RESUMEN
BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.
Asunto(s)
Miosina Tipo I , Síndrome Nefrótico , Animales , Humanos , Ratones , Mutación , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Síndrome Nefrótico/genética , EsteroidesRESUMEN
AIMS/HYPOTHESIS: Obesity and diabetes increase the risk of developing cardiovascular diseases and heart failure. These metabolic disorders are generally reflected by natriuretic peptide system deficiency. Since brain natriuretic peptide (BNP) is known to influence metabolism and cardioprotection, we investigated the effect of chronic exogenous BNP treatment on adverse myocardial consequences related to obesity and diabetes. METHODS: Ten-week-old C57BL/KsJ-db/db obese diabetic mice (db/db) and their lean control littermates (db/+) were treated with BNP (0.6 µg kg(-1) h(-1)) or saline for 12 weeks (n = 10/group). Serial blood and tomography analysis were performed. Cardiac function was determined by echocardiography, and biochemical and histological heart and fat analyses were also performed. RESULTS: BNP treatment resulted in an average increase in plasma BNP levels of 70 pg/ml. An improvement in the metabolic profile of db/db mice was observed, including a reduction in fat content, increased insulin sensitivity, improved glucose tolerance and lower blood glucose, despite increased food intake. db/db mice receiving saline displayed both early systolic and diastolic dysfunction, whereas these functional changes were prevented by BNP treatment. The cardioprotective effects of BNP were attributed to the inhibition of cardiomyocyte apoptosis, myocardial fibrosis, cardiac hypertrophy and the AGE-receptor for AGE (RAGE) system as well as normalisation of cardiac AMP-activated protein kinase and endothelial nitric oxide synthase activities. CONCLUSIONS/INTERPRETATION: Our results indicate that chronic BNP treatment at low dose improves the metabolic profile and prevents the development of myocardial dysfunction in db/db mice.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Péptido Natriurético Encefálico/uso terapéutico , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones ObesosRESUMEN
In the abdominal aortocaval (AV) fistula model of heart failure, we have shown that the acute doubling of cardiac mature mast cell (MC) density involved the maturation, but not proliferation, of a resident population of immature cardiac MCs. An increase in stem cell factor (SCF) may be responsible for this MC maturation process. Thus, the purpose of this study was to determine if: 1) myocardial SCF levels are increased following the initiation of cardiac volume overload; 2) the incubation of left ventricular (LV) tissue slices with SCF results in an increase in mature MC density; and 3) chemical degranulation of mature cardiac MCs in LV tissue slices results in an increase in SCF and mature MC density via MC chymase. Male rats with either sham or AV fistula surgery were studied at 6h and 1 and 3 days post-surgery. LV slices from normal male rat hearts were incubated for 16h with media alone or media containing one of the following: 1) recombinant rat SCF (20 ng/ml) to determine the effects of SCF on MC maturation; 2) the MC secretagogue compound 48/80 (20 µg/ml) to determine the effects of MC degranulation on SCF levels and mature MC density; 3) media containing compound 48/80 and anti-SCF (5 µg/ml) to block the effects of SCF; 4) chymase (100 nM) to determine the effects of chymase on SCF; and 5) compound 48/80 and chymostatin (chymase inhibitor, 10 µM) to block the effects of MC chymase. In AV fistula animals, myocardial SCF was significantly elevated above that in the sham group at 6h and 1 day post fistula by 2 and 1.8 fold, respectively, and then returned to normal by 3 days; this increase slightly preceded significant increases in MC density. Incubation of LV slices with SCF resulted in a doubling of mature MC density and this was concomitant with a significant decrease in the number of immature mast cells. Incubation of LV slices with compound 48/80 increased media SCF levels and mature MC density and with anti-SCF and chymostatin prevented these compound 48/80-induced increases. Incubation with chymase increased media SCF levels and mature MC density. These findings indicate that activated mature cardiac mast cells are responsible, in a paracrine fashion, for the increase in mature MC density post AV fistula by rapidly increasing SCF levels via the release of chymase.
