RESUMEN
AIMS: We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(/) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(/) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS: HypoE/SR-BI(/) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS: Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.
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Apolipoproteínas E/deficiencia , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Clorhidrato de Fingolimod/uso terapéutico , Inmunosupresores/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Receptores Depuradores de Clase B/biosíntesis , Animales , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/mortalidad , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , Infarto del Miocardio/metabolismo , Infarto del Miocardio/mortalidad , Tasa de Supervivencia/tendenciasRESUMEN
FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61(h/h)/SRB1(-/- mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg(-1)·d(-1)) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61(h/h)/SRB1(-/-) mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg(-1)·d(-1)). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-ß and reduced IFN-γ expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-ß and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.
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Apolipoproteínas E/genética , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Infarto del Miocardio/tratamiento farmacológico , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Animales , Cardiotónicos/administración & dosificación , Cardiotónicos/farmacología , Enfermedad de la Arteria Coronaria/fisiopatología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacología , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , Glicoles de Propileno/administración & dosificación , Esfingosina/administración & dosificación , Esfingosina/farmacología , Tasa de Supervivencia , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Exposure to second hand smoke (SHS) is believed to cause lung cancer. Pathological angiogenesis is a requisite for tumor growth. Lewis lung cancer cells were injected subcutaneously into mice, which were then exposed to sidestream smoke (SHS) or clean room air and administered vehicle, cerivastatin, or mecamylamine. SHS significantly increased tumor size, weight, capillary density, VEGF and MCP-1 levels, and circulating endothelial progenitor cells (EPC). Cerivastatin (an inhibitor of HMG-coA reductase) or mecamylamine (an inhibitor of nicotinic acetylcholine receptors) suppressed the effect of SHS to increase tumor size and capillary density. Cerivastatin reduced MCP-1 levels, whereas mecamylamine reduced VEGF levels and EPC. These studies reveal that SHS promotes tumor angiogenesis and growth. These effects of SHS are associated with increases in plasma VEGF and MCP-1 levels, and EPC, mediated in part by isoprenylation and nicotinic acetylcholine receptors.
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Carcinoma Pulmonar de Lewis/etiología , Neovascularización Patológica/etiología , Nicotina/toxicidad , Contaminación por Humo de Tabaco/efectos adversos , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Quimiocina CCL2/metabolismo , Células Endoteliales/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Mecamilamina/metabolismo , Ratones , Piridinas/metabolismo , Receptores Nicotínicos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro- and anti-apoptotic MAP kinase signaling in cardiac myocyte (CM) and non-myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post-MI remodeling involved dramatic changes in the phosphorylation of both stress-activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti-apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p-ERK localized to fibrotic areas populated primarily by non-myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non-myocytes in different regions of the post-MI myocardium revealed signaling patterns that imply cell type-specific changes in pro- and anti-apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type-specific manipulation of p38 and ERK activation.
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Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis , Western Blotting , Cardiomiopatías/metabolismo , Células Cultivadas , Ecocardiografía , Ensayo de Inmunoadsorción Enzimática , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Epinephrine (EPI) is an important neurotransmitter and hormone. Its role in regulating cardiovascular function at rest and with stress is unclear, however. METHODS AND RESULTS: An epinephrine-deficient mouse model was generated in which the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase was knocked out (KO). Blood pressure and heart rate were monitored by telemetry at rest and during graded treadmill exercise. Cardiac structure and function were evaluated by echocardiography in mice under 1 of 2 conditions: unstressed and lightly anesthetized or restrained and awake. In KO mice, resting cardiovascular function, including blood pressure, heart rate, and cardiac output, was the same as that in wild-type mice, and the basal norepinephrine plasma level was normal. However, inhibition of sympathetic innervation with the ganglion blocker hexamethonium caused a 54% smaller decrease in blood pressure in KO mice, and treadmill exercise caused an 11% higher increase in blood pressure, both suggesting impaired vasodilation in KO mice. Interestingly, phenylethanolamine N-methyltransferase KO did not change the heart rate response to ganglionic blockade and exercise. By echocardiography, KO mice had an increased ratio of left ventricular posterior wall thickness to internal dimensions but did not have cardiac hypertrophy, suggesting concentric remodeling in the KO heart. Finally, in restrained, awake KO mice, heart rate and ejection fraction remained normal, but cardiac output was significantly reduced because of diminished end-diastolic volume. CONCLUSION: Our data suggest that epinephrine is required for normal blood pressure and cardiac filling responses to stress but is not required for tachycardia during stress or normal cardiovascular function at rest.
