RESUMO
The stressed right ventricle (RV) is particularly susceptible to producing and accumulating reactive oxygen species, leading to extracellular matrix deposition and secretion of natriuretic peptides. The role of specific enzymes with antioxidative capacity, like glutathione peroxidase 3 (GPx3), in RV pathogenesis is currently unknown. Here, we use a murine model of pulmonary artery banding (PAB) to study the role of GPx3 in isolated RV pathology. Compared with wild-type (WT) mice undergoing PAB surgery, GPx3-deficient PAB mice presented with higher RV systolic pressure and higher LV eccentricity indices. PAB-induced changes in Fulton's Index, RV free wall thickness, and RV fractional area change were more pronounced in GPx3-deficient mice compared with WT controls. Adverse RV remodeling was enhanced in GPx3-deficient PAB animals, evidenced by increased RV expression levels of connective tissue growth factor (CTGF), transforming growth factor-ß (TGF-ß), and atrial natriuretic peptide (ANP). In summary, GPx3 deficiency exacerbates maladaptive RV remodeling and causes signs of RV dysfunction.
Assuntos
Glutationa Peroxidase , Disfunção Ventricular Direita , Remodelação Ventricular , Animais , Camundongos , Ventrículos do Coração/patologia , Artéria Pulmonar/patologia , Fator de Crescimento Transformador beta/metabolismo , Função Ventricular Direita , Glutationa Peroxidase/metabolismoRESUMO
BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.
Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Bufanolídeos/farmacologia , Cardiomiopatias/prevenção & controle , Fármacos Cardiovasculares/farmacologia , Fibroblastos/efeitos dos fármacos , Fenantridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diástole , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Endogâmicos Dahl , Selenoproteína P/genética , Selenoproteína P/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
BACKGROUND: The arrangement of myofibers in the heart is highly complex and must be replicated by injected cells to produce functional myocardium. A novel approach to characterize the microstructural response of the myocardium to ischemia and cell therapy, with the use of serial diffusion tensor magnetic resonance imaging tractography of the heart in vivo, is presented. METHODS AND RESULTS: Validation of the approach was performed in normal (n=6) and infarcted mice (n=6) as well as healthy human volunteers. Mice (n=12) were then injected with bone marrow mononuclear cells 3 weeks after coronary ligation. In half of the mice the donor and recipient strains were identical, and in half the strains were different. A positive response to cell injection was defined by a decrease in mean diffusivity, an increase in fractional anisotropy, and the appearance of new myofiber tracts with the correct orientation. A positive response to bone marrow mononuclear cell injection was seen in 1 mouse. The response of the majority of mice to bone marrow mononuclear cell injection was neutral (9/12) or negative (2/12). The in vivo tractography findings were confirmed with histology. CONCLUSIONS: Diffusion tensor magnetic resonance imaging tractography was able to directly resolve the ability of injected cells to generate new myofiber tracts and provided a fundamental readout of their regenerative capacity. A highly novel and translatable approach to assess the efficacy of cell therapy in the heart is thus presented.
Assuntos
Transplante de Medula Óssea/métodos , Imagem de Tensor de Difusão/métodos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Isquemia Miocárdica/patologia , Isquemia Miocárdica/terapia , Animais , Anisotropia , Modelos Animais de Doenças , Voluntários Saudáveis , Imageamento Tridimensional/métodos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologiaRESUMO
Lipids are a diverse collection of macromolecules essential for normal physiology, but the tissue distribution and function for many individual lipid species remain unclear. Here, we report a mass spectrometry survey of lipid abundance across 18 mouse tissues, detecting ~1,000 mass spectrometry features, of which we identify 179 lipids from the glycerolipids, glycerophospholipids, lysophospholipids, acylcarnitines, sphingolipids, and cholesteryl ester classes. Our data reveal tissue-specific organization of lipids and can be used to generate testable hypotheses. For example, our data indicate that circulating triglycerides positively and negatively associated with future diabetes in humans are enriched in mouse adipose tissue and liver, respectively, raising hypotheses regarding the tissue origins of these diabetes-associated lipids. We also integrate our tissue lipid data with gene expression profiles to predict a number of substrates of lipid-metabolizing enzymes, highlighting choline phosphotransferases and sterol O-acyltransferases. Finally, we identify several tissue-specific lipids not present in plasma under normal conditions that may be of interest as biomarkers of tissue injury, and we show that two of these lipids are released into blood following ischemic brain injury in mice. This resource complements existing compendia of tissue gene expression and may be useful for integrative physiology and lipid biology.