Asunto(s)
Fístula Arterio-Arterial/cirugía , Ventrículos Cardíacos/efectos de los fármacos , Mastocitos/citología , Factor de Células Madre/metabolismo , Factor de Células Madre/farmacología , Animales , Procedimientos Quirúrgicos Cardíacos , Recuento de Células , Degranulación de la Célula/efectos de los fármacos , Proliferación Celular , Quimasas/antagonistas & inhibidores , Quimasas/farmacología , Corazón , Insuficiencia Cardíaca/patología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Miocardio/patología , Oligopéptidos/farmacología , Ratas , Ratas Sprague-Dawley , Remodelación Ventricular/fisiologíaRESUMEN
The temporal myocardial remodeling induced by chronic ventricular volume overload in male rats was examined. Specifically, left ventricular (LV) cardiomyocyte length and width, sarcomere length, and number of nuclei were measured in male rats (n = 8 to 17) at 1, 3, 5, 7, 21, 35, and 56 days after creation of an infrarenal aortocaval fistula. In contrast to previously published reports of progressive increases in cardiomyocyte length and cross-sectional area at 5 days post-fistula and beyond in female hearts, cardiomyocyte length and width did not increase significantly in males during the first 35 days of volume overload. Furthermore, a significant decrease in cardiomyocyte length relative to age-matched controls, together with a reduced number of sarcomeres per cell, was noted in male hearts at 5 days post-fistula. There was a concurrent increase in the percentage of mononucleated cardiomyocytes from 11.6% to 18% at 5 days post-fistula. These initial differences could not be attributed to cardiomyocyte proliferation, and treatment with a microtubule stabilizing agent prevented them from occurring. The subsequent significant increase in LV weight without corresponding increases in cardiomyocyte dimensions is indicative of hyperplasia. Thus, these findings indicate hyperplasia resulting from cytokinesis of cardiomyocytes is a key mechanism, independent of hypertrophy, that contributes to the significant increase in LV mass in male hearts subjected to chronic volume overload.
Asunto(s)
Agua Corporal , Cardiomegalia/patología , Aumento de la Célula , Miocardio/patología , Miocitos Cardíacos/patología , Análisis de Varianza , Animales , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Hiperplasia/patología , Masculino , Microscopía Electrónica de Rastreo , Tamaño de los Órganos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
In the rat, oxytocin (OT) produces dose-dependent diuretic and natriuretic responses. Post-translational enzymatic conversion of the OT biosynthetic precursor forms both mature and C-terminally extended peptides. The plasma concentrations of these C-terminally extended peptides (OT-G; OT-GK and OT-GKR) are elevated in newborns and pregnant rats. Intravenous injection of OT-GKR to rats inhibits diuresis, whereas injection of amidated OT stimulates diuresis. Since OT and OT-GKR show different effects on the urine flow, we investigated whether OT-GKR modulates renal action by inhibition of the arginine-vasopressin (AVP) receptor V2 (V2R), the receptor involved in renal water reabsorption. Experiments were carried out in the 8-week-old Wistar rats receiving intravenous (iv) injections of vehicle, OT, OT-GKR or OT+OT-GKR combination. OT (10 µmol/kg) increased urine outflow by 40% (P<0.01) and sodium excretion by 47% (P<0.01). Treatment with OT-GKR (10 µmol/kg) decreased diuresis by 50% (P<0.001), decreased sodium excretion by 50% (P<0.05) and lowered potassium by 42% (P<0.05). OT antagonist (OTA) reduced diuresis and natriuresis exerted by OT, whereas the anti-diuretic effect of OT-GKR was unaffected by OTA. The treatment with V2R antagonist (V2A) in the presence and absence of OT induced diuresis, sodium and potassium outflow. V2A in the presence of OT-GKR only partially increased diuresis and natriuresis. Autoradiography and molecular docking analysis showed potent binding of OT-GKR to V2R. Finally, the release of cAMP from CHO cells overexpressing V2 receptor was induced by low concentration of AVP (EC50:4.2e-011), at higher concentrations of OT (EC50:3.2e-010) and by the highest concentrations of OT-GKR (EC50:1.1e-006). OT-GKR potentiated cAMP release when combined with AVP, but blocked cAMP release when combined with OT. These results suggest that OT-GKR by competing for the OT renal receptor (OTR) and binding to V2R in the kidney, induces anti-diuretic, anti-natriuretic, and anti-kaliuretic effects.