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Presión Sanguínea/fisiología , Fenómenos Fisiológicos Cardiovasculares , Epinefrina/uso terapéutico , Frecuencia Cardíaca/fisiología , Feniletanolamina N-Metiltransferasa/deficiencia , Estrés Fisiológico , Animales , Mapeo Cromosómico , Clonación Molecular , Prueba de Esfuerzo , Biblioteca Genómica , Genotipo , Pruebas de Función Cardíaca , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , TaquicardiaRESUMEN
The cardioprotective effectiveness of low-dose pyrroloquinoline quinone (PQQ, 3 mg/kg) was compared with metoprolol, a beta(1)-selective adrenoceptor antagonist. Rats underwent 30 minutes of left anterior descending coronary artery occlusion and 2 hours of reperfusion. Metoprolol and/or PQQ were given at the onset of reperfusion to mimic clinical treatment. Metoprolol and/or PQQ reduced infarct size and protected against ischemia-induced left ventricular dysfunction after 2 hours of reperfusion. Combined therapy augmented left ventricular developed pressure at the end of reperfusion. Metoprolol or PQQ alone enhanced mitochondrial respiratory ratios in ischemic and nonischemic myocardium. Although the PQQ/metoprolol combination therapy increased respiratory ratio values, the effects were small when compared with PQQ alone. Only PQQ decreased lipid peroxidation. Metoprolol and/or PQQ given at the onset of reperfusion reduce infarct size and improve cardiac function. Combination therapy further reduces infarct size. PQQ is superior to metoprolol in protecting mitochondria from ischemia/reperfusion oxidative damage.
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Antagonistas Adrenérgicos beta/uso terapéutico , Cardiotónicos/uso terapéutico , Metoprolol/uso terapéutico , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/patología , Cofactor PQQ/uso terapéutico , Antagonistas Adrenérgicos beta/administración & dosificación , Animales , Antioxidantes/farmacología , Peso Corporal/efectos de los fármacos , Cardiotónicos/administración & dosificación , Creatina Quinasa/sangre , Interacciones Farmacológicas , Hemodinámica/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Metoprolol/administración & dosificación , Mitocondrias Cardíacas/efectos de los fármacos , Infarto del Miocardio/mortalidad , Daño por Reperfusión Miocárdica/mortalidad , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Cofactor PQQ/administración & dosificación , Ratas , Ratas Sprague-Dawley , Fibrilación Ventricular/patologíaRESUMEN
OBJECTIVES: We previously showed that an angiotensin-converting enzyme inhibitor (captopril) or an angiotensin receptor blocker (losartan) reduced infarct size and improved endothelial function in a rat model of ischaemia-reperfusion. The present study was undertaken to see if aspirin (ASA) antagonised the beneficial effects of captopril or losartan. METHODS: One hundred and fourteen Sprague-Dawley rats were randomised into six groups; Control, ASA, captopril, losartan, ASA+captopril, and ASA+losartan. ASA, captopril or losartan were given at a concentration of 40 mg/kg/day in drinking water. After six weeks of pre-treatment, the rats were subjected to 17 minutes of left anterior descending coronary artery occlusion and 120 minutes of reperfusion, with haemodynamic and ECG monitoring. During the reperfusion period, the effective refractory period (ERP), ventricular fibrillation threshold (VFT) and bleeding time (BT) were measured. In fresh aortic rings precontracted with phenylephrine, endothelium-dependent and -independent relaxations were assessed using acetylcholine and nitroglycerin. RESULTS: Haemodynamic changes were not different between the groups. Serum ASA concentrations were 0.5, 1.1 and 0.6 mg/dl in the ASA, ASA+captopril and ASA+losartan groups, respectively, and BT was prolonged (p<0.01). ASA alone reduced endothelium-dependent relaxation (-29+8 vs. -69+11%, p<0.01), but did not change endothelium-independent relaxation. ASA did not affect endothelial relaxation induced by acetylcholine in the presence of either captopril or losartan. Angiotensin I and ERP were elevated by captopril and losartan. Angiotensin II and VFT were elevated by losartan. ASA with captopril, captopril and losartan equally reduced infarct size, compared with control (39+3, 39+4, and 39+5 vs. 53+3%, all p<0.05). CONCLUSIONS: Captopril and losartan had similar cardiovascular protective effects in a rat model of ischaemia-reperfusion. Aspirin did not attenuate the cardiovascular protective effects of captopril or losartan.