Assuntos
Estruturas Animais/química , Metabolismo dos Lipídeos , Lipídeos/análise , Metaboloma , Adiposidade , Estruturas Animais/metabolismo , Animais , Cromatografia Líquida , Análise por Conglomerados , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Distribuição TecidualRESUMO
RATIONALE: High-sensitivity in vivo phenotyping of cardiac function is essential for evaluating genes of interest and novel therapies in small animal models of cardiovascular disease. Transthoracic echocardiography is the principal method currently used for assessing cardiac structure and function; however, standard echocardiographic techniques are relatively insensitive to early or subtle changes in cardiac performance, particularly in mice. OBJECTIVE: To develop and validate an echocardiographic strain imaging methodology for sensitive and rapid cardiac phenotyping in small animal models. METHODS AND RESULTS: Herein, we describe a modified echocardiographic technique that uses speckle-tracking based strain analysis for the noninvasive evaluation of cardiac performance in adult mice. This method is found to be rapid, reproducible, and highly sensitive in assessing both regional and global left ventricular (LV) function. Compared with conventional echocardiographic measures of LV structure and function, peak longitudinal strain and strain rate were able to detect changes in adult mouse hearts at an earlier time point following myocardial infarction and predicted the later development of adverse LV remodeling. Moreover, speckle-tracking based strain analysis was able to clearly identify subtle improvement in LV function that occurred early in response to standard post-myocardial infarction cardiac therapy. CONCLUSIONS: Our results highlight the utility of speckle-tracking based strain imaging for detecting discrete functional alterations in mouse models of cardiovascular disease in an efficient and comprehensive manner. Echocardiography speckle-tracking based strain analysis represents a method for relatively high-throughput and sensitive cardiac phenotyping, particularly in evaluating emerging cardiac agents and therapies in mice.
Assuntos
Ecocardiografia/métodos , Contração Miocárdica/fisiologia , Infarto do Miocárdio/diagnóstico por imagem , Fenótipo , Disfunção Ventricular Esquerda/diagnóstico por imagem , Função Ventricular Esquerda/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/fisiopatologia , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação Ventricular/fisiologiaRESUMO
AMP-activated protein kinase (AMPK) is a master metabolic switch that plays an important role in energy homeostasis at the cellular and whole body level, hence a promising drug target. AMPK is a heterotrimeric complex composed of catalytic α-subunit and regulatory ß- and γ-subunits with multiple isoforms for each subunit. It has been shown that AMPK activity is increased in cardiac hypertrophy and failure but it is unknown whether changes in subunit composition of AMPK contribute to the altered AMPK activity. In this study, we determined the protein expression pattern of AMPK subunit isoforms during cardiac development as well as during cardiac hypertrophy and heart failure in mouse heart. We also compared the findings in failing mouse heart to that of the human failing hearts in order to determine whether the mouse heart is a good model of AMPK in human diseases. In mouse developmental hearts, AMPK was highly expressed in the fetal stages and fell back to the adult level after birth. In the failing mouse heart, there was a significant increase in α2, ß2, and γ2 subunits both at the mRNA and protein levels. In contrary, we found significant increases in the protein level of α1, ß1 and γ2c subunits in human failing hearts with no change in the mRNA level. We also compared isoform-specific AMPK activity in the mouse and human failing hearts. Consistent with the literature, in the failing mouse heart, the α2 complexes accounted for ~2/3 of total AMPK activity while the α1 complexes accounted for the remaining 30-35%. In the human hearts, however, the contribution of α1-AMPK activity was significantly higher (>40%) in the non-failing hearts, and it further increased to 50% in the failing hearts. Thus, the human hearts have a greater amount of α1-AMPK activity compared to the rodent hearts. In summary, the protein level and the isoform distribution of AMPK in the heart change significantly during normal development as well as in heart failure. These observations provide a basis for future development of therapeutic strategies for targeting AMPK.