Asunto(s)
Diuresis , Natriuresis , Oxitocina/metabolismo , Animales , Autorradiografía , Unión Competitiva , Células CHO , Línea Celular , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Electrólitos/metabolismo , Humanos , Riñón/metabolismo , Simulación del Acoplamiento Molecular , Péptidos/metabolismo , Ratas , Ratas Wistar , Receptores de Vasopresinas/metabolismo , Micción , Vasopresinas/metabolismoRESUMEN
The early events leading to the establishment of left ventricular hypertrophy associated to pressure overload (PO) are not well characterized. To explore these early events, aortic banding (AB) was performed in rats to induce left ventricle (LV) PO. Animals were sacrificed after 24, 48 h or 14 days. An echocardiogram was performed before the procedure and at sacrifice. LVs were preserved for the evaluation of fibrosis, angiotensin II (AT) receptors expression and stress-related MAP kinases (ERK 1/2, JNK and p38) pathways. We observed that concentric LV hypertrophy was established after only 14 days. Collagen I and fibronectin gene expressions were decreased the first 2 days after AB induction whereas AT receptors mRNA levels were sharply increased. ERK 1/2 and JNK activities in LV homogenates were decreased 24 h after AB but came back to normal after 14 days. p38 activity however was stable during the period studied. We also evaluated the presence of two phosphorylated transcription factors related to JNK signaling pathway (ATF-2 and c-Jun) in cardiomyocyte nuclei. The proportion of LV cell nuclei positive for these two activated transcription factors was significantly reduced in AB rats compared to sham. These results suggest that the early response of the LV to acute PO is to attenuate the expression of some pro-fibrotic and pro-hypertrophic signaling pathways and possibly AT signaling by decreasing ERK 1/2 and JNK relative activities.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Presión Sanguínea , Hipertrofia Ventricular Izquierda/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Miocitos Cardíacos/metabolismo , Remodelación Ventricular , Animales , Estenosis de la Válvula Aórtica/complicaciones , Estenosis de la Válvula Aórtica/fisiopatología , Núcleo Celular/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis/etiología , Fibrosis/metabolismo , Fibrosis/patología , Expresión Génica , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Ratas , Ratas Wistar , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMEN
BACKGROUND AND AIM OF THE STUDY: Chronic aortic regurgitation (AR) induces left ventricular (LV) hypertrophy and eventually LV dysfunction. While the effects of chronic AR on the left ventricle are well known, the effects of acute AR have not been adequately evaluated. It was hypothesized that the LV tissues would be rapidly remodeled by acute AR, and that the renin-angiotensin system would be involved in that acute remodeling. METHOD: The early LV adaptations to acute AR were evaluated serially over a period of 14 days, using a rat model. Adaptations were evaluated in vivo by echocardiography, and in vitro on explanted heart tissue after one, two, or 14 days. RESULTS: After 14 days, the left ventricle of AR rats was already significantly hypertrophied and dilated (end-diastolic diameter +16% (p <0.05) versus sham; LV mass +16% (p <0.01) versus sham). A short and transient increase in fractional shortening was observed during the first 48 h after AR induction. The cardiomyocyte cross-sectional area and perivascular fibrosis were significantly increased after 14 days of AR. The number of fibronectin-positive cells in LV sections rapidly increased, as did the fibronectin protein and mRNA content of LV crude homogenates. The expression of pro-matrix metalloproteinase 2 was clearly abnormal after two days. Significant shifts in the expression of angiotensin II receptors were also detected as early as one 1 day. CONCLUSION: Significant macroscopic and microscopic abnormalities were present in the left ventricle of rats with acute AR, soon after its induction. Considerable hypertrophy, perivascular fibrosis and extracellular matrix (ECM) remodeling were present after only 14 days. These results suggest that, in AR, the myocytes and ECM are affected significantly at a very early stage of the disease.
Asunto(s)
Insuficiencia de la Válvula Aórtica/complicaciones , Insuficiencia de la Válvula Aórtica/fisiopatología , Modelos Animales de Enfermedad , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/fisiopatología , Remodelación Ventricular , Enfermedad Aguda , Animales , Insuficiencia de la Válvula Aórtica/metabolismo , Presión Sanguínea/fisiología , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Frecuencia Cardíaca/fisiología , Hipertrofia Ventricular Izquierda/metabolismo , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Wistar , Receptores de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiología , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: Hypertension (HT) and aortic valve regurgitation (AR) often coexist but the specific impacts of AR + HT on the left ventricle (LV) are still unknown. The best treatment strategy for this combination of diseases is also unclear. The objectives of this study were 1) to evaluate LV function, remodeling and 2) to assess the effects of the angiotensin-converting enzyme (ACE) inhibitor captopril (C) in rats with AR +/- HT in spontaneously hypertensive rats (SHR). METHODS: Animals were grouped as follows: normotensive (NT) Wistar-Kyoto, NT + AR, hypertensive SHR (HT), and HT + AR receiving or not captopril (150 mg/kg/d). Hearts were evaluated in vivo by echocardiography and harvested for tissue analysis after 6 months of evolution. RESULTS: The HT + AR rats had the worst LV hypertrophy (LVH), subendocardial fibrosis, and lowest ejection fraction. Captopril normalized BP in HT and HT + AR, but could not prevent LVH in HT + AR as well as it did in isolated HT. The LV ejection fraction remained below normal in HT + AR + captopril compared to HT alone + captopril. Cardiomyocyte hypertrophy remained in HT + AR + captopril but was normalized in HT + captopril. Subendocardial fibrosis was reduced by captopril in HT + AR. CONCLUSIONS: The AR + HT rats had the most severe myocardial abnormalities. High dose captopril was effective to slow LVH and preserve normal LV ejection fraction in isolated HT or AR, but was less effective when both pathologies were combined. Prohypertrophic stimuli clearly remain active in HT + AR despite ACE inhibition. These results suggest that a very aggressive medical treatment strategy may be required to optimize LV protection when AR and HT co-exist.
