RESUMEN
Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.
Asunto(s)
Drosophila , Proteínas Musculares , Animales , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas Musculares/metabolismo , Estructura Terciaria de Proteína , Sarcómeros/química , Sarcómeros/metabolismoRESUMEN
Introduction: Heart failure remains one of the largest clinical challenges in the United States. Researchers have continually searched for more effective heart failure treatments that target the cardiac sarcomere but have found few successes despite numerous expensive cardiovascular clinical trials. Among many reasons, the high failure rate of cardiovascular clinical trials may be partly due to incomplete characterization of a drug candidate's complex interaction with cardiac physiology.Areas covered: In this review, the authors address the issue of preclinical cardiovascular studies of sarcomere-targeting heart failure therapies. The authors consider inherent tradeoffs made between mechanistic transparency and physiological fidelity for several relevant preclinical techniques at the atomic, molecular, heart muscle fiber, whole heart, and whole-organism levels. Thus, the authors suggest a comprehensive, bottom-up approach to preclinical cardiovascular studies that fosters scientific rigor and hypothesis-driven drug discovery.Expert opinion: In the authors' opinion, the implementation of hypothesis-driven drug discovery practices, such as the bottom-up approach to preclinical cardiovascular studies, will be imperative for the successful development of novel heart failure treatments. However, additional changes to clinical definitions of heart failure and current drug discovery culture must accompany the bottom-up approach to maximize the effectiveness of hypothesis-driven drug discovery.
Asunto(s)
Fármacos Cardiovasculares/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Sarcómeros/metabolismo , Animales , Desarrollo de Medicamentos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos , Insuficiencia Cardíaca/fisiopatología , HumanosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Leonurus japonicus Houtt. has a long history in the treatment of cardiovascular diseases. Stachydrine hydrochloride, the main bioactive ingredient extracted from Leonurus japonicus Houtt., has been shown to have cardioprotective effects. However, the underlying mechanisms of stachydrine hydrochloride haven't been comprehensively studied so far. AIM OF THE STUDY: The aim of this study was to investigate the protective role of stachydrine hydrochloride in heart failure and elucidate its possible mechanisms of action. MATERIALS AND METHODS: In vivo, transverse aorta constriction was carried out in C57BL/6J mice, and thereafter, 7.2â¯mg/kg telmisartan (a selective AT1R antagonist as positive control) and 12â¯mg/kg stachydrine hydrochloride was administered daily intragastrically for 4 weeks. Cardiac function was evaluated by assessing morphological changes as well as echocardiographic and haemodynamic parameters. In vitro, neonatal rat cardiomyocytes or adult mice cardiomyocytes were treated with stachydrine hydrochloride and challenged with phenylephrine (α-AR agonist). Ventricular myocytes were isolated from the hearts of C57BL/6J mice by Langendorff crossflow perfusion system. Intracellular calcium was measured by an ion imaging system. The length and movement of sarcomere were traced to evaluate the systolic and diastolic function of single myocardial cells. RESULTS: Stachydrine hydrochloride improved the cardiac function and calcium transient amplitudes, and inhibited the SR leakage and the amount of sparks in cardiac myocytes isolated from TAC mice. We also demonstrated that stachydrine hydrochloride could ameliorated phenylephrine-induced enhance in sarcomere contraction, calcium transients and calcium sparks. Moreover, our data shown that stachydrine hydrochloride blocked the hyper-phosphorylation of CaMKII, RyR2, PLN, and prevented the disassociation of FKBP12.6 from RyR2. CONCLUSION: Our results suggest that stachydrine hydrochloride exerts beneficial therapeutic effects against heart failure. These cardioprotective effects may be associated with the regulation of calcium handling by stachydrine hydrochloride through inhibiting the hyper-phosphorylation of CaMKII.
