent flying squirrel. Therefore, we suggest that the compounds responsible for fluorescence in flying squirrels may also be present in non-fluorescent mammal fur. Some currently unexplained factor likely leads to excitation of fluorophores in flying squirrel fur. A recently suggested hypothesis that fluorescence in mammals is widely caused by porphyrins is consistent with our findings.
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Pelo Animal/química , Corantes Fluorescentes/química , Pigmentos Biológicos/química , Sciuridae/fisiologia , Animais , Voo Animal/fisiologia , Corantes Fluorescentes/isolamento & purificação , Espectrometria de Massas/métodos , Pigmentos Biológicos/isolamento & purificaçãoRESUMO
INTRODUCTION: Previous studies have established a baseline of minimal reproducibility in the social science and biomedical literature. Clinical research is especially deficient in factors of reproducibility. Surgical journals contain fewer clinical trials than non-surgical areas of medicine, suggesting that it should be easier to reproduce the outcomes of surgical literature. METHODS: In this study, we evaluated a broad range of indicators related to transparency and reproducibility in a random sample of 387 articles published in Surgery journals between 2014 and 2018. RESULTS: A small minority of our sample made available their materials (5.3%, 95% C.I. 2.4%-8.2%), protocols (1.2%, 0-2.5%), data (2.5%, 0.7%-4.2%), or analysis scripts (0.04%). Four studies were adequately pre-registered. No studies were explicit replications of previous literature. Most studies (58%), declined to provide a funding statement, while conflicts of interest were declared in a small fraction (9.3%). Most have not been cited by systematic reviews (83%) or meta-analyses (87%), and most were only accessible to paying subscribers (59%). CONCLUSIONS: The transparency of the surgical literature could improve with adherence to baseline standards of reproducibility.
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Pesquisa Biomédica , Publicações , Reprodutibilidade dos Testes , Projetos de PesquisaRESUMO
Anthracyclines, such as doxorubicin, are commonly prescribed antineoplastic agents that cause irreversible cardiac injury. Doxorubicin cardiotoxicity is initiated by increased oxidative stress in cardiomyocytes. Oxidative stress enhances intracellular matrix metalloproteinase-2 (MMP-2) by direct activation of its full-length isoform and (or) de novo expression of an N-terminal-truncated isoform (NTT-MMP-2). As MMP-2 is localized to the sarcomere, we tested whether doxorubicin activates intracellular MMP-2 in neonatal rat ventricular myocytes (NRVM) and whether it thereby proteolyzes two of its identified sarcomeric targets, α-actinin and troponin I. Doxorubicin increased oxidative stress within 12 h as indicated by reduced aconitase activity. This was associated with a twofold increase in MMP-2 protein levels and threefold higher gelatinolytic activity. MMP inhibitors ARP-100 or ONO-4817 (1 µM) prevented doxorubicin-induced MMP-2 activation. Doxorubicin also increased the levels and activity of MMP-2 secreted into the conditioned media. Doxorubicin upregulated the mRNA expression of both full-length MMP-2 and NTT-MMP-2. α-Actinin levels remained unchanged, whereas doxorubicin downregulated troponin I in an MMP-independent manner. Doxorubicin induces oxidative stress and stimulates a robust increase in MMP-2 expression and activity in NRVM, including NTT-MMP-2. The sarcomeric proteins α-actinin and troponin I are, however, not targeted by MMP-2 under these conditions.
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Doxorrubicina/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Aconitato Hidratase/metabolismo , Actinina/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Éteres Fenílicos/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfonas/farmacologia , Troponina I/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Primary ubiquinone (UQ) deficiency is an important subset of mitochondrial disease that is caused by mutations in UQ biosynthesis genes. To guide therapeutic efforts we sought to estimate the number of individuals who are born with pathogenic variants likely to cause this disorder. We used the NCBI ClinVar database and literature reviews to identify pathogenic genetic variants that have been shown to cause primary UQ deficiency, and used the gnomAD database of full genome or exome sequences to estimate the frequency of both homozygous and compound heterozygotes within seven genetically-defined populations. We used known population sizes to estimate the number of afflicted individuals in these populations and in the mixed population of the USA. We then performed the same analysis on predicted pathogenic loss-of-function and missense variants that we identified in gnomAD. When including only known pathogenic variants, our analysis predicts 1,665 affected individuals worldwide and 192 in the USA. Adding predicted pathogenic variants, our estimate grows to 123,789 worldwide and 1,462 in the USA. This analysis predicts that there are many undiagnosed cases of primary UQ deficiency, and that a large proportion of these will be in developing regions of the world.