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Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Captopril/farmacología , Infarto del Miocardio/tratamiento farmacológico , Angiotensina I/sangre , Angiotensina II/sangre , Antagonistas de Receptores de Angiotensina , Animales , Antiarrítmicos/farmacología , Antiinflamatorios no Esteroideos/sangre , Aspirina/sangre , Tiempo de Sangría , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Femenino , Hemodinámica , Losartán/farmacología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Ratas , Ratas Sprague-Dawley , Periodo Refractario Electrofisiológico/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Fibrilación Ventricular/tratamiento farmacológico , Fibrilación Ventricular/patologíaRESUMEN
INTRODUCTION: Both second hand smoke (SHS) and the renin-angiotensin system (RAS) contribute to endothelial dysfunction and increased infarct size in a rat ischaemia-reperfusion model. However, the potential interaction between SHS and the RAS is unknown. METHODS: Eighty-four rats were randomised into four groups: group C was a normal control; L was given 40 mg/kg/day of losartan in drinking water; SC and SL were exposed to SHS (smoking chamber) and given regular water or 40 mg/kg/day of losartan in drinking water, respectively. After six weeks of pre-treatment, rats were subjected to 17 minutes of left coronary artery occlusion and 2 hours of reperfusion with haemodynamic and ECG monitoring. RESULTS: Haemodynamics were not significantly different among the four groups. Losartan increased the threshold for ventricular fibrillation (p=0.0001) and reduced spontaneous ventricular arrhythmias (p=0.002) during ischaemia-reperfusion, while SHS did not (p=0.713, 0.110), and there was no interaction between losartan and SHS. The maximal endothelium-dependent vasorelaxation induced by a calcium ionophore (A23187) was increased by losartan (p=0.007). Myocardial infarct size was smaller in the losartan groups (p=0.032), larger in the SHS groups (p=0.0001), and there was no significant interaction. CONCLUSION: In conclusion, losartan decreased infarct size and increased endothelium-dependent vasorelaxation. SHS exposure impaired endothelial function and increased infarct size. The effects of losartan and SHS were consistently independent of each other. These results suggest that the RAS does not contribute to the adverse effects of SHS.
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Endotelio Vascular/fisiopatología , Infarto del Miocardio/fisiopatología , Sistema Renina-Angiotensina/fisiología , Contaminación por Humo de Tabaco/efectos adversos , Angiotensina II/sangre , Animales , Antihipertensivos/farmacología , Arritmias Cardíacas/fisiopatología , Electrocardiografía/efectos de los fármacos , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/patología , Femenino , Hemodinámica/fisiología , Losartán/farmacología , Linfocinas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Infarto del Miocardio/patología , Nicotina/sangre , Ratas , Ratas Sprague-Dawley , Periodo Refractario Electrofisiológico/efectos de los fármacos , Renina/sangre , Sistema Renina-Angiotensina/efectos de los fármacos , Daño por Reperfusión/fisiopatología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Fibrilación Ventricular/fisiopatologíaRESUMEN
Matrix metalloproteinase-2 (MMP-2) is increasingly recognized as a major contributor to progressive cardiac injury within the setting of ischemia-reperfusion injury and ischemic ventricular remodeling. A common feature of these conditions is an increase in oxidative stress, a process that engages multiple pro-inflammatory and innate immunity cascades. We recently reported on the identification and characterization of an intracellular isoform of MMP-2 generated by oxidative stress-mediated activation of an alternative promoter located within the first intron of the MMP-2 gene. Transcription from this site generates an N-terminal truncated 65 kDa isoform of MMP-2 (NTT-MMP-2) that lacks the secretory sequence and the inhibitory prodomain region. The NTT-MMP-2 isoform is intracellular, enzymatically active and localizes in part to mitochondria. Expression of the NTT-MMP-2 isoform triggers Nuclear Factor of Activated T-cell (NFAT) and NF-κB signaling with the expression of a highly defined innate immunity transcriptome, including Interleukin-6, MCP-1, IRF-7 and pro-apoptotic transcripts. To determine the functional significance of the NTT-MMP-2 isoform in vivo we generated cardiac-specific NTT-MMP-2 transgenic mice. These mice developed progressive cardiomyocyte and ventricular hypertrophy associated with systolic heart failure. Further, there was evidence for cardiomyocyte apoptosis and myocardial infiltration with mononuclear cells. The NTT-MMP-2 transgenic hearts also demonstrated more severe injury following ex vivo ischemia-reperfusion injury. We conclude that a novel intracellular MMP-2 isoform induced by oxidant stress directly contributes, in the absence of superimposed injury, to cardiomyocyte hypertrophy. inflammation, systolic heart failure and enhanced susceptibility to ischemia-reperfusion injury.