Asunto(s)
Aorta/fisiopatología , Presión Arterial , Señalización del Calcio/efectos de los fármacos , Fármacos Cardiovasculares/farmacología , Insuficiencia Cardíaca/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Prolina/análogos & derivados , Función Ventricular Izquierda/efectos de los fármacos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Aorta/cirugía , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Fosforilación , Prolina/farmacología , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Telmisartán/farmacologíaRESUMEN
Androgen therapy provides cardiovascular benefits for hypogonadism. However, myocardial hypertrophy, fibrosis, and infarction have been reported in testosterone or androgenic anabolic steroid abuse. Therefore, better understanding of the factors leading to adverse results of androgen abuse is needed. The aim of the present study was to examine the impact of high dose of androgen treatment on cardiac biology, and whether exposure duration modulates this response. Male rats were treated with 10 mg/kg testosterone, three times a week, for either 4 or 12 weeks; vehicle injections served as controls. Four weeks of testosterone treatment induced an increase in ventricular wall thickness, indicative of concentric hypertrophy, as well as increased ejection fraction; in contrast, both parameters were blunted following 12 weeks of high-dose testosterone treatment. Cardiac myocyte contractile parameters were assessed in isolated electrically stimulated myocytes (sarcomere and intracellular calcium dynamics), and in chemically permeabilized isolated myocardium (myofilament force development and tension-cost). High-dose testosterone treatment for 4 weeks was associated with increased myocyte contractile parameters, while 12 weeks treatment induced significant depression of these parameters, mirroring the cardiac pump function results. In conclusion, chronic administration of high-dose testosterone initially induces increased cardiac function. However, this initial beneficial impact is followed by significant depression of cardiac pump function, myocyte contractility, and cardiac myofilament function. Our results indicate that chronic high-testosterone usage is of limited use and may, instead, induce significant cardiac dysfunction.
Asunto(s)
Andrógenos/farmacología , Corazón/efectos de los fármacos , Contracción Miocárdica , Testosterona/farmacología , Andrógenos/administración & dosificación , Andrógenos/efectos adversos , Animales , Calcio/metabolismo , Células Cultivadas , Corazón/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Sarcómeros/fisiología , Testosterona/administración & dosificación , Testosterona/efectos adversosRESUMEN
Friedreich's ataxia (FA) is caused by reduced levels of frataxin, a highly conserved mitochondrial protein. There is currently no effective treatment for this disease, which is characterized by progressive neurodegeneration and cardiomyopathy, the latter being the most common cause of death in patients. We previously developed a Drosophila melanogaster cardiac model of FA, in which the fly frataxin is inactivated specifically in the heart, leading to heart dilatation and impaired systolic function. Methylene Blue (MB) was highly efficient to prevent these cardiac dysfunctions. Here, we used this model to screen in vivo the Prestwick Chemical Library, comprising 1280 compounds. Eleven drugs significantly reduced the cardiac dilatation, some of which may possibly lead to therapeutic applications in the future. The one with the strongest protective effects was paclitaxel, a microtubule-stabilizing drug. In parallel, we characterized the histological defects induced by frataxin deficiency in cardiomyocytes and observed strong sarcomere alterations with loss of striation of actin fibers, along with full disruption of the microtubule network. Paclitaxel and MB both improved these structural defects. Therefore, we propose that frataxin inactivation induces cardiac dysfunction through impaired sarcomere assembly or renewal due to microtubule destabilization, without excluding additional mechanisms. This study is the first drug screening of this extent performed in vivo on a Drosophila model of cardiac disease. Thus, it also brings the proof of concept that cardiac functional imaging in adult Drosophila flies is usable for medium-scale in vivo pharmacological screening, with potent identification of cardioprotective drugs in various contexts of cardiac diseases.
Asunto(s)
Cardiotónicos/análisis , Cardiotónicos/uso terapéutico , Drosophila melanogaster/fisiología , Evaluación Preclínica de Medicamentos , Ataxia de Friedreich/tratamiento farmacológico , Actinas/metabolismo , Animales , Cardiotónicos/farmacología , Modelos Animales de Enfermedad , Ataxia de Friedreich/patología , Proteínas de Unión a Hierro/metabolismo , Azul de Metileno/farmacología , Azul de Metileno/uso terapéutico , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miosinas/metabolismo , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Reproducibilidad de los Resultados , Sarcómeros/metabolismo , Bibliotecas de Moléculas Pequeñas , FrataxinaRESUMEN
Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 µm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca2+ sensitivity for 2.5 vs. 2.0 µm sarcomere length fibers (pCa50 = 5.68 ± 0.01 vs. 5.60 ± 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca2+ sensitivity of force was attenuated, though the Ca2+ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by â¼15% as sarcomere length increased, due to a slower MgADP release rate (11.2 ± 0.5 vs. 13.5 ± 0.7 s-1). In the presence of dextran, cross-bridge detachment was â¼20% slower at 2.5 vs. 2.0 µm sarcomere length due to a slower MgADP release rate (10.1 ± 0.6 vs. 12.9 ± 0.5 s-1). However, osmotic compression of fibers at either 2.5 or 2.0 µm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing.
