RESUMEN
Modulation of endothelial nitric oxide synthase (eNOS) activation is recognized as a main trigger of the cardioprotective effects of exercise training on heart vulnerability to ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes and the contribution of each one to such cardioprotection has never been challenged. The aim of this study was to investigate the role of eNOS from the cardiomyocytes vs. the endothelium in the exercise cardioprotection. Male Wistar rats were assigned to a chronic aerobic training (Ex) (vs. sedentary group; Sed) and we investigated the role of eNOS in the effects of exercise on sensitivity to IR or anoxia-reoxygenation (A/R) at whole heart, isolated cardiomyocytes and left coronary artery (LCA) levels. We observed that exercise increased eNOS activation (Ser1177 phosphorylation) and protein S-nitrosylation in whole heart but not at cardiomyocyte level, suggesting the specific target of endothelial cells by exercise. Consistently, in isolated cardiomyocytes submitted to the A/R procedure, exercise reduced cell death and improved cells contractility, but independently of the eNOS pathway. Next, to evaluate the contribution of endothelial cells in exercise cardioprotection, LCA were isolated before and after an IR procedure performed on Langendorff hearts. Exercise improved basal relaxation sensitivity to acetylcholine and markedly reduced the alteration of endothelium-dependent coronary relaxation induced by IR. Furthermore, inactivation of coronary endothelial cells activity just before IR, obtained with a bolus of Triton X-100, totally suppressed cardioprotective effects of exercise on both left ventricular functional recovery after IR and infarct size, whereas no effect of Triton X-100 was observed in Sed group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in the eNOS-dependent cardioprotection of exercise.
Asunto(s)
Endotelio/metabolismo , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Condicionamiento Físico Animal , Animales , Modelos Animales de Enfermedad , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Óxido Nítrico/metabolismo , Fosforilación , Ratas , Transducción de SeñalRESUMEN
Clenbuterol is a ß2 -adrenergic receptor agonist known to induce skeletal muscle hypertrophy and a slow-to-fast phenotypic shift. The aim of the present study was to test the effects of chronic clenbuterol treatment on contractile efficiency and explore the underlying mechanisms, i.e. the muscle contractile machinery and calcium-handling ability. Forty-three 6-week-old male Wistar rats were randomly allocated to one of six groups that were treated with either subcutaneous equimolar doses of clenbuterol (4 mg kg(-1) day(-1) ) or saline solution for 9, 14 or 21 days. In addition to the muscle hypertrophy, although an 89% increase in absolute maximal tetanic force (Po ) was noted, specific maximal tetanic force (sPo) was unchanged or even depressed in the slow twitch muscle of the clenbuterol-treated rats (P < 0.05). The fit of muscle contraction and relaxation force kinetics indicated that clenbuterol treatment significantly reduced the rate constant of force development and the slow and fast rate constants of relaxation in extensor digitorum longus muscle (P < 0.05), and only the fast rate constant of relaxation in soleus muscle (P < 0.05). Myofibrillar ATPase activity increased in both relaxed and activated conditions in soleus (P < 0.001), suggesting that the depressed specific tension was not due to the myosin head alteration itself. Moreover, action potential-elicited Ca(2+) transients in flexor digitorum brevis fibres (fast twitch fibres) from clenbuterol-treated animals demonstrated decreased amplitude after 14 days (-19%, P < 0.01) and 21 days (-25%, P < 0.01). In conclusion, we showed that chronic clenbuterol treatment reduces contractile efficiency, with altered contraction and relaxation kinetics, but without directly altering the contractile machinery. Lower Ca(2+) release during contraction could partially explain these deleterious effects.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Clenbuterol/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/metabolismo , Hipertrofia/inducido químicamente , Hipertrofia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/metabolismo , Ratas , Ratas WistarRESUMEN