Assuntos
Adenilato Quinase/metabolismo , Expressão Gênica , Insuficiência Cardíaca/enzimologia , Adenilato Quinase/genética , Adulto , Idoso , Animais , Cardiomiopatia Dilatada/enzimologia , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Isquemia Miocárdica/enzimologia , Miocárdio/enzimologia , Adulto JovemRESUMO
BACKGROUND: The signaling mechanisms that regulate the recruitment of bone marrow (BM)-derived cells to the injured heart are not well known. Notch receptors mediate binary cell fate determination and may regulate the function of BM-derived cells. However, it is not known whether Notch1 signaling in BM-derived cells mediates cardiac repair after myocardial injury. METHODS AND RESULTS: Mice with postnatal cardiac-specific deletion of Notch1 exhibit infarct size and heart function after ischemic injury that is similar to that of control mice. However, mice with global hemizygous deletion of Notch1 (N1(±)) developed larger infarct size and worsening heart function. When the BM of N1(±) mice were transplanted into wild-type (WT) mice, infarct size and heart function were worsened and neovascularization in the infarct border area was reduced compared with WT mice transplanted with WT BM. In contrast, transplantation of WT BM into N1(±) mice lessened the myocardial injury observed in N1(±) mice. Indeed, hemizygous deletion of Notch1 in BM-derived cells leads to decreased recruitment, proliferation, and survival of mesenchymal stem cells (MSC). Compared with WT MSC, injection of N1(±) MSC into the infarcted heart leads to increased myocardial injury whereas injection of MSC overexpressing Notch intracellular domain leads to decreased infarct size and improved cardiac function. CONCLUSIONS: These findings indicate that Notch1 signaling in BM-derived cells is critical for cardiac repair and suggest that strategies that increase Notch1 signaling in BM-derived MSC could have therapeutic benefits in patients with ischemic heart disease.
Assuntos
Células da Medula Óssea/citologia , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Receptor Notch1/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de SinaisRESUMO
Tissue-specific progenitor cells contribute to local cellular regeneration and maintain organ function. Recently, we have determined that cardiac side-population (CSP) cells represent a distinct cardiac progenitor cell population, capable of in vitro differentiation into functional cardiomyocytes. The response of endogenous CSP to myocardial injury, however, and the cellular mechanisms that maintain this cardiac progenitor cell pool in vivo remain unknown. In this report we demonstrate that local progenitor cell proliferation maintains CSP under physiologic conditions, with little contribution from extracardiac stem cell sources. Following myocardial infarction in adult mice, however, CSP cells are acutely depleted, both within the infarct and noninfarct areas. CSP pools are subsequently reconstituted to baseline levels within 7 days after myocardial infarction, through both proliferation of resident CSP cells, as well as through homing of bone marrow-derived stem cells (BMC) to specific areas of myocardial injury and immunophenotypic conversion of BMC to adopt a CSP phenotype. We, therefore, conclude that following myocardial injury, cardiac progenitor cell populations are acutely depleted and are reconstituted to normal levels by both self-proliferation and selective homing of BMC. Understanding and enhancing such processes hold enormous potential for therapeutic myocardial regeneration.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Animais , Proliferação de Células , Antígenos Comuns de Leucócito/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/terapia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , RegeneraçãoRESUMO
OBJECTIVE: Female gender is associated with reduced tolerance against acute ischemic events and a higher degree of left ventricular hypertrophy under chronic pressure overload. We tested whether female and male rats with left ventricular hypertrophy present the same susceptibility to demand ischemia. METHODS: Hearts from hypertrophied female and male salt-resistant and salt-sensitive Dahl rats (n=8 per group) underwent 30min of demand ischemia induced by rapid pacing (7Hz) and an 85% reduction of basal coronary blood flow, followed by 30min of reperfusion on an isovolumic red cell perfused Langendorff model. RESULTS: In female hearts, high-salt diet induced a pronounced hypertrophy of the septum (2.38+/-0.09 vs 2.17+/-0.08mm; p<0.01), whereas male hearts showed the greatest increase in the anterior/posterior wall of the left ventricle (LV) (3.19+/-0.22 vs 2.01+/-0.16mm; p<0.05) compared with salt-resistant controls. At baseline, LV-developed pressure/g LV was significantly higher in female than male hearts (200+/-13 and 196+/-14 vs 161+/-10 and 152+/-15mmHgg(-1); p<0.01), independent of hypertrophy, indicating greater contractility in females. During ischemia, LV-developed pressure decreased in all groups; at the end of reperfusion, hypertrophied female and male hearts showed higher developed pressures independent of gender (148+/-3 and 130+/-8 vs 100+/-7 and 85+/-6mmHg; p<0.01). In contrast, diastolic pressure was more pronounced in female than in male hypertrophied hearts during ischemia and reperfusion (24+/-3 vs 12+/-2mmHg; p<0.01). CONCLUSIONS: In the pressure overload model of the Dahl salt-sensitive rat, female gender is associated with a more pronounced concentric hypertrophy, whereas male hearts develop a more eccentric type of remodeling. Although present at baseline, after ischemia/reperfusion systolic function is gender-independent but more determined by hypertrophy. In contrast, diastolic function is gender-dependent and aggravated by hypertrophy, leading to pronounced diastolic dysfunction. We can conclude that in the malignant setting of demand ischemia/reperfusion gender differences in hypertrophied hearts are unmasked: female hypertrophied hearts are more susceptible to ischemia/reperfusion than males. To determine whether in female hypertensive patients with acute coronary syndromes, diastolic dysfunction could contribute to the worse clinical course, further experimental and clinical studies are needed.