Asunto(s)
Insuficiencia de la Válvula Aórtica/complicaciones , Insuficiencia de la Válvula Aórtica/tratamiento farmacológico , Modelos Animales de Enfermedad , Hipertensión/complicaciones , Hipertensión/tratamiento farmacológico , Animales , Insuficiencia de la Válvula Aórtica/patología , Insuficiencia de la Válvula Aórtica/fisiopatología , Captopril/uso terapéutico , Fibrosis Endomiocárdica/patología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Hipertensión/patología , Hipertensión/fisiopatología , Hipertrofia/fisiopatología , Masculino , Tamaño de los Órganos , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Endogámicas WKYRESUMEN
BACKGROUND AND AIM OF THE STUDY: Aortic valve regurgitation (AR) can result in heart failure from chronic overloading of the left ventricle. As little is known of gender-specific responses of the left ventricle to this condition, the study aim was to compare left ventricular (LV) remodeling in male and female rats with severe AR. In order to assess the impact of estrogens on LV remodeling in AR, the effect of ovariectomy (OVX) was also evaluated. METHODS: AR was created in adult Wistar rats (females (control or OVX) and males). Animals were followed for 26 weeks and compared to sham-operated groups. Heart function was evaluated in vivo using echocardiography, and the hearts were subsequently harvested for tissue analysis. RESULTS: The LV ejection fraction was decreased similarly in both sexes. Despite similar echocardiographic AR severity, females had higher indexed cardiac output and the largest increase in LV weight, cardiomyocyte hypertrophy and eccentric remodeling. No differences were observed between control and OVX females. Ovariectomy had no significant impact on any of the parameters monitored. CONCLUSION: Female rats developed more LV remodeling in response to chronic AR than males. AR appears to impose a greater LV workload on females due to their smaller body and heart size. Hormonal status did not have any impact on LV remodeling in this experimental model.
Asunto(s)
Insuficiencia de la Válvula Aórtica/fisiopatología , Remodelación Ventricular , Animales , Insuficiencia de la Válvula Aórtica/diagnóstico por imagen , Colágeno/genética , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Fibronectinas/genética , Masculino , Tamaño de los Órganos , Ovariectomía , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres SexualesRESUMEN
BACKGROUND: Past studies have suggested that the adrenergic system becomes abnormally activated in chronic volume overload, such as in severe aortic valve regurgitation (AR). However, the effectiveness of agents directed against this adrenergic activation has never been adequately tested in chronic AR. We therefore tested the effects of metoprolol treatment on the left ventricular (LV) function and remodeling in severe chronic AR in rats. METHODS AND RESULTS: Severe AR was created in adult male Wistar rats by retrograde puncture of the aortic leaflets under echocardiographic guidance. Two weeks later, some animals received metoprolol treatment (25 mg/kg) orally for 24 weeks, and some were left untreated. LV dimensions, ejection fraction, and filling parameters were evaluated by echocardiography. Hearts were harvested at 1, 2, 14, and 180 days for the evaluation of hypertrophy, beta-adrenergic receptor status, and extracellular matrix remodeling. We found that metoprolol treatment prevented LV dilatation and preserved the ejection fraction and filling parameters compared with untreated animals. Metoprolol increased the expression of beta1-adrenoreceptor mRNA and reduced G protein receptor kinase 2 levels. Collagen I and III mRNA levels were reduced. Cardiac myocyte hypertrophy was also prevented. CONCLUSIONS: In our experimental model of severe AR, metoprolol treatment had a significant beneficial global effect on LV remodeling and function. These results suggest that the adrenergic system is important in the development of volume-overload cardiomyopathy in AR and that adrenergic-blocking agents may play a role in the treatment of this disease.