Exercise is an efficient strategy for myocardial protection against ischemia-reperfusion (IR) injury. Although endothelial nitric oxide synthase (eNOS) is phosphorylated and activated during exercise, its role in exercise-induced cardioprotection remains unknown. This study investigated whether modulation of eNOS activation during IR could participate in the exercise-induced cardioprotection against IR injury. Hearts isolated from sedentary or exercised rats (5 weeks training) were perfused with a Langendorff apparatus and IR performed in the presence or absence of NOS inhibitors [N-nitro-L-arginine methyl ester, L-NAME or N5-(1-iminoethyl)-L-ornithine, L-NIO] or tetrahydrobiopterin (BH4). Exercise training protected hearts against IR injury and this effect was abolished by L-NAME or by L-NIO treatment, indicating that exercise-induced cardioprotection is eNOS dependent. However, a strong reduction of eNOS phosphorylation at Ser1177 (eNOS-PSer1177) and of eNOS coupling during early reperfusion was observed in hearts from exercised rats (which showed higher eNOS-PSer1177 and eNOS dimerization at baseline) in comparison to sedentary rats. Despite eNOS uncoupling, exercised hearts had more S-nitrosylated proteins after early reperfusion and also less nitro-oxidative stress, indexed by lower malondialdehyde content and protein nitrotyrosination compared to sedentary hearts. Moreover, in exercised hearts, stabilization of eNOS dimers by BH4 treatment increased nitro-oxidative stress and then abolished the exercise-induced cardioprotection, indicating that eNOS uncoupling during IR is required for exercise-induced myocardial cardioprotection. Based on these results, we hypothesize that in the hearts of exercised animals, eNOS uncoupling associated with the improved myocardial antioxidant capacity prevents excessive NO synthesis and limits the reaction between NO and O2·- to form peroxynitrite (ONOOâ»), which is cytotoxic.
Asunto(s)
Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Western Blotting , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Ratas , Ratas WistarRESUMEN
INTRODUCTION: Cyclooxygenase-2 (COX2) is an enzyme that plays a role in different stages of the carcinogenic process and has prognostic and predictive values that have not yet been established. The objective of this study was to evaluate the role of COX2 overexpression in advanced squamous cell carcinoma of the larynx that has been treated with a phonation conservation protocol. MATERIALS AND METHODS: This study included a retrospective analysis of 59 patients with resectable tumours that were treated with chemotherapy (CT) and/or radiation therapy (RT). The expression levels of COX2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor (VEGFR-2) in collected biopsy specimens were determined via immunohistochemistry. RESULTS: Forty-four percent of the included samples demonstrated overexpression of COX2. In the statistical analysis, COX2 overexpression did not correlate with other clinical or treatment efficacy prognostic factors; however, the median global survival (OS) of patients whose tumours expressed COX2 was 79 months, whereas COX2-negative patients had a median OS of only 38 months, although this finding did not reach statistical significance. The other analysed biological parameters did not demonstrate a significant relationship with COX2. CONCLUSIONS: COX2 overexpression was a common finding in our study. The results obtained did not reveal relationships with established prognostic categories; however, the difference in survival between patients with and without COX2 expression justifies the need for future prospective studies that utilise a larger patient sample size (AU)
Asunto(s)
Humanos , Masculino , Femenino , Activación Enzimática , Activación Enzimática/inmunología , Activación Enzimática/efectos de la radiación , Activación Enzimática/genética , Activación Enzimática/fisiologíaRESUMEN
INTRODUCTION: Cyclooxygenase-2 (COX2) is an enzyme that plays a role in different stages of the carcinogenic process and has prognostic and predictive values that have not yet been established. The objective of this study was to evaluate the role of COX2 overexpression in advanced squamous cell carcinoma of the larynx that has been treated with a phonation conservation protocol. MATERIALS AND METHODS: This study included a retrospective analysis of 59 patients with resectable tumours that were treated with chemotherapy (CT) and/or radiation therapy (RT). The expression levels of COX2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor (VEGFR-2) in collected biopsy specimens were determined via immunohistochemistry. RESULTS: Forty-four percent of the included samples demonstrated overexpression of COX2. In the statistical analysis, COX2 overexpression did not correlate with other clinical or treatment efficacy prognostic factors; however, the median global survival (OS) of patients whose tumours expressed COX2 was 79 months, whereas COX2-negative patients had a median OS of only 38 months, although this finding did not reach statistical significance. The other analysed biological parameters did not demonstrate a significant relationship with COX2. CONCLUSIONS: COX2 overexpression was a common finding in our study. The results obtained did not reveal relationships with established prognostic categories; however, the difference in survival between patients with and without COX2 expression justifies the need for future prospective studies that utilise a larger patient sample size.