Assuntos
Hipertensão/complicações , Hipertrofia Ventricular Esquerda/etiologia , Traumatismo por Reperfusão Miocárdica/etiologia , Caracteres Sexuais , Animais , Circulação Coronária , Diástole , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Ácido Láctico/biossíntese , Masculino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ratos , Ratos Sprague-Dawley , SístoleRESUMO
Extracellular nucleic acids are proinflammatory molecules that have been implicated in a diverse range of diseases. We report here the development of a multivalent nucleic acid scavenging nanoprobe, where the fluorochrome thiazole orange (TO) is conjugated to a polymeric 40 kDa dextran carrier. Dextran-TO (Dex-TO) has nanomolar affinity for mammalian and bacterial nucleic acids and attenuates the production of inflammatory cytokines from activated macrophages exposed to DNA and RNA. Mice with myocardial ischemia reperfusion that were treated with Dex-TO showed a decrease in myocardial macrophage infiltration at 24 hours (p<0.05) and a decrease in infarct size (18% ± 9%, p<0.01) on day 7. Dex-TO allows sites of injury to be identified with fluorescence imaging, while simultaneously exerting an anti-inflammatory and cytoprotective effect. Dex-TO could be of significant diagnostic and therapeutic (theranostic) utility in a broad range of conditions including ischemia, trauma, burns, sepsis and autoimmune disease.
Assuntos
Anti-Inflamatórios/administração & dosagem , Citoproteção , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Nanoestruturas/administração & dosagem , Ácidos Nucleicos/metabolismo , Animais , Benzotiazóis/administração & dosagem , Dextranos/administração & dosagem , Camundongos , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/patologia , Imagem Óptica , Quinolinas/administração & dosagem , Nanomedicina Teranóstica/métodos , Resultado do TratamentoRESUMO
BACKGROUND: Increased rates of glucose uptake and glycolysis have been repeatedly observed in cardiac hypertrophy and failure. Although these changes have been considered part of the fetal gene reactivation program, the functional significance of increased glucose utilization in hypertrophied and failing myocardium is poorly understood. METHODS AND RESULTS: We generated transgenic (TG) mice with cardiac-specific overexpression of insulin-independent glucose transporter GLUT1 to recapitulate the increases in basal glucose uptake rate observed in hypertrophied hearts. Isolated perfused TG hearts showed a greater rate of basal glucose uptake and glycolysis than hearts isolated from wild-type littermates, which persisted after pressure overload by ascending aortic constriction (AAC). The in vivo cardiac function in TG mice, assessed by echocardiography, was unaltered. When subjected to AAC, wild-type mice exhibited a progressive decline in left ventricular (LV) fractional shortening accompanied by ventricular dilation and decreased phosphocreatine to ATP ratio and reached a mortality rate of 40% at 8 weeks. In contrast, TG-AAC mice maintained LV function and phosphocreatine to ATP ratio and had <10% mortality. CONCLUSIONS: We found that increasing insulin-independent glucose uptake and glycolysis in adult hearts does not compromise cardiac function. Furthermore, we demonstrate that increasing glucose utilization in hypertrophied hearts protects against contractile dysfunction and LV dilation after chronic pressure overload.