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Insuficiencia Cardíaca Sistólica/enzimología , Inflamación/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Miocitos Cardíacos/patología , Animales , Insuficiencia Cardíaca Sistólica/genética , Inflamación/genética , Factor 7 Regulador del Interferón/metabolismo , Interleucina-6/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Transgénicos , Isoformas de Proteínas/metabolismoRESUMEN
INTRODUCTION: Myxomatous mitral valve "degeneration" with prolapse (MVP) is the most frequent form of nonischemic mitral valve disease. In myxomatous valves, interstitial cells express extracellular matrix-degrading enzymes and it has been postulated that matrix metalloproteinases (MMPs) contribute to these changes. METHODS: We generated mice with cardiac-specific expression of constitutively active MMP-2 under the control of the alpha-myosin heavy chain promoter. RESULTS: These mice are normal at 4-6 months of age; at 12-14 months the mitral valves and chordae tendineae exhibit severe myxomatous change with echocardiographic MVP. Myxomatous change was also evident to a lesser extent in the aortic valves. Myxomatous changes were heterogeneous and limited to the left side of the heart with major disorganization of collagen bundles within the lamina fibrosa. Alcian blue/PAS-stained valves revealed massive accumulation of acidic glycosoaminoglycans within the lamina spongiosa, consistent with valvular interstitial cell differentiation to a chondrocytic phenotype. Cells with the histologic features of hypertrophied chondrocytes were found within the chordae tendineae and the tips of the mitral papillary muscles. CONCLUSION: This report demonstrates that increased activity of a single enzyme, MMP-2, within a transgenic context reproduces many of the features of the human MVP syndrome. The cardiac-specific MMP-2 transgenic mouse potentially provides a unique experimental platform for the evaluation of nonsurgical therapies based on the underlying pathophysiology of this disease.
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Modelos Animales de Enfermedad , Metaloproteinasa 2 de la Matriz/biosíntesis , Prolapso de la Válvula Mitral/enzimología , Prolapso de la Válvula Mitral/genética , Animales , Ecocardiografía , Humanos , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Transgénicos , Válvula Mitral/patología , Prolapso de la Válvula Mitral/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway, known to determine the fate and growth of various cell types, can enhance cardiac myocyte survival in vitro and provide cardioprotection in acute ex vivo heart preparations. However, the relevance of these findings to chronic cardiac pathology has never been demonstrated. We hypothesized that S1P signaling is impaired during chronic remodeling of the uninfarcted ventricle during the evolution of post-myocardial infarction (MI) cardiomyopathy and that a therapeutic enhancement of S1P signaling would ameliorate ventricular dysfunction. SphK expression and activity were measured in the remote, uninfarcted myocardium (RM) of C57Bl/6 mice subjected to coronary artery ligation. The mRNA expression of S1P receptor isoforms was also measured, as was the activation of the downstream S1P receptor mediators. A cardioprotective role for S1P(1) receptor agonism was tested via the administration of the S1P(1)-selective agonist SEW2871 during and after MI. As a result, the expression data suggested that a dramatic reduction in SphK activity in the RM early after MI may reflect a combination of posttranscriptional and posttranslational modulation. SphK activity continued to decline gradually during chronic post-MI remodeling, when S1P(1) receptor mRNA also fell below baseline. The S1P(1)-specific agonism with oral SEW2871 during the first 2-wk after MI reduced apoptosis in the RM and resulted in improved myocardial function, as reflected in the echocardiographic measurement of fractional shortening. In conclusion, these results provide the first documentation of alterations in S1P-mediated signaling during the in situ development of cardiomyopathy and suggest a possible therapeutic role for the pharmacological S1P receptor agonism in the post-MI heart.