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Ciclooxigenasa 2/metabolismo , Neoplasias Laríngeas/tratamiento farmacológico , Fonación/efectos de los fármacos , Adulto , Anciano , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/fisiopatología , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Neoplasias Laríngeas/patología , Neoplasias Laríngeas/fisiopatología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Since Claude Bernard first demonstrated in the 19th century that carbon monoxide (CO) poisoning occurs through hemoglobin binding, CO has proven to be more than simply a toxic gas, and to possess complex biological properties. In this review, we highlight the dual nature of CO in cardiovascular function, from endogenous and therapeutic properties to harmful aspects. Focussing on exposure to low environmental CO levels, the most common but least studied form of exposure, we summarize the pathophysiological effects of CO in vivo and in vitro, from cardiac disorders to phenotypic remodelling of cardiomyocytes, based on clinical observations and experimental studies. While acute exposure to low CO levels is considered beneficial and cardioprotective, prolonged exposure appears deleterious, mainly due to alterations in redox status, ion homeostasis, intracellular Ca(2+) handling, and sympathovagal balance. We emphasize that, despite its fascinating therapeutic potential at low levels, regular exposure to CO may have significant consequences on cardiovascular health and must be considered a cardiovascular risk factor.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Monóxido de Carbono/toxicidad , Exposición a Riesgos Ambientales/efectos adversos , Cardiopatías/inducido químicamente , Animales , Fenómenos Electrofisiológicos , Cardiopatías/epidemiología , Cardiopatías/terapia , Humanos , Población UrbanaRESUMEN
Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16- PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Calcio/metabolismo , Infarto del Miocardio/metabolismo , Miofibrillas/metabolismo , Remodelación Ventricular/efectos de los fármacos , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Diástole , Modelos Animales de Enfermedad , Acoplamiento Excitación-Contracción/efectos de los fármacos , Masculino , Ratones , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/mortalidad , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Sustained urban carbon monoxide (CO) exposure exacerbates heart vulnerability to ischemia-reperfusion via deleterious effects on the antioxidant status and Ca(2+) homeostasis of cardiomyocytes. The aim of this work was to evaluate whether moderate exercise training prevents these effects. Wistar rats were randomly assigned to a control group and to CO groups, living during 4 wk in simulated urban CO pollution (30-100 parts/million, 12 h/day) with (CO-Ex) or sedentary without exercise (CO-Sed). The exercise procedure began 4 wk before CO exposure and was maintained twice a week in standard filtered air during CO exposure. On one set of rats, myocardial ischemia (30 min) and reperfusion (120 min) were performed on isolated perfused rat hearts. On another set of rats, myocardial antioxidant status and Ca(2+) handling were evaluated following environmental exposure. As a result, exercise training prevented CO-induced myocardial phenotypical changes. Indeed, exercise induced myocardial antioxidant status recovery in CO-exposed rats, which is accompanied by a normalization of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a expression and then of Ca(2+) handling. Importantly, in CO-exposed rats, the normalization of cardiomyocyte phenotype with moderate exercise was associated with a restored sensitivity of the myocardium to ischemia-reperfusion. Indeed, CO-Ex rats presented a lower infarct size and a significant decrease of reperfusion arrhythmias compared with their sedentary counterparts. To conclude, moderate exercise, by preventing CO-induced Ca(2+) handling and myocardial antioxidant status alterations, reduces heart vulnerability to ischemia-reperfusion.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Señalización del Calcio/efectos de los fármacos , Monóxido de Carbono/toxicidad , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Resistencia Física , Animales , Antioxidantes/metabolismo , Catalasa/metabolismo , Muerte Celular , Glutatión Peroxidasa/metabolismo , Exposición por Inhalación , Masculino , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/inducido químicamente , Infarto del Miocardio/metabolismo , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/inducido químicamente , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Fibrilación Ventricular/inducido químicamente , Fibrilación Ventricular/metabolismo , Fibrilación Ventricular/prevención & controlRESUMEN