Asunto(s)
Adenosina Difosfato/metabolismo , Contracción Muscular/efectos de los fármacos , Miosinas/metabolismo , Sarcómeros/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Calcio/farmacología , Dextranos/farmacología , Relación Dosis-Respuesta a Droga , Elasticidad/efectos de los fármacos , Cinética , Locomoción/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Sarcómeros/efectos de los fármacos , Sarcómeros/fisiología , Viscosidad/efectos de los fármacosRESUMEN
BACKGROUND: Reactive oxygen species, such as superoxide, are being increasingly recognized as key components of a vast array of signaling pathways. Angiotensin II is a well-recognized stimulus for superoxide production through NADPH oxidase activation and opening of the mitochondrial ATP-sensitive potassium channels (mKATP). A role for this mechanism has been proposed to explain several physiological effects of the peptide. The aim of this study was to evaluate the involvement of this mechanism in the inotropic response to 100nmol/L angiotensin II. METHODS: Sarcomere shortening and intracellular pH (BCECF-epifluorescence technique) were evaluated in isolated cat ventricular myocytes placed in a perfusion chamber on the stage of an inverted microscope. Myocardial superoxide production was evaluated by the lucigenin quimioluminiscence method. RESULTS: Angiotensin II (100nmol/L) increased~70% sarcomere shortening, effect that was only partially prevented by NADPH oxidase inhibition, mKATP channel blockade or inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1). Moreover, angiotensin II stimulates NHE-1 activity by a NADPH oxidase-dependent mechanism. Myocardial superoxide production was also increased by angiotensin II, and this action was completely prevented either by NADPH oxidase inhibition or mKATP channel blockade. CONCLUSIONS: The positive inotropic response to 100nmol/L angiotensin II is due to both ROS/NHE-1 dependent and independent pathways, this being a point of divergence with the signaling previously described to be triggered by lower concentrations of angiotensin II (i.e.: 1nmol/L).
Asunto(s)
Angiotensina II/farmacología , Cardiotónicos/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Superóxidos/metabolismo , Animales , Gatos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ventrículos Cardíacos/metabolismo , Concentración de Iones de Hidrógeno , Miocitos Cardíacos/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Transducción de Señal/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD.
Asunto(s)
Músculo Esquelético/patología , Mioblastos/fisiología , Fenilbutiratos/farmacología , Plectina/deficiencia , Animales , Diferenciación Celular , Células Cultivadas , Desmina/metabolismo , Evaluación Preclínica de Medicamentos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Filamentos Intermedios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/tratamiento farmacológico , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Fenilbutiratos/uso terapéutico , Plectina/genética , Estabilidad Proteica , Sarcómeros/metabolismo , Sarcómeros/patología , Regulación hacia ArribaRESUMEN
Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²âº transient amplitude. Effects were primarily observed at 5 and 10 µM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²âº transient amplitudes. At 10 µM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²âº handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.
