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RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.
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Cardiomiopatía Dilatada/metabolismo , Diabetes Mellitus Experimental/metabolismo , Metabolismo Energético , Factores de Transcripción de Tipo Kruppel/metabolismo , Miocitos Cardíacos/metabolismo , PPAR alfa/metabolismo , Sepsis/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/fisiopatología , Línea Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Ácidos Grasos/metabolismo , Genotipo , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , PPAR alfa/genética , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/metabolismo , Sepsis/genética , Sepsis/fisiopatología , Transducción de Señal , Transportador 2 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Factores de Tiempo , Transcripción Genética , Activación Transcripcional , Transfección , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
Animal studies suggest that hypertension leads to cardiac tissue hypothyroidism, a condition that can by itself lead to heart failure. We have previously shown that short-term thyroid hormone treatment in Spontaneously Hypertensive Heart Failure (SHHF) rats near heart failure is beneficial. This study tested the hypothesis that therapeutic, long-term T3 treatment in SHHF rats can prevent or attenuate cardiac dysfunction. Female SHHF rats were treated orally with a physiological T3 dose (0.04 µg/ml) from 12 to 24 mo of age. Age-matched female SHHF and Wistar-Kyoto rats served as hypertensive and normotensive controls, respectively. SHHF rats had reduced serum free thyroid hormone levels and cardiac tissue T3 levels, LV dysfunction, and elevated LV collagen content compared with normotensive controls. Restoration of serum and cardiac tissue thyroid hormone levels in T3-treated rats was associated with no change in heart rate, but strong trends for improvement in LV systolic function and collagen levels. For instance, end-systolic diameter, fractional shortening, systolic wall stress, and LV collagen levels were no longer significantly different from controls. In conclusion, longstanding hypertension in rats led to chronic low serum and cardiac tissue thyroid hormone levels. Long-term treatment with low-dose T3 was safe. While cardiac dysfunction could not be completely prevented in the absence of antihypertensive treatment, T3 may offer additional benefits as an adjunct therapy with possible improvement in diastolic function.
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Colágeno/efectos de los fármacos , Insuficiencia Cardíaca/etiología , Ventrículos Cardíacos/efectos de los fármacos , Corazón/efectos de los fármacos , Hipertensión/complicaciones , Hipotiroidismo/etiología , Triyodotironina/farmacología , Disfunción Ventricular Izquierda/etiología , Función Ventricular Izquierda/efectos de los fármacos , Animales , Colágeno/metabolismo , Femenino , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Hipertensión/metabolismo , Hipotiroidismo/metabolismo , Miosinas/efectos de los fármacos , Miosinas/metabolismo , Distribución Aleatoria , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Tiroxina/metabolismo , Disfunción Ventricular Izquierda/metabolismoRESUMEN
Thyroid dysfunction is common in individuals with diabetes mellitus (DM) and may contribute to the associated cardiac dysfunction. However, little is known about the extent and pathophysiological consequences of low thyroid conditions on the heart in DM. DM was induced in adult female Sprague Dawley (SD) rats by injection of nicotinamide (N; 200 mg/kg) followed by streptozotocin (STZ; 65 mg/kg). One month after STZ/N, rats were randomized to the following groups (N = 10/group): STZ/N or STZ/N + 0.03 µg/mL T3; age-matched vehicle-treated rats served as nondiabetic controls (C). After 2 months of T3 treatment (3 months post-DM induction), left ventricular (LV) function was assessed by echocardiography and LV pressure measurements. Despite normal serum thyroid hormone (TH) levels, STZ/N treatment resulted in reductions in myocardial tissue content of THs (T3 and T4: 39% and 17% reduction versus C, respectively). Tissue hypothyroidism in the DM hearts was associated with increased DIO3 deiodinase (which converts THs to inactive metabolites) altered TH transporter expression, reexpression of the fetal gene phenotype, reduced arteriolar resistance vessel density, and diminished cardiac function. Low-dose T3 replacement largely restored cardiac tissue TH levels (T3 and T4: 43% and 10% increase versus STZ/N, respectively), improved cardiac function, reversed fetal gene expression and preserved the arteriolar resistance vessel network without causing overt symptoms of hyperthyroidism. We conclude that cardiac dysfunction in chronic DM may be associated with tissue hypothyroidism despite normal serum TH levels. Low-dose T3 replacement appears to be a safe and effective adjunct therapy to attenuate and/or reverse cardiac remodeling and dysfunction induced by experimental DM.