Asunto(s)
Antagonistas Adrenérgicos beta/uso terapéutico , Insuficiencia de la Válvula Aórtica/tratamiento farmacológico , Metoprolol/uso terapéutico , Animales , Insuficiencia de la Válvula Aórtica/diagnóstico por imagen , Insuficiencia de la Válvula Aórtica/genética , Insuficiencia de la Válvula Aórtica/fisiopatología , Catecolaminas/análisis , Tamaño de la Célula/efectos de los fármacos , Enfermedad Crónica , Evaluación Preclínica de Medicamentos , Fibronectinas/análisis , Frecuencia Cardíaca/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/prevención & control , Masculino , Metoprolol/farmacología , Miocitos Cardíacos/química , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Tamaño de los Órganos , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 1/biosíntesis , Receptores Adrenérgicos beta 1/genética , Volumen Sistólico , Ultrasonografía , Regulación hacia Arriba/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Dobutamine stress echocardiography is used to investigate a wide variety of heart diseases in humans. Dobutamine stress echocardiography has also been used in animal models of heart disease despite the facts that the normal response of healthy rat hearts to this type of pharmacological stress testing is unknown. This study was performed to assess this normal response. METHODS: 15 normal adult male Wistar rats were evaluated. Increasing doses of dobutamine were infused intravenously under continuous imaging of the heart by a 12 MHz ultrasound probe. RESULTS: Dobutamine stress echocardiography reduced gradually LV diastolic and systolic dimensions. Ejection fraction increased by a mean of +24% vs. baseline. Heart rate increased progressively without reaching a plateau. Changes in LV dimensions and ejection fraction reached a plateau after a mean of 4 minutes at a constant infusion rate. CONCLUSION: DSE can be easily performed in rats. The normal response is an increase in heart rate and ejection fraction and a decrease in LV dimensions. A plateau in echocardiographic measurements is obtained after 4 minutes of a constant infusion rate in most animals.
Asunto(s)
Dobutamina/administración & dosificación , Ecocardiografía/métodos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/diagnóstico por imagen , Función Ventricular Izquierda/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Prueba de Esfuerzo , Frecuencia Cardíaca/efectos de los fármacos , Infusiones Intravenosas , Masculino , Ratas , Ratas Wistar , Volumen Sistólico/efectos de los fármacosRESUMEN
Oxytocin (OT) is involved in the regulation of energy metabolism and in the activation of cardioprotective mechanisms. We evaluated whether chronic treatment with OT could prevent the metabolic and cardiac abnormalities associated with diabetes and obesity using the db/db mice model. Four-week-old male db/db mice and their lean nondiabetic littermates (db/+) serving as controls were treated with OT (125 ng/kg · h) or saline vehicle for a period of 12 weeks. Compared with db/+ mice, the saline-treated db/db mice developed obesity, hyperglycemia, and hyperinsulinemia. These mice also exhibited a deficient cardiac OT/natriuretic system and developed systolic and diastolic dysfunction resulting from cardiomyocyte hypertrophy, fibrosis, and apoptosis. These abnormalities were associated with increased reactive oxygen species (ROS) production, inflammation, and suppressed 5'-adenosine monophosphate kinase signaling pathway. The db/db mice displayed reduced serum levels of adiponectin and adipsin and elevated resistin. OT treatment increased circulating OT levels, significantly reduced serum resistin, body fat accumulation (19%; P<.001), fasting blood glucose levels by (23%; P<.001), and improved glucose tolerance and insulin sensitivity. OT also normalized cardiac OT receptors, atrial natriuretic peptide, and brain natriuretic peptide, expressions and prevented systolic and diastolic dysfunction as well as cardiomyocyte hypertrophy, fibrosis, and apoptosis. Furthermore, OT reduced cardiac oxidative stress and inflammation and normalized the 5'-adenosine monophosphate-activated protein kinase signaling pathway. The complete normalization of cardiac structure and function by OT treatment in db/db mice contrasted with only partial improvement of hyperglycemia and hyperinsulinemia. These results indicate that chronic treatment with OT partially improves glucose and fat metabolism and reverses abnormal cardiac structural remodeling, preventing cardiac dysfunction in db/db mice.