Asunto(s)
Calcio/metabolismo , Contracción Muscular/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Clorhidrato de Raloxifeno/farmacología , Tamoxifeno/análogos & derivados , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Femenino , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Ratas , Ratas Sprague-Dawley , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Sarcómeros/fisiología , Tamoxifeno/farmacologíaRESUMEN
It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG) and that exposure of zinc ion (Zn2+) to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after ß-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% ß-myosin heavy chain expression in the heart. ß-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR) at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our results suggest that the relaxing effects of Zn2+ on cardiomyocyte function are more pronounced in the HG state due an insulin-dependent effect of enhancing removal of cytosolic Ca2+ via SERCA2a or NCX or by reducing Ca2+ influx via L-type channel or Ca2+ leak through the RyR. Investigations into the effects of Zn2+ on these mechanisms are now underway.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosinas Ventriculares/metabolismo , Zinc/metabolismo , Animales , Glucemia/metabolismo , Calcio/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/fisiopatología , Diástole , Regulación de la Expresión Génica , Hipotiroidismo/metabolismo , Hipotiroidismo/fisiopatología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Necrosis , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Factores de TiempoRESUMEN
Chemical approaches are widely used in directed differentiation of embryonic stem (ES) cells. In our search for novel lead compounds that could facilitate cardiomyogenesis of ES cells, we designed a two-step screening system based on P19 embryonic carcinoma and mouse ES cells. Application of this system to a quinazoline compound library including 2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines and 2,6-disubstituted 4-anilinoquinazoline led us to the discovery of compound 62, which exhibits a stable cardiomyogenic effect on both P19 and mouse ES cells at a concentration of 0.1 µM. An EGFR inhibition assay and molecular docking studies confirmed 62 as a potent EGFR inhibitor with a tyrosine kinase IC(50) value of 101 nM. However, major differences in cardiomyogenic activity were observed between iressa and 62, indicating that other molecular events are also involved in compound 62-induced cardiomyogenesis of ES cells.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/citología , Quinazolinas/química , Quinazolinas/farmacología , Actinina/metabolismo , Animales , Factor Natriurético Atrial/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Receptores ErbB/antagonistas & inhibidores , Concentración 50 Inhibidora , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas/síntesis química , Sarcómeros/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
1. Evidence is accumulating for a role for Ca²âº signalling in the differentiation and development of embryonic skeletal muscle. 2. Imaging of intact, normally developing transgenic zebrafish that express the protein component of the Ca²âº-sensitive complex aequorin, specifically in skeletal muscle, show that two distinct periods of spontaneous synchronised Ca²âº transients occur in the trunk: one at approximately 17.5-19.5 h post-fertilization (h.p.f.; termed signalling period SP1) and the other after approximately 23 h.p.f. (termed SP2). These periods of intense Ca²âº signalling activity are separated by a quiet period. 3. Higher-resolution confocal imaging of embryos loaded with the fluorescent Ca²âº reporter calcium green-1 dextran shows that the Ca²âº signals are generated almost exclusively in the slow muscle cells, the first muscle cells to differentiate, with distinct nuclear and cytoplasmic components. 4. Here, we show that coincidental with the SP1 Ca²âº signals, dystrophin becomes localized to the vertical myoseptae of the myotome. Introduction of a dmd morpholino (dmd-MO) resulted in no dystrophin being expressed in the vertical myoseptae, as well as a disruption of myotome morphology and sarcomere organization. In addition, the Ca²âº signalling signatures of dmd-MO-injected embryos or homozygous sapje mutant embryos were abnormal such that the frequency, amplitude and timing of the Ca²âº signals were altered compared with controls. 5. Our new data suggest that, in addition to a structural role, dystrophin may function in the regulation of [Ca²âº](i) during the early stages of slow muscle cell differentiation when the Ca²âº signals generated in these cells coincide with the first spontaneous contractions of the trunk.
Asunto(s)
Señalización del Calcio , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Desarrollo de Músculos , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Lenta/patología , Distrofias Musculares/metabolismo , Animales , Animales Modificados Genéticamente , Señalización del Calcio/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Citoplasma/efectos de los fármacos , Citoplasma/patología , Desarrollo Embrionario/efectos de los fármacos , Mediciones Luminiscentes/métodos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente/métodos , Morfolinos/farmacología , Contracción Muscular/efectos de los fármacos , Desarrollo de Músculos/efectos de los fármacos , Fibras Musculares de Contracción Lenta/efectos de los fármacos , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofias Musculares/embriología , Distrofias Musculares/patología , Mutación , Especificidad de Órganos , Transporte de Proteínas/efectos de los fármacos , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Sarcómeros/patología , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In this study, 20 young steers received no beta-agonist (C), 100 animals all received zilpaterol hydrochloride (Z), with 1 group only receiving Z while the other 4 groups received zilpaterol and vitamin D3 at the following levels (IU/animal/day) and durations before slaughter: 7 million for 3 days (3D7M); 7 million for 6 days (6D7M); 7 million for 6 days with 7 days no supplementation (6D7M7N) and 1 million for 9 days (9D1M). Left carcass sides were electrically stimulated (ES) and the right side not stimulated (NES). Samples were aged for 3 or 14 days post mortem. Parameters included Warner-Bratzler shear force (WBSF), myofibril filament length, sarcomere length and calpastatin and calpain enzyme activity. Both ES and prolonged aging reduced WBSF (P<0.001). 6D7M, 6D7M7N and Z remained significantly tougher than C (P<0.001), while 3D7M and 9D1M improved WBSF under NES conditions. ES is more effective to alleviate beta-agonist induced toughness than high vitamin D3 supplements.