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Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Transducción de Señal/fisiología , Esfingolípidos/metabolismo , Remodelación Ventricular/fisiología , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oxadiazoles/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Mensajero/metabolismo , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/metabolismo , Tiofenos/farmacologíaRESUMEN
BACKGROUND: Experimental human stem cell transplantation to the heart has begun, but the mechanisms underlying benefits seen in preclinical models, both at the site of cell injection and at more distant regions, remain uncertain. We hypothesize that these benefits can be best understood first at the level of key intracellular signaling cascades in the host myocardium, which can be responsible for functional and structural preservation of the heart. STUDY DESIGN: Western blot and ELISA were used to assess key pathways that regulate cardiac myocyte survival and hypertrophy in both the infarct/borderzone and remote myocardium of C57/B6 mouse hearts subjected to coronary artery ligation, with subsequent injection of either vehicle or bone marrow-derived adult mesenchymal stem cells (MSC). RESULTS: Improved left ventricular function with MSC transplantation was associated with a relative preservation of Akt phosphorylation (activation) and of phosphorylation of downstream mediators of cell survival and hypertrophy. There was no substantial difference in activation of mitogen-activated protein kinase p38, and activation of the antiapoptotic mitogen-activated protein kinase extracellular signal-regulated kinase was lower at 1 week after MSC treatment, but rose beyond controls by week 2. Similar changes were observed in both the infarct/borderzone and the remote myocardium. CONCLUSION: Stem cell transplantation in the post-MI murine myocardium is associated with preservation of Akt signaling. Together with a possible later increase in extracellular signal-regulated kinase activation, this signaling change might be responsible for cardioprotection. Additional focused investigation might identify elements in transplantation regimens that optimize this mechanism of benefit, and that can increase the likelihood of human clinical success.
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Trasplante de Células Madre Mesenquimatosas , Proteínas Quinasas Activadas por Mitógenos/fisiología , Miocardio/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Función Ventricular Izquierda , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Increased signaling by G(i)-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted G(i)-coupled receptor (Ro1). Activation of G(i) signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist nor-binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum Ca(2+)-ATPase (P < 0.05); thereafter, there was progressive impairment of both Ca(2+) handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic Ca(2+) concentration was reduced to 0.61 +/- 0.08 vs. 0.91 +/- 0.07 microM for control (n = 6-8; P < 0.05), diastolic Ca(2+) concentration was elevated to 0.41 +/- 0.07 vs. 0.23 +/- 0.06 microM for control (n = 6-8; P < 0.01), and the decline phase of the Ca(2+) transient (time from peak to 50% decline) was slowed to 0.25 +/- 0.02 s vs. 0.13 +/- 0.02 s for control (n = 6-8; P < 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 (P < 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, Ca(2+)-saturated myofilament force in skinned trabeculae was reduced to 21 +/- 2 vs. 38 +/- 0.1 mN/mm(2) for controls (n = 3; P < 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of G(i)-coupled receptors leads to impaired Ca(2+) handling and myofilament injury, contributing to impaired ventricular pump function and heart failure.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Miocardio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides kappa/metabolismo , Transducción de Señal , Citoesqueleto de Actina/ultraestructura , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , AMP Cíclico/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Contracción Miocárdica , Miocardio/enzimología , Miocardio/ultraestructura , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Pirrolidinas/farmacología , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides kappa/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Función Ventricular IzquierdaRESUMEN
Matrix metalloproteinases (MMPs) are central to the development and progression of dysfunctional ventricular remodeling after tissue injury. We studied 6 month old heterozygous mice with cardiac-specific transgenic expression of active MMP-2 (MMP-2 Tg). MMP-2 Tg hearts showed no substantial gross alteration of cardiac phenotype compared to age-matched wild-type littermates. However, buffer perfused MMP-2 Tg hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion had a larger infarct size and greater depression in contractile performance compared to wild-type hearts. Importantly, cardioprotection mediated by ischemic preconditioning (IPC) was completely abolished in MMP-2 Tg hearts, as shown by abnormalities in mitochondrial ultrastructure and impaired respiration, increased lipid peroxidation, cell necrosis and persistently reduced recovery of contractile performance during post-ischemic reperfusion. We conclude that MMP-2 functions not only as a proteolytic enzyme but also as a previously unrecognized active negative regulator of mitochondrial function during superimposed oxidative stress.