Asunto(s)
Cardiomiopatías/prevención & control , Diabetes Mellitus Tipo 2/complicaciones , Hiperinsulinismo/complicaciones , Obesidad/complicaciones , Oxitocina/uso terapéutico , Adiponectina/sangre , Animales , Glucemia/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético/efectos de los fármacos , Hiperinsulinismo/metabolismo , Resistencia a la Insulina/fisiología , Masculino , Ratones , Obesidad/metabolismo , Oxitocina/farmacología , Resistina/sangreRESUMEN
Oxytocin (OT) stimulates cardioprotection. Here we investigated heart-derived H9c2 cells in simulated ischemia-reperfusion (I-R) experiments in order to examine the mechanism of OT protection. I-R was induced in an anoxic chamber for 2 hours and followed by 2 h of reperfusion. In comparison to normoxia, I-R resulted in decrease of formazan production by H9c2 cells to 63.5 ± 1.7% (MTT assay) and in enhanced apoptosis from 1.7 ± 0.3% to 2.8 ± 0.4% (Tunel test). Using these assays it was observed that treatment with OT (1-500 nM) exerted significant protection during I-R, especially when OT was added at the time of ischemia or reperfusion. Using the CM-H2DCFDA probe we found that OT triggers a short-lived burst in reactive oxygen species (ROS) production in cells but reduces ROS production evoked by I-R. In cells treated with OT, Western-blot revealed the phosphorylation of Akt (Thr 308, p-Akt), eNOS and ERK 1/2. Microscopy showed translocation of p-Akt and eNOS into the nuclear and perinuclear area and NO production in cells treated with OT. The OT-induced protection against I-R was abrogated by an OT antagonist, the Pi3K inhibitor Wortmannin, the cGMP-dependent protein kinase (PKG) inhibitor, KT5823, as well as soluble guanylate cyclase (GC) inhibitor, ODQ, and particulate GC antagonist, A71915. In conditions of I-R, the cells with siRNA-mediated reduction in OT receptor (OTR) expression responded to OT treatment by enhanced apoptosis. In conclusion, the OTR protected H9c2 cells against I-R, especially if activated at the onset of ischemia or reperfusion. The OTR-transduced signals include pro-survival kinases, such as Akt and PKG.
Asunto(s)
Cardiotónicos/farmacología , Miocitos Cardíacos/fisiología , Oxitocina/farmacología , Hipoxia de la Célula , Línea Celular , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos , Mioblastos/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Oxitocina/metabolismoRESUMEN
INTRODUCTION: The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. METHODS: MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. RESULTS: Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. CONCLUSIONS: Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Medios de Cultivo Condicionados/farmacología , Trasplante de Células Madre Mesenquimatosas , Piel/lesiones , Cicatrización de Heridas/fisiología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Quimiocina CXCL10/biosíntesis , Cobalto/farmacología , Femenino , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Regeneración Tisular Dirigida/métodos , Caballos , Interferón gamma/farmacología , Interleucina-8/biosíntesis , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/biosíntesis , Células Madre Mesenquimatosas/fisiología , Mitomicina/farmacología , Modelos Animales , Enfermedades de la Piel/terapia , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND AND AIM OF THE STUDY: Experimental aortic regurgitation (AR) induced by retrograde perforation of the aortic leaflets has been used as an animal model of volume overload, but causes high mortality. In the past, AR was induced under hemodynamic guidance, but echocardiography can be used to guide the investigator as well as grade AR severity and monitor left ventricular function. The study aim was to assess the value of echocardiography in experimental AR in rats. METHODS: Sixty-six Wistar rats (bodyweight 250-275 g) underwent perforation of the aortic leaflets via a right transcarotid approach to induce moderate to severe AR. Transthoracic echocardiographic guidance was used to assess catheter location, degree of AR, ventricular function and complications. RESULTS: Echocardiographic images were easily obtained and of excellent quality (M-mode, two-dimensional and complete Doppler evaluation). Catheter location, movement and guidance during aortic valve perforation were straightforward, and AR gradation and ventricular function easily assessed. Procedural complications and causes of death were identified. Had hemodynamic criteria been used, 24% of AR cases would have been misgraded. With echocardiography, the overall mortality rate was 17% (<10% in the last 25 animals). CONCLUSION: Echocardiographic guidance is mandatory for protocols of experimental AR. Using this technique, the procedure is simplified, and is more accurate, more reproducible and safer for the animals.