Asunto(s)
Colecalciferol/administración & dosificación , Suplementos Dietéticos , Estimulación Eléctrica , Carne/análisis , Compuestos de Trimetilsililo/administración & dosificación , Alimentación Animal , Animales , Calcio/sangre , Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Bovinos , Masculino , Miofibrillas/metabolismo , Hormona Paratiroidea/sangre , Proteolisis/efectos de los fármacos , Sarcómeros/metabolismoRESUMEN
The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised â¼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.
Asunto(s)
Citoesqueleto de Actina/fisiología , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Miosinas/genética , Animales , Western Blotting , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Permeabilidad de la Membrana Celular/fisiología , Separación Celular , ADN Complementario/biosíntesis , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Inmunohistoquímica , Inmunoprecipitación , Miocardio/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Miosinas/biosíntesis , Miosinas/química , Conformación Proteica , Ratas , Sarcómeros/metabolismoRESUMEN
Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label. Under physiological conditions, a close association of gelsolin with the thin filaments was observed for both muscle types. The preferential localization of particles in the I- and AI-region indicated that gelsolin was distributed randomly over the whole length of the thin filaments. Preincubation of muscle strips with Ringer solution containing 0.5 mM EGTA resulted in a significantly different distribution pattern; gold particles were now localized preferentially in the cell periphery close to the sarcolemma, with significantly decreased abundance in the centre of the cell. Compared with the muscle under physiological conditions, the number of gold particles over sarcomeric structures was significantly reduced. Thus, binding of gelsolin to the thin filaments is apparently reversible in vivo and depends on the presence of calcium ions. We assume a functional role for gelsolin in the actin turnover processes in invertebrate muscle systems.
Asunto(s)
Gelsolina/análisis , Miofibrillas/química , Nephropidae/química , Actinas/análisis , Actinas/metabolismo , Animales , Western Blotting , Calcio/análisis , Calcio/metabolismo , Citoesqueleto/metabolismo , Electroforesis en Gel de Poliacrilamida , Gelsolina/inmunología , Gelsolina/ultraestructura , Microscopía Inmunoelectrónica , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Nephropidae/anatomía & histología , Nephropidae/ultraestructura , Sarcómeros/química , Sarcómeros/metabolismo , Sarcómeros/ultraestructuraRESUMEN
Cytokinetics Inc is developing CK-1827452, an agent that directly activates myosin, for use in the treatment of heart failure. The reaction of myosin cross-bridges from the thick myofilaments of cardiac sarcomeres with actin molecules from the thin myofilaments induces the muscle force and shortening that is responsible for systolic pressure development and ejection. Preclinical in vitro studies with isolated proteins investigated the mode of action of CK-1827452, demonstrating the ability of the drug to prolong the duration of time that myosin motors remain in a force-generating reaction with actin. Studies with isolated field-stimulated cardiac myocytes demonstrated the critical property of enhancing the extent of myocyte shortening, with no effect on the Ca2+ transient. Thus, CK-1827452 represents a novel myosin activator that acts independently of Ca2+, with an advantage compared with inotropic agents that elevate cAMP, enhance Ca2+ fluxes and have the potential to induce arrhythmias. Phase II clinical trials in patients with heart failure treated with intravenous and/or oral formulations of CK-1827452 support the hypothesis that the direct activation of myosin is a well-tolerated and viable approach to improving cardiac function. CK-1827452 represents a new and promising class of pure sarcomeric activators with a novel mechanism that could be an improvement over existing agents acting in part through this mechanism.