Asunto(s)
Metaloproteinasa 2 de la Matriz/biosíntesis , Mitocondrias Cardíacas/fisiología , Miocardio/enzimología , Animales , Creatina Quinasa/metabolismo , Heterocigoto , Precondicionamiento Isquémico Miocárdico , Peroxidación de Lípido , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Transgénicos , Mitocondrias Cardíacas/enzimología , Contracción Miocárdica , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Miocardio/ultraestructura , NecrosisRESUMEN
Although enhanced cardiac matrix metalloproteinase (MMP)-2 synthesis has been associated with ventricular remodeling and failure, whether MMP-2 expression is a direct mediator of this process is unknown. We generated transgenic mice expressing active MMP-2 driven by the alpha-myosin heavy chain promoter. At 4 mo MMP-2 transgenic hearts demonstrated expression of the MMP-2 transgene, myocyte hypertrophy, breakdown of Z-band registration, lysis of myofilaments, disruption of sarcomere and mitochondrial architecture, and cardiac fibroblast proliferation. Hearts from 8-mo-old transgenic mice displayed extensive myocyte disorganization and dropout with replacement fibrosis and perivascular fibrosis. Older transgenic mice also exhibited a massive increase in cardiac MMP-2 expression, representing recruitment of endogenous MMP-2 synthesis, with associated expression of MMP-9 and membrane type 1 MMP. Increases in diastolic [control (C) 33 +/- 3 vs. MMP 51 +/- 12 microl; P = 0.003] and systolic (C 7 +/- 2 vs. MMP 28 +/- 14 microl; P = 0.003) left ventricular (LV) volumes and relatively preserved stroke volume (C 26 +/- 4 vs. MMP 23 +/- 3 microl; P = 0.16) resulted in markedly decreased LV ejection fraction (C 78 +/- 7% vs. MMP 48 +/- 16%; P = 0.0006). Markedly impaired systolic function in the MMP transgenic mice was demonstrated in the reduced preload-adjusted maximal power (C 240 +/- 84 vs. MMP 78 +/- 49 mW/microl(2); P = 0.0003) and decreased end-systolic pressure-volume relation (C 7.5 +/- 1.5 vs. MMP 4.7 +/- 2.0; P = 0.016). Expression of active MMP-2 is sufficient to induce severe ventricular remodeling and systolic dysfunction in the absence of superimposed injury.
Asunto(s)
Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Miocardio/enzimología , Sístole/fisiología , Disfunción Ventricular/fisiopatología , Remodelación Ventricular/fisiología , Animales , Células COS , Chlorocebus aethiops , Diástole/fisiología , Fibroblastos/citología , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Miocardio/patología , Ratas , Transcripción Genética/fisiología , Troponina I/metabolismo , Disfunción Ventricular/metabolismo , Disfunción Ventricular/patologíaRESUMEN
BACKGROUND: Sphingosine kinase (SKase) has been implicated in the protection of hearts from ischemia/reperfusion injury. This hypothesis was further examined. MATERIAL/METHODS: Changes in SKase activity and cardiac function (left ventricular developed pressure, LVDP, and infarct size) in response to ischemia and reperfusion were studied in adult rat hearts by the ex vivo Langendorff method. Following initial equilibration or preconditioning, there was 45 min no-flow ischemia and then 45 min of reperfusion. RESULTS: SKase activity declined 61% during ischemia and did not recover upon reperfusion. LVDP also did not recover upon reperfusion and the infarct size was 47%. A short 30 min period of ischemia was associated with variable recovery of SKase activity that directly correlated with LVDP recovery. Preconditioning of hearts reduced the decrease in SKase activity during ischemia by half, and upon reperfusion activity returned to normal. The LVDP recovered 79% and infarct size was small. Preconditioned hearts had higher S-1-P levels after ischemia/reperfusion relative to non-preconditioned hearts. The decline in SKase activity during ischemia of preconditioned hearts could not be mimicked in vitro by treatment with protein phosphatases. Attempts to alter activity of SKase from control, preconditioned, ischemic, or reperfused hearts by phosphorylation with ERK1/2 were unsuccessful. Treatment of non-preconditioned hearts at reperfusion with 100 nM S-1-P improved recovery of LVDP. The SKase inhibitor dimethylsphingosine blocked hemodynamic recovery in preconditioned hearts. CONCLUSIONS: The data support a role for SKase activity in recovery of hemodynamic function after ischemic injury and also in the cardioprotective effect of preconditioning.