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Ataxia/epidemiologia , Ataxia/genética , Doenças Mitocondriais/epidemiologia , Doenças Mitocondriais/genética , Debilidade Muscular/epidemiologia , Debilidade Muscular/genética , Ubiquinona/deficiência , Bases de Dados de Ácidos Nucleicos , Exoma , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , Fenótipo , Ubiquinona/genética , Ubiquinona/fisiologia , Sequenciamento do ExomaRESUMO
Mouse and Caenorhabditis elegans mutants with altered life spans are being used to investigate the aging process and how genes determine life span. The survival of a population can be modeled by the Gompertz function, which comprises two parameters. One of these parameters ("G") describes the rate at which mortality accelerates with age and is often described as the "rate of aging." The other parameter ("A") may correspond to the organism's baseline vulnerability to deleterious effects of disease and the environment. We show that, in mice, life-span-extending mutations systematically fail to affect the age-dependent acceleration of mortality (G), but instead affect only baseline vulnerability (A). This remains true even when comparing strains maintained under identical environmental conditions. In contrast, life-span-extending mutations in C. elegans were associated with decreases in G These observations on mortality rate kinetics suggest that the mechanisms of aging in mammals might fundamentally differ from those in nematodes.
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Longevidade/genética , Modelos Genéticos , Mutação , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Camundongos , Especificidade da EspécieRESUMO
Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration.
Assuntos
Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Oncostatina M/toxicidade , Sarcômeros/efeitos dos fármacos , Actinina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores de Metaloproteinases de Matriz/farmacologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Sarcômeros/enzimologia , Sarcômeros/patologia , Fatores de Tempo , Troponina I/metabolismo , Regulação para CimaRESUMO
Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in intra- and extra-cellular matrix remodeling resulting from oxidative stress injury to the heart. MMP-2 was the first MMP to be localized to the nucleus; however, its biological functions there are unclear. We hypothesized that MMP-2 is present in the nucleus under normal physiological conditions but increases during myocardial ischemia-reperfusion (I/R) injury-induced oxidative stress, proteolyzing nuclear structural proteins. Lamins are intermediate filament proteins that provide structural support to the nucleus and are putative targets of MMP-2. To identify lamin susceptibility to MMP-2 proteolysis, purified lamin A or B was incubated with MMP-2 in vitro. Lamin A, but not lamin B, was proteolysed by MMP-2 into an approximately 50kDa fragment, which was also predicted by in silico cleavage site analysis. Immunofluorescent confocal microscopy and subcellular fractionation showed MMP-2 both in the cytosol and nuclei of neonatal rat ventricular myocytes. Rat hearts were isolated and perfused by the Langendorff method aerobically, or subjected to I/R injury in the presence or absence of o-phenanthroline, an MMP inhibitor. Nuclear fractions extracted from I/R hearts showed increased MMP-2 activity, but not protein level. The level of troponin I, a known sarcomeric target of MMP-2, was rescued in I/R hearts treated with o-phenanthroline, demonstrating the efficacy of MMP inhibition. However, lamin A or B levels remained unchanged in I/R hearts. MMP-2 has a widespread subcellular distribution in cardiomyocytes, including a significant presence in the nucleus. The increase in nuclear MMP-2 activity seen during stunning injury here, indicates yet unknown biological actions, other than lamin proteolysis, which may require more severe ischemia to effect.
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Metaloproteinase 2 da Matriz/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Matriz Nuclear/metabolismo , Animais , Espaço Intracelular/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Masculino , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Transporte Proteico , Proteólise , RatosRESUMO
Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.
Assuntos
Actinina/metabolismo , Mitose , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Actinina/fisiologia , Animais , Fracionamento Celular , Conectina/metabolismo , Conectina/fisiologia , Citosol/metabolismo , Citosol/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Fosforilação , Inibidores de Proteases/farmacologia , Ratos , Ratos Sprague-Dawley , Sarcômeros/efeitos dos fármacos , Sarcômeros/ultraestruturaRESUMO
Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.â¢Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kitsâ¢The protocol can be applied to tissue samples or cultured cells without changing buffer componentsâ¢Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C.