Myocardial damages due to ischemia-reperfusion (I/R) are recognized to be the result of a complex interplay between genetic and environmental factors. Epidemiological studies suggested that, among environmental factors, carbon monoxide (CO) urban pollution can be linked to cardiac diseases and mortality. The aim of this work was to evaluate the impact of exposure to CO pollution on cardiac sensitivity to I/R. Regional myocardial I/R was performed on isolated perfused hearts from rats exposed for 4 wk to air enriched with CO (30-100 ppm). Functional variables, reperfusion ventricular arrhythmias (VA) and cellular damages (infarct size, lactate dehydrogenase release) were assessed. Sarcomere length shortening and Ca(2+) handling were evaluated in intact isolated cardiomyocytes during a cellular anoxia-reoxygenation protocol. The major results show that prolonged CO exposure worsens myocardial I/R injuries, resulting in increased severity of postischemic VA, impaired recovery of myocardial function, and increased infarct size (60 +/- 5 vs. 33 +/- 2% of ischemic zone). The aggravating effects of CO exposure on I/R could be explained by a reduced myocardial enzymatic antioxidant status (superoxide dismutase -45%; glutathione peroxidase -49%) associated with impaired intracellular Ca(2+) handling. In conclusion, our results are consistent with the idea that chronic CO pollution dramatically increases the severity of myocardial I/R injuries.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Contaminación del Aire/efectos adversos , Monóxido de Carbono/toxicidad , Daño por Reperfusión Miocárdica/patología , Contaminantes Atmosféricos/análisis , Animales , Antioxidantes/metabolismo , Arritmias Cardíacas/fisiopatología , Calcio/fisiología , Monóxido de Carbono/análisis , Vasos Coronarios/fisiología , Glutatión Peroxidasa/metabolismo , Ventrículos Cardíacos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Miocardio/enzimología , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Wistar , Sarcómeros/efectos de los fármacos , Sarcómeros/ultraestructura , Superóxido Dismutasa/metabolismoRESUMEN
OBJECTIVE: Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level. METHODS: Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL). RESULTS: Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch- dependent Ca2+ sensitization of the myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca2+ sensitivity. In contrast, high-altitude training decreased Ca2+ sensitivity of the myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data. CONCLUSIONS: Training at sea level increased the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed myofilament response to Ca2+, especially in the Endo layer. This led to a reduction in this transmural gradient that may contribute to the lack of improvement in LV function via the Frank-Starling mechanism.
Asunto(s)
Mal de Altura/fisiopatología , Altitud , Miocitos Cardíacos/patología , Condicionamiento Físico Animal , Citoesqueleto de Actina/metabolismo , Mal de Altura/patología , Animales , Fenómenos Biomecánicos , Calcio/metabolismo , Ecocardiografía , Técnicas In Vitro , Masculino , Contracción Miocárdica , Cadenas Pesadas de Miosina/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Volumen SistólicoRESUMEN
We studied the effect of titin-based passive force on the length dependence of activation of cardiac myocytes to explore whether titin may play a role in the generation of systolic force. Force-pCa relations were measured at sarcomere lengths (SLs) of 2.0 and 2.3 microm. Passive tension at 2.3 microm SL was varied from approximately 1 to approximately 10 mN/mm(2) by adjusting the characteristics of the stretch imposed on the passive cell before activation. Relative to 2.0 microm SL, the force-pCa curve at 2.3 microm SL and low passive tension showed a leftward shift (pCa(50) [change in pCa at half-maximal activation]) of 0.09+/-0.02 pCa units while at 2.3 microm SL and high passive tension the shift was increased to 0.25+/-0.03 pCa units. Passive tension also increased pCa(50) at reduced interfilament lattice spacing achieved with dextran. We tested whether titin-based passive tension influences the interfilament lattice spacing by measuring the width of the myocyte and by using small-angle x-ray diffraction of mouse left ventricular wall muscle. Cell width and interfilament lattice spacing varied inversely with passive tension, in the presence and absence of dextran. The passive tension effect on length-dependent activation may therefore result from a radial titin-based force that modulates the interfilament lattice spacing.