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Terapia de Reemplazo de Hormonas , Miocardio/metabolismo , Hormonas Tiroideas/uso terapéutico , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Femenino , Hemodinámica , Miocardio/patología , Ratas Sprague-Dawley , Hormonas Tiroideas/sangre , Hormonas Tiroideas/farmacología , Remodelación Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Cytomegalovirus (CMV) is the most frequent cause of congenital infection and ≈20% of all infected neonates present or will develop sensorineural hearing loss. Targeted congenital CMV (cCMV) screening in newborns who failed universal newborn hearing screening has been proposed as a strategy to identify neonates with both hearing loss and cCMV infection who could benefit from antiviral treatment implemented within the first month of life. OBJECTIVES: To evaluate the feasibility and performance of cCMV targeted screening in a French setting. METHODS: Neonates were recruited in 5 maternity centers in greater Paris. A saliva sample for CMV polymerase chain reaction (PCR) testing was collected in neonates who failed newborn hearing screening. Outcomes including CMV PCR result and confirmation of hearing loss by an otorhinolaryngologist specialist were documented. RESULTS: Two-hundred thirty-six newborns were included and a saliva sample was collected in 98% (231/236) of them. The result of CMV PCR was available at a median of 9 days (7-10 days) of life and in 96% of cases within the first month of life. Two neonates were infected with CMV. The result of the otorhinolaryngologist assessment was available in 75% (178/236) of cases at a median of 16 days (9-26 days). Hearing loss was confirmed in 2.8% (5/178). The 2 infected neonates had hearing loss confirmed at 5 and 8 days of life and were treated with valganciclovir at days 9 and 16, respectively. CONCLUSIONS: The result of this study confirms that targeted cCMV screening is feasible in these French settings.
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Infecciones por Citomegalovirus , Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Citomegalovirus/genética , Infecciones por Citomegalovirus/congénito , Femenino , Audición , Pérdida Auditiva/diagnóstico , Pérdida Auditiva Sensorineural/diagnóstico , Humanos , Recién Nacido , Tamizaje Neonatal , Embarazo , SalivaRESUMEN
Recent studies in various rodent models of pathologic ventricular hypertrophy report the re-expression of deiodinase type 3 (D3) in cardiomyocytes. D3 inactivates thyroid hormone (T3) and is mainly expressed in tissues during development. The stimulation of D3 activity in ventricular hypertrophy and subsequent heart failure is associated with severe impairment of cardiac T3 signaling. Hypoxia-induced signaling appears to drive D3 expression in the hypertrophic cardiomyocyte, but other signaling cascades implicated in hypertrophy are also capable of stimulating transcription of the DIO3 gene. Many cardiac genes are transcriptionally regulated by T3 and impairment of T3 signaling will not only reduce energy turnover, but also lead to changes in gene expression that contribute to contractile dysfunction in pathologic remodeling. Whether stimulation of D3 activity and the ensuing local T3-deficiency is an adaptive response of the stressed heart or part of the pathologic signaling network leading to heart failure, remains to be established.
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Cardiomegalia/metabolismo , Yoduro Peroxidasa/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal , Hormonas Tiroideas/metabolismo , Animales , Cardiomegalia/complicaciones , Cardiomegalia/fisiopatología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Yoduro Peroxidasa/genética , RatasRESUMEN
Dual peroxisome proliferator-activated receptor (PPAR)α/γ agonists that were developed to target hyperlipidemia and hyperglycemia in type 2 diabetes patients, caused cardiac dysfunction or other adverse effects. We studied the mechanisms that underlie the cardiotoxic effects of a dual PPARα/γ agonist, tesaglitazar, in wild type and diabetic (leptin receptor deficient - db/db) mice. Mice treated with tesaglitazar-containing chow or high fat diet developed cardiac dysfunction despite lower plasma triglycerides and glucose levels. Expression of cardiac peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which promotes mitochondrial biogenesis, had the most profound reduction among various fatty acid metabolism genes. Furthermore, we observed increased acetylation of PGC1α, which suggests PGC1α inhibition and lowered sirtuin 1 (SIRT1) expression. This change was associated with lower mitochondrial abundance. Combined pharmacological activation of PPARα and PPARγ in C57BL/6 mice reproduced the reduction of PGC1α expression and mitochondrial abundance. Resveratrol-mediated SIRT1 activation attenuated tesaglitazar-induced cardiac dysfunction and corrected myocardial mitochondrial respiration in C57BL/6 and diabetic mice but not in cardiomyocyte-specific Sirt1-/- mice. Our data shows that drugs, which activate both PPARα and PPARγ lead to cardiac dysfunction associated with PGC1α suppression and lower mitochondrial abundance likely due to competition between these two transcription factors.