Asunto(s)
Daño por Reperfusión Miocárdica/prevención & control , Miocardio/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Técnicas In Vitro , Precondicionamiento Isquémico Miocárdico , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Ratas , Fracciones Subcelulares/enzimología , Presión VentricularRESUMEN
As pyrroloquinoline quinone (PQQ) is a redox cofactor in mammals, we asked if it is cardioprotective. Rats were subjected to 2 h of left anterior descending (LAD) coronary artery ligation without reperfusion (model 1, ischemia). In model 2 (ischemia/reperfusion), rats were subjected to 17 or 30 min of LAD occlusion and 2 h of reperfusion. PQQ (15-20 mg/kg) was given i.p., either 30 min before LAD occlusion (Pretreatment) or i.v. at the onset of reperfusion (Treatment). In model 1, PQQ reduced infarct size (10.0 +/- 1.5 vs 19.1 +/- 2.1%, P < 0.01). In model 2, either PQQ Pretreatment or Treatment also reduced infarct size (18.4 +/- 2.3 and 25.6 +/- 3.5% vs 38.1 +/- 2.6%, P < 0.01). PQQ resulted in higher LV developed pressure and LV (+)dP/dt after 1-2 h of reperfusion (P < 0.05), and fewer ventricular fibrillation episodes. PQQ dose (5-20 mg/kg) was inversely related to infarct size. PQQ reduced myocardial tissue levels of malondialdehyde (MDA), an indicator of lipid peroxidation (316 +/- 88 vs 99 +/- 14 nmol/g, P < 0.01). PQQ given either as Pretreatment or as Treatment at the onset of reperfusion is highly effective in reducing infarct size and improving cardiac function in a dose-related manner in rat models of ischemia and ischemia/reperfusion. The optimal dose in this study, which exhibited neither renal nor hepatic toxicity, was 15 mg/kg, but lower doses may also be efficacious. We conclude that PQQ, which appears to act as a free radical scavenger in ischemic myocardium, is a highly effective cardioprotective agent.
Asunto(s)
Modelos Animales de Enfermedad , Pruebas de Función Cardíaca/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Cofactor PQQ/uso terapéutico , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Malondialdehído/química , Malondialdehído/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/química , Miocardio/metabolismo , Cofactor PQQ/química , Cofactor PQQ/farmacología , Ratas , Fibrilación Ventricular/tratamiento farmacológico , Fibrilación Ventricular/fisiopatología , Fibrilación Ventricular/prevención & controlRESUMEN
Concern about the effects of anesthesia on physiological measurements led us to develop methodology to assess left ventricular (LV) pressure in conscious mice. Polyethylene-50 tubing filled with heparinized saline was implanted in the LV cavity through its apex via an abdominal approach and exteriorized to the back of the animal. This surgery was done under anesthesia with either an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg) (K+X) in 11 mice or isoflurane (ISF; 1.5 vol%) by inhalation in 14 mice. Postoperatively, mice were trained daily to lie quietly head first in a plastic cone. LV pressure, the first derivative of LV pressure (dP/dt), and heart rate (HR) in the conscious state were compared between the two groups at 3 days and 1 wk after recovery from surgery using a 1.4-Fr Millar catheter inserted into the LV through the tubing, with the mice lying quietly in the plastic cone. Acutely during anesthesia, K+X decreased HR (from 698 to 298 beats/min), LV systolic pressure (from 107 to 65 mmHg), and maximal dP/dt (dP/dt(max)) (from 15,724 to 4,445 mmHg/s), all P < 0.01. Similar but less marked negative chronotropic and inotropic effects were seen with ISF. HR and dP/dt(max) were decreased significantly in K+X mice 3 days after surgery compared with those anesthetized with ISF (655 vs. 711 beats/min, P < 0.05; 14,448 vs. 18,048 mmHg/s, P < 0.001) but increased to the same level as in ISF mice 1 wk after surgery. In ISF mice, recovery of function occurred rapidly and there were no differences in LV variables between 3 days and 1 wk. LV pressure and dP/dt can be measured in conscious mice with a micromanometer catheter inserted through tubing implanted permanently in the LV apex. Anesthesia with either K+X or, to a lesser extent, ISF, depressed LV function acutely. This depression of function persisted for 3 days after surgery with K+X (but not ISF) and did not recover completely until 1 wk postanesthesia.