Assuntos
Insuficiência Cardíaca/prevenção & controle , Proteínas de Transporte de Monossacarídeos/genética , Miocárdio/metabolismo , Trifosfato de Adenosina/análise , Animais , Aorta , Transporte Biológico , Constrição , Ecocardiografia , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Hipertrofia Ventricular Esquerda/complicações , Camundongos , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/metabolismo , Contração Miocárdica , Miocárdio/patologia , Técnicas de Cultura de Órgãos , Fosfocreatina/análise , Pressão , Análise de Sobrevida , Remodelação VentricularRESUMO
Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure(1-3). Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction(4). To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential. A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software. Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC. Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.
Assuntos
Arteriopatias Oclusivas/diagnóstico por imagem , Arteriopatias Oclusivas/fisiopatologia , Ecocardiografia/métodos , Coração/fisiopatologia , Artéria Pulmonar/fisiopatologia , Animais , Modelos Animais de Doenças , Coração/anatomia & histologia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Função Ventricular DireitaRESUMO
BACKGROUND: Autophagy is a biological process during which cells digest organelles in their cytoplasm and recycle the constituents. The impact of autophagy in the heart, however, remains unclear in part because of the inability to noninvasively image this process in living animals. METHODS AND RESULTS: Here, we report the use of fluorescence molecular tomography and a cathepsin-activatable fluorochrome to image autophagy in the heart in vivo after ischemia/reperfusion and rapamycin (RAP) therapy. We show that cathepsin-B activity in the lysosome is upregulated by RAP and that this allows the expanded lysosomal compartment in autophagy to be imaged in vivo with fluorescence molecular tomography. We further demonstrate that the delivery of diagnostic nanoparticles to the lysosome by endocytosis is enhanced during autophagy. The upregulation of autophagy by RAP was associated with a 23% reduction (P<0.05) of apoptosis in the area at risk and a 45% reduction in final infarct size (19.6±5.6% of area at risk with RAP versus 35.9±9.1% of area at risk without RAP; P<0.05). CONCLUSIONS: The ability to perform noninvasive tomographic imaging of autophagy in the heart has the potential to provide valuable insights into the pathophysiology of autophagy, particularly its role in cardiomyocyte salvage. Although additional data are needed, our study supports the investigation of RAP therapy in patients with acute coronary syndromes.
Assuntos
Autofagia/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Imagem Óptica/métodos , Sirolimo/farmacologia , Tomografia/métodos , Animais , Apoptose/efeitos dos fármacos , Catepsina B/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Corantes Fluorescentes , Pós-Condicionamento Isquêmico , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microesferas , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Cintilografia , Fatores de Tempo , Microtomografia por Raio-XRESUMO
BACKGROUND: Current techniques to image cell death in the myocardium are largely nonspecific. We report the use of a novel DNA-binding gadolinium chelate (Gd-TO) to specifically detect the exposed DNA in acutely necrotic (ruptured) cells in vivo. METHODS AND RESULTS: In vivo MRI was performed in 20 mice with myocardial infarction (MI). The mice were injected with Gd-TO or Gd-DTPA at varying time points after MI. MRI was performed 2 hours after probe injection, to avoid nonspecific signal from the late gadolinium enhancement effect. Cell rupture (Gd-TO uptake) was present within 2 hours of infarction but peaked 9 to 18 hours after the onset of injury. A significant increase in the longitudinal relaxation rate (R(1)) in the infarct was seen in mice injected with Gd-TO within 48 hours of MI, but not in those injected more than 72 hours after MI (R(1)=1.24±0.08 and 0.92±0.03 s(-1), respectively, P<0.001). Gd-DTPA, unlike Gd-TO, washed completely out of acute infarcts within 2 hours of injection (P<0.001). The binding of Gd-TO to exposed DNA in acute infarcts was confirmed with fluorescence microscopy. CONCLUSIONS: Gd-TO specifically binds to acutely necrotic cells and can be used to image the mechanism and chronicity of cell death in injured myocardium. Cell rupture in acute MI begins early but peaks many hours after the onset of injury. The ruptured cells are efficiently cleared by the immune system and are no longer present in the myocardium 72 hours after injury.