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Insuficiencia Cardíaca/metabolismo , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Peroxisomas/metabolismo , Sirtuina 1/metabolismo , Alcanosulfonatos/efectos adversos , Animales , Glucemia , Línea Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , PPAR alfa/agonistas , PPAR gamma/agonistas , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Fenilpropionatos/efectos adversos , Receptores de Leptina/metabolismo , Sirtuina 1/genética , Factores de Transcripción , TranscriptomaRESUMEN
Cardiac metabolism affects systemic energetic balance. Previously, we showed that Krüppel-like factor (KLF)-5 regulates cardiomyocyte PPARα and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (αMHC-KLF5-/-) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT). Alterations in cardiac expression of the mediator complex subunit 13 (Med13) modulates obesity. αMHC-KLF5-/- mice had reduced cardiac Med13 expression likely because KLF5 upregulates Med13 expression in cardiomyocytes. We then investigated potential mechanisms that mediate cross-talk between cardiomyocytes and WAT. High fat diet-fed αMHC-KLF5-/- mice had increased levels of cardiac and plasma FGF21, while food intake, activity, plasma leptin, and natriuretic peptides expression were unchanged. Consistent with studies reporting that FGF21 signaling in WAT decreases sumoylation-driven PPARγ inactivation, αMHC-KLF5-/- mice had less SUMO-PPARγ in WAT. Increased diet-induced obesity found in αMHC-KLF5-/- mice was absent in αMHC-[KLF5-/-;FGF21-/-] double knockout mice, as well as in αMHC-FGF21-/- mice that we generated. Thus, cardiomyocyte-derived FGF21 is a component of pro-adipogenic crosstalk between heart and WAT.
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Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Animales , Peso Corporal , Dieta Alta en Grasa , Femenino , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Leptina/sangre , Masculino , Complejo Mediador/genética , Complejo Mediador/metabolismo , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Obesidad/etiología , Transducción de SeñalRESUMEN
Background: We have shown that thyroid hormones (THs) are cardioprotective and can be potentially used as safe therapeutic agents for diabetic cardiomyopathy and permanent infarction. However, no reliable, clinically translatable protocol exists for TH treatment of myocardial ischemia-reperfusion (IR) injury. We hypothesized that modified low-dose triiodo-L-thyronine (T3) therapy would confer safe therapeutic benefits against IR injury. Methods: Adult female rats underwent left coronary artery ligation for 60 min or sham surgeries. At 2 months following surgery and T3 treatment (described below), the rats were subjected to functional, morphological, and molecular examination. Results: Following surgery, the rats were treated with T3 (8 µg/kg/day) or vehicle in drinking water ad libitum following IR for 2 months. Oral T3 significantly improved left ventricular (LV) contractility, relaxation, and relaxation time constant, and decreased beta-myosin heavy chain gene expression. As it takes rats ~6 h post-surgery to begin drinking water, we then investigated whether modified T3 dosing initiated immediately upon reperfusion confers additional improvement. We injected an intraperitoneal bolus of T3 (12 µg/kg) upon reperfusion, along with low-dose oral T3 (4.5 µg/kg/day) in drinking water for 2 months. Continuous T3 therapy (bolus + low-dose oral) enhanced LV contractility compared with oral T3 alone. Relaxation parameters were also improved compared to vehicle. Importantly, these were accomplished without significant increases in hypertrophy, serum free T3 levels, or blood pressure. Conclusions: This is the first study to provide a safe cardiac therapeutic window and optimized, clinically translatable treatment-monitoring protocol for myocardial IR using commercially available and inexpensive T3. Low-dose oral T3 therapy supplemented with bolus treatment initiated upon reperfusion is safer and more efficacious.
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BACKGROUND: A large body of evidence suggests that thyroid hormones (THs) are beneficial for the treatment of cardiovascular disorders. We have shown that 3 days of triiodo-L-thyronine (T3) treatment in myocardial infarction (MI) rats increased left ventricular (LV) contractility and decreased myocyte apoptosis. However, no clinically translatable protocol is established for T3 treatment of ischemic heart disease. We hypothesized that low-dose oral T3 will offer safe therapeutic benefits in MI. METHODS AND RESULTS: Adult female rats underwent left coronary artery ligation or sham surgeries. T3 (~6 µg/kg/day) was available in drinking water ad libitum immediately following MI and continuing for 2 month(s) (mo). Compared to vehicle-treated MI, the oral T3-treated MI group at 2 mo had markedly improved anesthetized Magnetic Resonance Imaging-based LV ejection fraction and volumes without significant negative changes in heart rate, serum TH levels or heart weight, indicating safe therapy. Remarkably, T3 decreased the incidence of inducible atrial tachyarrhythmias by 88% and improved remodeling. These were accompanied by restoration of gene expression involving several key pathways including thyroid, ion channels, fibrosis, sympathetic, mitochondria and autophagy. CONCLUSIONS: Low-dose oral T3 dramatically improved post-MI cardiac performance, decreased atrial arrhythmias and cardiac remodeling, and reversed many adverse changes in gene expression with no observable negative effects. This study also provides a safe and effective treatment/monitoring protocol that should readily translate to humans.