Asunto(s)
Cardiotónicos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Miocardio/metabolismo , Miosinas/metabolismo , Sarcómeros/efectos de los fármacos , Enfermedad Aguda , Cardiotónicos/administración & dosificación , Cardiotónicos/efectos adversos , Cardiotónicos/farmacología , Enfermedad Crónica , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Sarcómeros/metabolismo , Resultado del TratamientoRESUMEN
Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.
Asunto(s)
Endotelinas/química , Troponina I/química , Citoesqueleto de Actina/metabolismo , Adenoviridae/genética , Alanina/química , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Sitios de Unión , Calcio/metabolismo , Células Cultivadas , Análisis por Conglomerados , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Endotelinas/metabolismo , Concentración de Iones de Hidrógeno , Modelos Estadísticos , Células Musculares/citología , Mutagénesis , Contracción Miocárdica , Miocardio/citología , Miocardio/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Serina/química , Treonina/química , Factores de Tiempo , Troponina I/metabolismoRESUMEN
Projectin is a giant protein related to twitchin and titin/connectin, that is found in arthropod striated muscle. The complete sequence of a 1 MDa projectin from Drosophila muscle was recently deduced from a thorough analysis of the genomic DNA (Southgate and Ayme-Southgate, 2001). Here we report the complete sequence for projectin from crayfish claw closer muscle (8625 residues; 962,634 Da). The N-terminal sequence contains 12 unique 19-residue repeats rich in glutamic acid (E) and lysine (K). This region, termed the EK region, is clearly distinguishable from the PEVK-like domain of Drosophila projectin. The sequence of crayfish flexor projectin differs from that of closer muscle projectin in that there is a 114-residue deletion and a 35-residue insertion in the N-terminal region. Immunofluorescence microscopy demonstrated that projectin is mainly localized within the sarcomeric A band in both closer and flexor muscles, although the N-terminal region was shown to extrude into the I band region. In the closer muscles, invertebrate connectin (D-titin) connects the Z line to the edge of the A band (Fukuzawa et al., 2001). We have shown that invertebrate connectin is also present in flexor muscle sarcomeres, although in very low abundance.
Asunto(s)
Astacoidea/genética , Proteínas Musculares/genética , Músculos/metabolismo , Sarcómeros/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Astacoidea/metabolismo , Proteínas de Caenorhabditis elegans , Proteínas de Unión a Calmodulina/metabolismo , ADN Complementario/genética , Ácido Glutámico/metabolismo , Lisina/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sarcómeros/genéticaRESUMEN
The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.
Asunto(s)
Calcineurina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Musculares/metabolismo , Actinina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Calcineurina/genética , Proteínas Portadoras/genética , Chlorocebus aethiops , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica , Corazón/embriología , Humanos , Ratones , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Estructura Terciaria de Proteína , Conejos , Sarcómeros/metabolismo , Factores de TiempoRESUMEN
The assembly of stable cytoskeletal structures from dynamically recycled molecules requires developmental and spatial regulation of protein interactions. In muscle, titin acts as a molecular ruler organizing the actin cytoskeleton via interactions with many sarcomeric proteins, including the crosslinking protein alpha-actinin. An interaction between the C-terminal domain of alpha-actinin and titin Z-repeat motifs targets alpha-actinin to the Z-disk. Here we investigate the cellular regulation of this interaction. alpha-actinin is a rod shaped head-to-tail homodimer. In contrast to C-terminal fragments, full-length alpha-actinin does not bind Z-repeats. We identify a 30-residue Z-repeat homologous sequence between the actin-binding and rod regions of alpha-actinin that binds the C-terminal domain with nanomolar affinity. Thus, Z-repeat binding is prevented by this 'pseudoligand' interaction between the subunits of the alpha-actinin dimer. This autoinhibition is relieved upon binding of the Z-disk lipid phosphatidylinositol-bisphosphate to the actin-binding domain. We suggest that this novel mechanism is relevant to control the site-specific interactions of alpha-actinin during sarcomere assembly and turnover. The intramolecular contacts defined here also constrain a structural model for intrasterical regulation of all alpha-actinin isoforms.