Asunto(s)
Calcio/metabolismo , Proteínas Musculares/metabolismo , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Proteínas Quinasas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Animales , Calcio/farmacología , Células Cultivadas , Conectina , Dextranos/farmacología , Electroforesis en Gel de Poliacrilamida , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/ultraestructura , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/fisiología , Ratones , Proteínas Musculares/análisis , Proteínas Musculares/efectos de la radiación , Contracción Miocárdica/efectos de los fármacos , Miocardio/química , Miocardio/ultraestructura , Concentración Osmolar , Proteínas Quinasas/análisis , Proteínas Quinasas/efectos de la radiación , Sarcómeros/fisiología , Sarcómeros/ultraestructura , Estrés Mecánico , Tripsina/metabolismo , Tripsina/farmacología , Difracción de Rayos XRESUMEN
The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.
Asunto(s)
Secuencia Conservada , Fibronectinas/química , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Subfragmentos de Miosina/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/farmacología , Conectina , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Humanos , Inmunoglobulinas/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Musculares/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Conejos , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Alineación de Secuencia , Solventes/metabolismo , Relación Estructura-Actividad , Función VentricularRESUMEN
Small (N2B) and large (N2BA) cardiac titin isoforms are differentially expressed in a species-specific and heart location-specific manner. To understand how differential expression of titin isoforms may influence passive stiffness of cardiac muscle we investigated the mechanical properties of mouse left ventricular (MLV) wall muscle (expressing predominantly the small titin isoform), bovine left atrial (BLA) wall muscle (predominantly the large isoform), and bovine left ventricular (BLV) wall muscle (expressing small and large isoforms at similar levels). Results indicate that the overall passive muscle stiffness of the muscle types varies nearly ten-fold, with stiffness increasing in the following order: BLA, BLV and MLV. To investigate the basis of the variation in the overall muscle stiffness, the contributions of titin and collagen to muscle stiffness were determined. Results showed that increased muscle stiffness results from increases in both titin- and collagen-based passive stiffness, indicating that titin and collagen change in a co-ordinated fashion. The expression level of the small titin isoform correlates with titin's contribution to overall muscle stiffness, suggesting that differential expression of titin isoforms is an effective means to modulate the filling behavior of the heart.
Asunto(s)
Colágeno/metabolismo , Diástole , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Proteínas Quinasas/metabolismo , Animales , Western Blotting , Bovinos , Conectina , Electroforesis en Gel de Poliacrilamida , Corazón/fisiología , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Musculares/química , Isoformas de Proteínas , Proteínas Quinasas/química , Sarcómeros/metabolismoRESUMEN
Titin is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the sarcomere. As a result of differential splicing, length variants of titin are expressed in different skeletal and cardiac muscles. Here we first briefly review some of our previous work that has revealed that titin develops force in sarcomeres either stretched beyond their slack length (passive force) or shortened to below the slack length (restoring force) and that titin's force underlies a large fraction of the diastolic force of cardiac muscle. Next we present our mechanical and immunoelectron microscopical (IEM) studies of skeletal and cardiac muscles that express titin isoforms. The previously deduced molecular properties of titin were used to model titin's extensible region in the sarcomere as serially linked WLCs: rigid segments (containing folded Ig/Fn domains) and more flexible segments (PEVK segment). The model was tested on skeletal muscle fibers that express titin isoforms with tandem Ig and PEVK length variants. The model adequately predicts titin's behavior along a wide sarcomere length range in skeletal muscle, but at long sarcome lengths (SLs), predicted forces are much higher than those determined experimentally. IEM reveals that this may result from Ig domain unfolding. Experiments were also performed on cardiac myocytes from mouse and cow that express predominantly a small cardiac titin isoform (N2B titin) or a large isoform (N2BA titin), respectively. The passive tension-SL relation of myocytes was found to increase more steeply with SL in mouse than in cow. IEM revealed an additional source of extensibility within both of these cardiac titins: the unique N2B sequence (absent in skeletal muscle). Furthermore, the PEVK segment of the N2BA isoform extended to a maximal length of approximately 200 nm, as opposed to approximately 60 nm for the N2B isoform. We propose that, along the physiological SL range, the long PEVK segment found in N2BA titins results in a low PEVK fractional extension and that this underlies the lower passive tensions of N2BA-expressing cow myocytes.