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Arritmias Cardíacas/prevención & control , Infarto del Miocardio/complicaciones , Triyodotironina/administración & dosificación , Administración Oral , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/fisiopatología , Femenino , Imagen por Resonancia Magnética , Infarto del Miocardio/fisiopatología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Triyodotironina/sangreRESUMEN
Over 5 million people in the United States suffer from the complications of heart failure (HF), which is a rapidly expanding health complication. Disorders that contribute to HF include ischemic cardiac disease, cardiomyopathies, and hypertension. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family. There are three PPAR isoforms: PPARα, PPARγ, and PPARδ. They can be activated by endogenous ligands, such as fatty acids, as well as by pharmacologic agents. Activators of PPARs are used for treating several metabolic complications, such as diabetes and hyperlipidemia that are directly or indirectly associated with HF. However, some of these drugs have adverse effects that compromise cardiac function. This review article aims to summarize the current basic and clinical research findings of the beneficial or detrimental effects of PPAR biology on myocardial function.
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BACKGROUND: Evidence indicates that cardiac hypothyroidism may contribute to heart failure progression. It is also known that heart failure is associated with an increased risk of atrial fibrillation (AF). Although it is established that hyperthyroidism increases AF incidence, the effect of hypothyroidism on AF is unclear. This study investigated the effects of different thyroid hormone levels, ranging from hypothyroidism to hyperthyroidism on AF inducibility in thyroidectomized rats. METHODS AND RESULTS: Thyroidectomized rats with serum-confirmed hypothyroidism 1 month after surgery were randomized into hypothyroid (N=9), euthyroid (N=9), and hyperthyroid (N=9) groups. Rats received placebo, 3.3-mg l-thyroxine (T4), or 20-mg T4 pellets (60-day release form) for 2 months, respectively. At the end of treatment, hypothyroid, euthyroid, and hyperthyroid status was confirmed. Hypothyroid animals showed cardiac atrophy and reduced cardiac systolic and diastolic functions, whereas hyperthyroid rats exhibited cardiac hypertrophy and increased cardiac function. Hypothyroidism and hyperthyroidism produced opposite electrophysiological changes in heart rates and atrial effective refractory period, but both significantly increased AF susceptibility. AF incidence was 78% in hypothyroid, 67% in hyperthyroid, and the duration of induced AF was also longer, compared with 11% in the euthyroid group (all P<0.05). Hypothyroidism increased atrial interstitial fibrosis, but connexin 43 was not affected. CONCLUSIONS: Both hypothyroidism and hyperthyroidism lead to increased AF vulnerability in a rat thyroidectomy model. Our results stress that normal thyroid hormone levels are required to maintain normal cardiac electrophysiology and to prevent cardiac arrhythmias and AF.
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Fibrilación Atrial/etiología , Hipertiroidismo/complicaciones , Hipotiroidismo/complicaciones , Animales , Fibrilación Atrial/diagnóstico por imagen , Fibrilación Atrial/fisiopatología , Modelos Animales de Enfermedad , Ecocardiografía , Técnicas Electrofisiológicas Cardíacas , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemodinámica , Inmunohistoquímica , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Hormonas Tiroideas/sangre , TiroidectomíaRESUMEN
Thyroid hormones (THs) play a pivotal role in regulating cardiovascular homeostasis. To provide a better understanding of the coordinated processes that govern cardiac TH bioavailability, this study investigated the influence of serum and cardiac TH status on the expression of TH transporters and cytosolic binding proteins in the myocardium. In addition, we sought to determine whether the administration of T(3) (instead of T(4)) improves the relationship between THs in serum and cardiac tissue and cardiac function over a short-term treatment period. Adult female Sprague Dawley rats were made hypothyroid by 7 weeks treatment with the antithyroid drug 6-n-propyl-2-thiouracil (PTU). After establishing hypothyroidism, rats were assigned to 1 of 5 graded T(3) dosages plus PTU for a 2-week dose-response experiment. Untreated, age-matched rats served as euthyroid controls. PTU was associated with depressed serum and cardiac tissue T(3) and T(4) levels, arteriolar atrophy, altered TH transporter and cytosolic TH binding protein expression, fetal gene reexpression, and cardiac dysfunction. Short-term administration of T(3) led to a mismatch between serum and cardiac tissue TH levels. Normalization of serum T(3) levels was not associated with restoration of cardiac tissue T(3) levels or cardiac function. In fact, a 3-fold higher T(3) dosage was necessary to normalize cardiac tissue T(3) levels and cardiac function. Importantly, this study provides the first comprehensive data on the relationship between altered TH status (serum and cardiac tissue), cardiac function, and the coordinated in vivo changes in cardiac TH membrane transporters and cytosolic TH binding proteins in altered TH states.