Assuntos
Gadolínio DTPA , Imageamento por Ressonância Magnética/métodos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Doença Aguda , Análise de Variância , Animais , Sítios de Ligação , Morte Celular/efeitos dos fármacos , DNA/metabolismo , Modelos Animais de Doenças , Gadolínio DTPA/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Biologia Molecular , Miócitos Cardíacos/fisiologia , Necrose/patologiaRESUMO
Progressive cardiac remodeling is characterized by subsequent chamber hypertrophy, enlargement, and pump dysfunction. It is also associated with increased cardiac fibrosis and matrix turnover. Interestingly, peroxisome proliferator-activated receptor (PPAR) alpha activators reduce cardiac hypertrophy, inflammation, and fibrosis. Little is known about the role of fenofibrates in mediating PPARalpha-independent effects in response to chronic pressure overload (PO). Wild-type and PPARalpha-deficient mice were subjected to chronic PO caused by ascending aortic constriction to test the role of fenofibrates in chronic, progressive cardiac remodeling by a PPARalpha-independent mechanism. Mice were randomized to regular chow or chow-containing fenofibrate (100 mg/kg of body weight per day) for 1 week before and 8 weeks after ascending aortic constriction. In the presence of PPARalpha, wild-type chronic PO mice, treated with fenofibrate, had improved cardiac remodeling. However, PO PPARalpha-deficient mice treated with fenofibrate had increased mortality, significantly adverse left ventricular end diastolic (3.4+/-0.1 versus 4.2+/-0.1 mm) and end systolic (1.5+/-0.2 versus 2.5+/-0.2 mm) dimensions, and fractional shortening (57+/-3% versus 40+/-3%). Fenofibrate also increased myocardial hypertrophy, cardiac fibrosis, and the ratio of matrix metalloproteinase-2/tissue inhibitor of matrix metalloproteinase-2 in PO PPARalpha-deficient mice. Fenofibrate inhibited matrix metalloproteinase activity in vitro and aldosterone-induced increases in extracellular signal-regulated kinase phosphorylation. Thus, fenofibrate improved cardiac remodeling in chronic PO mice. However, in PPARalpha-deficient mice, this chronic PO was exacerbated and associated with increased myocardial fibrosis and altered matrix remodeling. In the absence of PPARalpha, fenofibrates exerts deleterious, pleiotropic myocardial actions. This is an important observation, because PPARalpha agonists are considered possible inhibitory regulators of cardiac remodeling in the remodeled heart.
Assuntos
Fenofibrato/farmacologia , Hipertensão/fisiopatologia , Hipolipemiantes/farmacologia , Miocárdio/patologia , PPAR alfa/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Aldosterona/farmacologia , Animais , Células Cultivadas , Doença Crônica , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose , Hipertensão/mortalidade , Hipertensão/patologia , Estimativa de Kaplan-Meier , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Endogâmicos , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , PPAR alfa/deficiência , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fosforilação/efeitos dos fármacos , Fatores de Tempo , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
After myocardial infarction (MI), the left ventricle (LV) undergoes ventricular remodeling characterized by progressive global dilation, infarct expansion, and compensatory hypertrophy of the noninfarcted myocardium. Little attention has been given to the response of remodeling myocardium to additional hemodynamic overload. Studies have indicated that gender may influence remodeling and the response to both MI and hemodynamic overload. We therefore determined 1) structural and function consequences of superimposing hemodynamic overload (systemic hypertension) on remodeling myocardium after a MI and 2) the potential influence of gender on this remodeling response. Male and female Dahl salt-sensitive and salt-resistant rats underwent coronary ligation, resulting in similar degrees of MI. One week post-MI, all rats were placed on a high-salt diet. Four groups were then studied 4 wk after initiation of high-salt feeding: MI female, MI female + hypertension, MI male, and MI male + hypertension. Hypertension-induced pressure overload resulted in additional comparable degrees of myocardial hypertrophy in both females and males. In females, hypertension post-MI resulted in concentric hypertrophy with no additional cavity dilation and no measurable scar thinning. In contrast, in males, hypertension post-MI resulted in eccentric hypertrophy, further LV cavity dilation, and scar thinning. Physiologically, concentric hypertrophy in post-MI hypertensive females resulted in elevated contractile function, whereas eccentrically hypertrophied males had no such increase. Female gender influences favorably the remodeling and physiological response to hemodynamic overload after large MI.