Asunto(s)
Corazón/fisiología , Proteínas Musculares/fisiología , Proteínas Musculares/ultraestructura , Músculo Esquelético/fisiología , Miofibrillas/fisiología , Miofibrillas/ultraestructura , Proteínas Quinasas/fisiología , Proteínas Quinasas/ultraestructura , Animales , Conectina , Masculino , Ratones , Microscopía Electrónica , Fibras Musculares Esqueléticas/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Miocardio/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , ConejosRESUMEN
One of the most salient physiological characteristics of cardiac muscle is that a dilated heart pumps more vigorously, a phenomenon known as the Frank-Starling relationship (see Allen and Kentish, 1985). At least two cellular mechanisms participate in this phenomenon: the reduction of the interfilament lattice spacing which favors the formation of cross-bridges (Wang and Fuchs, 1995) and the increased affinity of troponin C (TnC) for calcium (Ca2+) (Babu et al., 1988). In the latter case, it has been established that TnC itself is not the length sensor (Moss et al., 1991). The intracellular structure(s) able to sense changes in cell length has always been challenged and is still not known. We previously observed on intact isolated cardiac cells that active tension is more closely related to passive tension than to sarcomere length per se (Cazorla et al., 1997). This might have some physiological implications in the working heart since we found that sub-epicardial cells are more supple than sub-endocardial cells. In the present work on skinned cells, we studied the relationship between different levels of passive tension (modulated by a mild trypsin digestion) and the shift in pCa50 of tension-pCa relations induced by a stretch of cells from 1.9 to 2.3 microns sarcomere length. A significant correlation was obtained between passive tension and the stretch-induced shift in pCa50, or stretch-sensitivity of the active force. These observations led us to assume that titin might play a role in sensing cell length to modulate the contractile activity. Besides, it is known that myocardial infarcted cells are less sensitive to stretch. We propose that, in such a rat model, alterations of titin might participate in heart failure.
Asunto(s)
Proteínas Musculares/fisiología , Contracción Miocárdica/fisiología , Proteínas Quinasas/fisiología , Sarcómeros/fisiología , Animales , Células Cultivadas , Conectina , Cobayas , Corazón/fisiología , Corazón/fisiopatología , Miocardio/ultraestructura , Ratas , Sarcómeros/ultraestructuraRESUMEN
Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.
Asunto(s)
Exones/genética , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Miofibrillas/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Conectina , Elasticidad , Genoma , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Quinasas/metabolismo , Conejos , Ratas , Porcinos , Transcripción GenéticaRESUMEN
In vivo the sub-epicardial myocardium (EPI) and sub-endocardial myocardium (ENDO) operate over different ranges of sarcomere length (SL). However, it has not been previously shown whether EPI and ENDO work upon different ranges of the same or differing length-tension curves. We have compared the SL-tension relationship of intact, single ventricular EPI and ENDO myocytes from rat and ferret hearts. Cells were attached to carbon fibres of known compliance in order to stretch them and to record force at rest (passive tension) and during contractions (active tension). In both species, ENDO cells were significantly stiffer (i.e. had steeper SL-passive tension relationships) than EPI cells. Ferret ENDO cells had significantly steeper SL-active tension relationships than EPI cells; rat cells tended to behave similarly but no significant regional differences in active properties were observed. There were no inter-species differences in the active and passive properties of EPI cells, but ferret ENDO cells displayed significantly steeper passive and active SL-tension relationships than rat ENDO. We conclude that in vivo, ferret EPI and ENDO myocytes will function over different ranges of different SL-tension curves. There is a close relationship between SL and active tension (the Frank-Starling law of the heart), and our observations suggest that regional differences in the response to ventricular dilation will depend on both the change in SL and differing regional slopes of the SL-active tension curves.