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Mitochondria play a crucial role in determining whole-body metabolism and exercise capacity. Genetic mouse models of mild mitochondrial dysfunction provide an opportunity to understand how mitochondrial function affects these parameters. MCLK1 (a.k.a. Coq7) is an enzyme implicated in the biosynthesis of ubiquinone (UQ; Coenzyme Q). Low levels of MCLK1 in Mclk1(+/-) heterozygous mutants lead to abnormal sub-mitochondrial distribution of UQ, impaired mitochondrial function, elevated mitochondrial oxidative stress, and increased lifespan. Here, we report that young Mclk1(+/-) males, but not females, show a significant decrease in whole-body metabolic rate as measured by indirect calorimetry. Such a sex-specific effect of mitochondrial dysfunction on energy metabolism has also been reported for heterozygous mice carrying a mutation for the gene encoding the "Rieske" protein of mitochondrial complex III (RISP(+/P224S)). We find that both Mclk1(+/-) and RISP(+/P224S) males are capable of restoring their defective metabolic rates by making significantly more voluntary use of a running wheel compared to wild type. However, this increase in voluntary activity does not reflect their exercise capacity, which we found to be impaired as revealed by a shorter treadmill distance run before exhaustion. In contrast to what is observed in Mclk1(+/-) and RISP(+/P224S) mutants, Sod2(+/-) mice with elevated oxidative stress and major mitochondrial dysfunction did not increase voluntary activity. Our study reveals a sex-specific effect on how impaired mitochondrial function impacts whole-body energy metabolism and locomotory behavior, and contributes to the understanding of the metabolic and behavioral consequences of mitochondrial disorders.
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Matrix metalloproteinase (MMPs) are long understood to be involved in remodeling of the extracellular matrix. However, over the past decade, it has become clear that one of the most ubiquitous MMPs, MMP-2, has numerous intracellular targets in cardiac myocytes. Notably, MMP-2 proteolyzes components of the sarcomere, and its intracellular activity contributes to ischemia-reperfusion injury of the heart. Together with the well documented role played by MMPs in the myocardial remodeling that occurs following myocardial infarction, this has led to great interest in targeting MMPs to treat cardiac ischemic injury. In this review we will describe the expanding understanding of intracellular MMP-2 biology, and how this knowledge may lead to improved treatments for ischemic heart injury. We also critically review the numerous preclinical studies investigating the effects of MMP inhibition in animal models of myocardial infarction and ischemia-reperfusion injury, as well as the recent clinical trials that are part of the effort to translate these results into clinical practice. Acknowledging the disappointing results of past clinical trials of MMP inhibitors for other diseases, we discuss the need for carefully designed preclinical and clinical studies to avoid mistakes that have been previously made. We conclude that inhibition of MMPs, and in particular MMP-2, shows promise as a therapy to prevent the progression from ischemic injury to heart failure. However, it is critical that the full breadth of MMP-2 biology be taken into account as such therapies are developed.
Assuntos
Fármacos Cardiovasculares/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miocárdio/enzimologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Humanos , Terapia de Alvo Molecular , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Matrix metalloproteinase-2 (MMP-2) has been extensively studied in the context of extracellular matrix remodeling but is also localized within cells and can be activated by prooxidants to proteolyze specific intercellular targets. Although there are reports of MMP-2 in mitochondria, a critical source of cellular oxidative stress, these studies did not take into account the presence within their preparations of the mitochondria-associated membrane (MAM), a subdomain of the endoplasmic reticulum (ER). We hypothesized that MMP-2 is situated in the MAM and therefore investigated its subcellular distribution between mitochondria and the MAM. Immunogold electron microscopy revealed MMP-2 localized in mitochondria of heart sections from mice. In contrast, immunofluorescence analysis of an MMP-2:HaloTag fusion protein expressed in HL-1 cardiomyocytes showed an ER-like distribution, with greater colocalization with an ER marker (protein disulfide isomerase) relative to the mitochondrial marker, MitoTracker red. Although MMP-2 protein and enzymatic activity were present in crude mitochondrial fractions, once these were separated into purified mitochondria and MAM, MMP-2 was principally associated with the latter. Thus, although mitochondria may contain minimal levels of MMP-2, the majority of MMP-2 previously identified as "mitochondrial" is in fact associated with the MAM. We also found that calreticulin, an ER- and MAM-resident Ca(2+) handling protein and chaperone, could be proteolyzed by MMP-2 in vitro. MAM-localized MMP-2 could therefore potentially impact mitochondrial function by affecting ER-mitochondrial Ca(2+) signaling via its proteolysis of calreticulin.