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Hipotiroidismo/tratamiento farmacológico , Hormonas Tiroideas/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Corazón/efectos de los fármacos , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Hormonas Tiroideas/sangre , Tiroxina/sangre , Tiroxina/uso terapéutico , Triyodotironina/sangre , Triyodotironina/uso terapéuticoRESUMEN
Similarities in cardiac gene expression in hypothyroidism and left ventricular (LV) pathological remodeling after myocardial infarction (MI) suggest a role for impaired cardiac thyroid hormone (TH) signaling in the development of heart failure. Increased ventricular activity of the TH-degrading enzyme type 3 deiodinase (D3) is recognized as a potential cause. In the present study, we investigated the cardiac expression and activity of D3 over an 8-wk period after MI in C57Bl/6J mice. Pathological remodeling of the noninfarcted part of the LV was evident from cardiomyocyte hypertrophy, interstitial fibrosis, and impairment of contractility. These changes were maximal and stable from the first week onward, as was the degree of LV dilation. A strong induction of D3 activity was found, which was similarly stable for the period examined. Plasma T(4) levels were transiently decreased at 1 wk after MI, but T(3) levels remained normal. The high D3 activity was associated with increased D3 mRNA expression at 1 but not at 4 and 8 wk after MI. Immunohistochemistry localized D3 protein to cardiomyocytes. In vivo measurement of TH-dependent transcription activity in cardiomyocytes using a luciferase reporter assay indicated a 48% decrease in post-MI mice relative to sham-operated animals, and this was associated with a 50% decrease in LV tissue T(3) concentration. In conclusion, pathological ventricular remodeling after MI in the mouse leads to high and stable induction of D3 activity in cardiomyocytes and a local hypothyroid condition.
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Hipotiroidismo/metabolismo , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Remodelación Ventricular/fisiología , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Femenino , Yoduro Peroxidasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Hormonas Tiroideas/metabolismoRESUMEN
Serine protease inhibitors form a diverse family of proteins of which most members inhibit target serine proteases. Neuroserpin is a member of this family. Here, we have characterized neuroserpin in the nonmammalian species Xenopus laevis and found a high degree of aminoacid sequence conservation, especially of the reactive center loop, of the Xenopus protein compared to mammalian and chicken neuroserpin sequences, suggesting a conserved target specificity. Neuroserpin mRNA and protein were expressed throughout Xenopus development, while in the adult frog high mRNA expression was found in neuronal and neuroendocrine tissues, and the reproductive organs, and the neuroserpin protein was detected mainly in brain and pituitary. More specifically, in Xenopus pituitary neuroserpin mRNA was expressed higher in the neurointermediate lobe than in the pars distalis. At the protein level, we detected a 55-kDa neuroserpin protein in the pars nervosa, two neuroserpin proteins of 44- and 50-kDa in the melanotrope cells of the pars intermedia, and a 46-kDa product in the pars distalis. On the basis of its relatively high degree of sequence conservation and its expression pattern, we conclude that Xenopus neuroserpin may play an important physiological role, e.g. as a serine protease inhibitor during development, and for proper neuronal and neuroendocrine cell functioning.
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Neuropéptidos/genética , Adenohipófisis/metabolismo , Inhibidores de Serina Proteinasa/genética , Serpinas/genética , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Pollos , Clonación Molecular , ADN Complementario/aislamiento & purificación , Drosophila , Expresión Génica , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Sistemas Neurosecretores/metabolismo , ARN Mensajero/análisis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/metabolismo , Serpinas/metabolismo , Distribución Tisular , Xenopus laevis/metabolismo , NeuroserpinaRESUMEN
We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.