Asunto(s)
Insuficiencia de la Válvula Aórtica/diagnóstico por imagen , Modelos Animales de Enfermedad , Ecocardiografía Doppler , Animales , Insuficiencia de la Válvula Aórtica/fisiopatología , Hemodinámica , Ratas , Ratas Wistar , Función Ventricular IzquierdaRESUMEN
BACKGROUND: Endothelial function in hypercholesterolemic rabbits is usually evaluated ex vivo on isolated aortic rings. In vivo evaluation requires invasive imaging procedures that cannot be repeated serially. AIM: We evaluated a non-invasive ultrasound technique to assess early endothelial function in rabbits and compare data with ex vivo measurements. METHODS: Twenty-four rabbits (fed with a cholesterol diet (0.5%) for 2 to 8 weeks) were given progressive infusions of acetylcholine (0.05-0.5 microg/kg/min) and their endothelial function was assessed in vivo by transcutaneous vascular ultrasound of the abdominal aorta. Ex vivo endothelial function was evaluated on isolated aortic rings and compared to in vivo data. RESULTS: Significant endothelial dysfunction was demonstrated in hypercholesterolemic animals as early as 2 weeks after beginning the cholesterol diet (aortic cross-sectional area variation: -2.9% vs. +4% for controls, p < 0.05). Unexpectedly, response to acetylcholine at 8 weeks was more variable. Endothelial function improved in 5 rabbits while 2 rabbits regained a normal endothelial function. These data corroborated well with ex vivo results. CONCLUSION: Endothelial function can be evaluated non-invasively in vivo by transcutaneous vascular ultrasound of the abdominal aorta in the rabbit and results correlate well with ex vivo data.
Asunto(s)
Aorta Abdominal/diagnóstico por imagen , Enfermedades de la Aorta/diagnóstico por imagen , Arteriosclerosis/diagnóstico por imagen , Endotelio Vascular/diagnóstico por imagen , Hipercolesterolemia/diagnóstico por imagen , Animales , Enfermedades de la Aorta/inducido químicamente , Arteriosclerosis/inducido químicamente , Colesterol en la Dieta , Dieta Aterogénica , Modelos Animales de Enfermedad , Hipercolesterolemia/inducido químicamente , Masculino , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
Increased numbers of mast cells have been reported in explanted human hearts with dilated cardiomyopathy and in animal models of experimentally induced hypertension, myocardial infarction, and chronic volume overload secondary to aortocaval fistula and mitral regurgitation. Accordingly, mast cells have been implicated to have a major role in the pathophysiology of these cardiovascular disorders. In vitro studies have verified that mast cell proteases are capable of activating collagenase, gelatinases and stromelysin. Recent results have shown that with chronic ventricular volume overload, there is an elevation in mast cell density, which is associated with a concomitant increase in matrix metalloproteinase (MMP) activity and extracellular matrix degradation. However, the role of the cardiac mast cell is not one dimensional, with evidence from hypertension and cardiac transplantation studies suggesting that they can also assume a pro-fibrotic phenotype in the heart. These adverse events do not occur in mast cell deficient rodents or when cardiac mast cells are pharmacologically prevented from degranulating. This review is focused on the regulation and dual roles of cardiac mast cells in: (i) activating MMPs and causing myocardial fibrillar collagen degradation and (ii) causing fibrosis in the stressed, injured or diseased heart. Moreover, there is strong evidence that premenopausal female cardioprotection may at least partly be due to gender differences in cardiac mast cells. This too will be addressed.
Asunto(s)
Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/fisiopatología , Mastocitos/patología , Mastocitos/fisiología , Remodelación Ventricular/fisiología , Animales , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Diferenciación Celular , Proliferación Celular , Complemento C5a/fisiología , Endotelina-1/fisiología , Femenino , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Trasplante de Corazón/patología , Trasplante de Corazón/fisiología , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/fisiología , Humanos , Hipertensión/patología , Hipertensión/fisiopatología , Técnicas In Vitro , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocarditis/patología , Miocarditis/fisiopatología , Neuropéptidos/fisiología , Especies Reactivas de Oxígeno/metabolismo , Caracteres SexualesRESUMEN
Aortic valve regurgitation (AR) imposes a severe volume overload to the left ventricle (LV), which results in dilation, eccentric hypertrophy, and eventually loss of function. Little is known about the impact of AR on LV gene expression. We, therefore, conducted a gene expression profiling study in the LV of rats with acute and severe AR. We identified 64 genes that were specifically upregulated and 29 that were downregulated out of 21,910 genes after 2 wk. Of the upregulated genes, a good proportion was related to the extracellular matrix. We subsequently studied a subset of 19 genes by quantitative RT-PCR (qRT-PCR) to see if the modulation seen in the LV after 2 wk persisted in the chronic phase (after 6 and 12 mo) and found that it did persist. Knowing that the adrenergic and renin-angiotensin systems are overactivated in our animal model, we were interested to see if blocking those systems using metoprolol (25 mg.kg(-1).day(-1)) and captopril (100 mg.kg(-1).day(-1)) would alter the expression of some upregulated LV genes in AR rats after 6 mo. By qRT-PCR, we observed that upregulations of LV mRNA levels encoding for procollagens type I and III, fibronectin, atrial natriuretic peptide, transforming growth factor-beta(2), and connective tissue growth factor were totally or partially reversed by this treatment. These observations provide a molecular rationale for a medical strategy aiming these systems in the medical treatment of AR and expand the paradigm in the study of this form of LV volume overload.