Assuntos
Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Miócitos Cardíacos/enzimologia , Animais , Sinalização do Cálcio , Calreticulina/metabolismo , Linhagem Celular , Retículo Endoplasmático/ultraestrutura , Membranas Intracelulares/ultraestrutura , Masculino , Metaloproteinase 2 da Matriz/deficiência , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/enzimologia , Membranas Mitocondriais/enzimologia , Miócitos Cardíacos/ultraestrutura , Proteólise , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Impairments of various aspects of mitochondrial function have been associated with increased lifespan in various model organisms ranging from Caenorhabditis elegans to mice. For example, disruption of the function of the 'Rieske' iron-sulfur protein (RISP) of complex III of the mitochondrial electron transport chain can result in increased lifespan in the nematode worm C. elegans. However, the mechanisms by which impaired mitochondrial function affects aging remain under investigation, including whether or not they require decreased electron transport. We have generated knock-in mice with a loss-of-function Risp mutation that is homozygous lethal. However, heterozygotes (Risp(+/P224S)) were viable and had decreased levels of RISP protein and complex III enzymatic activity. This decrease was sufficient to impair mitochondrial respiration and to decrease overall metabolic rate in males, but not females. These defects did not appear to exert an overtly deleterious effect on the health of the mutants, since young Risp(+/P224S) mice are outwardly normal, with unaffected performance and fertility. Furthermore, biomarkers of oxidative stress were unaffected in both young and aged animals. Despite this, the average lifespan of male Risp(+/P224S) mice was shortened and aged Risp(+/P224S) males showed signs of more rapidly deteriorating health. In spite of these differences, analysis of Gompertz mortality parameters showed that Risp heterozygosity decreased the rate of increase of mortality with age and increased the intrinsic vulnerability to death in both sexes. However, the intrinsic vulnerability was increased more dramatically in males, which resulted in their shortened lifespan. For females, the slower acceleration of age-dependent mortality results in significantly increased survival of Risp(+/P224S) mice in the second half of lifespan. These results demonstrate that even relatively small perturbations of the mitochondrial electron transport chain can have significant physiological effects in mammals, and that the severity of those effects can be sex-dependent.
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Longevidade , Mitocôndrias/metabolismo , Caracteres Sexuais , Alelos , Animais , Biomarcadores/metabolismo , Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Fertilidade/genética , Técnicas de Introdução de Genes , Heterozigoto , Longevidade/genética , Longevidade/fisiologia , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/genética , Mutação PuntualRESUMO
BACKGROUND: Mice with a single copy of Mclk1 (a.k.a. Coq7), a gene that encodes a mitochondrial enzyme required for the biosynthesis of ubiquinone and other functions, live longer than wild-type mice. The prolonged survival implies a decreased mortality from age-dependent lethal pathologies. Atherosclerosis is one of the main age-dependent pathologies in humans and can be modeled in mice that lack Apolipoprotein E (ApoE-/-) or mice that lack the Low Density Lipoprotein Receptor (LDLr-/-) in addition to being fed an atherosclerosis-inducing diet. We sought to determine if Mclk1 heterozygosity protects against atherosclerosis and dyslipidemia in these models. RESULTS: We found that Mclk1 heterozygosity did not protect against dyslipidemia, oxidative stress, or atherosclerosis in young (6 or 10 months) or older (18 months) mice. Furthermore, the absence of ApoE suppressed the lifespan-promoting effects of Mclk1 heterozygosity. CONCLUSION: These findings indicate that although Mclk1 heterozygosity can extend lifespan of mice, it does not necessarily protect against atherosclerosis. Moreover, in the presence of hyperlipidemia and chronic inflammation, Mclk1 heterozygosity is incapable of extending lifespan.