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Insuficiencia de la Válvula Aórtica/tratamiento farmacológico , Perfilación de la Expresión Génica , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Receptores Adrenérgicos beta/efectos de los fármacos , Sistema Renina-Angiotensina/efectos de los fármacos , Enfermedad Aguda , Animales , Insuficiencia de la Válvula Aórtica/complicaciones , Insuficiencia de la Válvula Aórtica/genética , Insuficiencia de la Válvula Aórtica/metabolismo , Captopril/farmacología , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/metabolismo , Masculino , Metoprolol/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
BACKGROUND: Aortic valve regurgitation (AR) imposes a pathologic volume overload to the left ventricle (LV), whereas aerobic exercise causes physiologic volume overloading. The impact of combining both LV volume overloads (pathologic and physiologic) is unknown. Considering the known beneficial effects of aerobic training on the cardiovascular system, we hypothesized that the positive effects would outweigh the negative ones and that exercise would improve the tolerance of the LV to AR. METHODS: Forty female adult Wistar rats were randomly divided in the following groups: 1) sham sedentary (SS), 2) sham trained (ST), 3) AR sedentary (ARS), and 4) AR trained (ART). Training consisted in treadmill running for 30 min five times per week at 20 m x s(-1) for 24 wk. In vivo follow-up was made by echocardiography and invasive intracardiac pressure measurements. Hearts were harvested for tissue analysis. RESULTS: Echocardiography revealed less LV dilation and hypertrophy in ART versus ARS as well as improved myocardial performance index. LV ejection fractions remained similar and within normal range in ART versus ARS. Invasive cardiac pressures yielded improved dP/dt- in ART versus ARS but similar dP/dt+. beta(1)-Adrenergic receptor mRNA expression was improved in the ART group versus ARS. CONCLUSION: Our data suggest that a moderate aerobic exercise program helps minimize LV dilation and hypertrophy and improves diastolic cardiac performance in heart submitted to chronic volume overload due to severe aortic valve regurgitation in this animal model.
Asunto(s)
Insuficiencia de la Válvula Aórtica/fisiopatología , Cardiomiopatía Hipertrófica/fisiopatología , Tolerancia al Ejercicio , Hipertrofia Ventricular Izquierda/fisiopatología , Animales , Insuficiencia de la Válvula Aórtica/diagnóstico por imagen , Insuficiencia de la Válvula Aórtica/prevención & control , Cardiomiopatía Hipertrófica/diagnóstico por imagen , Cardiomiopatía Hipertrófica/prevención & control , Modelos Animales de Enfermedad , Femenino , Hemodinámica , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/prevención & control , Ratas , UltrasonografíaRESUMEN
BACKGROUND: Aortic regurgitation (AR) is a disease of chronic left ventricular (LV) volume overload. Over time, AR will lead to LV dilatation, hypertrophy, and loss of function. There is currently no medical treatment proven effective to slow the evolution of this cardiomyopathy. Vasodilators were once thought to have protective effects, but recent publications have cast some doubts about their effectiveness. We hypothesized that drugs targeting the renin-angiotensin system should be more effective than those having no direct effect on the renin-angiotensin system. METHODS AND RESULTS: We designed a protocol comparing the effects of 3 vasodilators in a rat AR model (n=9 to 11 animals per group). The effects of a 6-month treatment of (1) nifedipine, (2) captopril, or (3) losartan were compared in male AR rats. Sham-operated and untreated AR animals were used as controls. Nifedipine-treated animals displayed hemodynamics, LV dilatation, hypertrophy, and loss of function similar to those of the untreated group. Both captopril and losartan were effective in improving hemodynamics, slow LV dilatation, hypertrophy, and dysfunction. Gene expression analysis confirmed the lack of effects of the nifedipine treatment at the molecular level. CONCLUSIONS: Using an animal model of severe AR, we found that vasodilators targeting the renin-angiotensin system were effective to slow the development of LV remodeling and to preserve LV function. As recently shown in the most recent human clinical trial, nifedipine was totally ineffective. Targeting the renin-angiotensin system seems a promising avenue in the treatment of this disease, and clinical trials should be carefully designed to re-evaluate the effectiveness of angiotensin I-converting enzyme inhibitors or angiotensin II receptor blockers in AR.