Asunto(s)
Endocardio/fisiología , Pericardio/fisiología , Animales , Tamaño de la Célula , Células Cultivadas , Endocardio/citología , Endocardio/metabolismo , Femenino , Hurones , Corazón , Ventrículos Cardíacos/citología , Masculino , Miocardio/citología , Pericardio/citología , Pericardio/metabolismo , RatasRESUMEN
Extension of the I-band segment of titin gives rise to part of the diastolic force of cardiac muscle. Previous studies of human cardiac titin transcripts suggested a series of differential splicing events in the I-band segment of titin leading to the so-called N2A and N2B isoform transcripts. Here we investigated titin expression at the protein level in a wide range of mammalian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B titin) and a larger isoform with both the N2B and N2A elements (N2BA titin). The expression ratio of large N2BA to small N2B titin isoforms was found to vary greatly in different species; eg, in the left ventricle the ratio is approximately 0.05 in mouse and approximately 1.5 in pig. Differences in the expression ratio were also found between atria and ventricles and between different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoforms takes place at the single-myocyte level. The diastolic properties of single cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On average, pig myocytes are significantly less stiff than mouse and rat myocytes. Gel analysis indicates that this result cannot be explained by varying amounts of titin in mouse and pig myocardium. Rather, low stiffness of pig myocytes can be explained by its high expression level of the large isoform: the longer extensible region of this isoform results in a lower fractional extension for a given sarcomere length and hence a lower force. Implications of our findings to cardiac function are discussed.
Asunto(s)
Corazón/fisiología , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Proteínas Quinasas/metabolismo , Animales , Western Blotting , Conectina , Diástole , Elasticidad , Electroforesis en Gel Bidimensional , Técnica del Anticuerpo Fluorescente , Ratones , Miocardio/citología , Isoformas de Proteínas/metabolismo , Ratas , PorcinosRESUMEN
We studied the effects of an external acute 10 min application of cytoskeletal interfering agents on cardiac L-type calcium current (ICa,L). We found that colchicine, taxol and cytochalasin D had no direct effect on the L-type calcium channel as indicated by the absence of effect on voltage-dependent parameters. Phalloidin induced a shift in the I-V curve which renders it difficult to use in excitation-contraction coupling studies. Microfilaments of actin did not seem to regulate cardiac ICa,L as indicated by the lack of effect of cytochalasin D on ICa,L amplitude and inactivation kinetics. On the contrary, microtubules seem to be involved in the calcium-dependent inactivation of ICa,L. This involvement might be direct, i.e. a physical link between the microtubules and some part of the channel protein, or it could be indirect, i.e. the calcium chelating properties and physical obstacle of microtubules in the space between the sarcolemma and the SR.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Miocardio/metabolismo , Animales , Células Cultivadas , Colchicina/farmacología , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Cobayas , Ventrículos Cardíacos/efectos de los fármacos , Paclitaxel/farmacología , Técnicas de Placa-Clamp , Faloidina/farmacologíaRESUMEN
The intrinsic cellular mechanisms by which length regulates myocardial contraction, the basis of the Frank-Starling relation, are uncertain. The aim of this work was to test the hypothesis that passive force, possibly via titin, participates in the modulation of Ca2+ sensitivity of cardiac contractile proteins induced by stretch. Titin degradation by a mild trypsin digestion modulated passive force induced by increasing from 1.9 to 2.3 microm sarcomere length in skinned rat cardiac cells. Force-pCa curves were established at these two sarcomere lengths after various durations of trypsin application that induced different passive force levels. They allowed us to evaluate myofilament Ca2+ sensitivity by the pCa of half-maximal activation (pCa50). In control conditions, stretching cells from 1.9 to 2.3 microm induced a leftward shift of pCa50 (DeltapCa50) of 0.39+/-0.03 pCa units (mean+/-SEM, n=8 cells), reflecting an increase in Ca2+ sensitivity of the contractile machinery. Passive force measured every 2 min decreased exponentially after the beginning of the trypsin application (t1/2 approximately 12 min). The first 30% decrease of passive force did not affect the stretch-induced variation in Ca2+ sensitivity. Then, with further decrease in passive force, DeltapCa50 decreased. At the lowest passive force investigated 20% of initial passive force, DeltapCa50 decreased by approximately 55%. These effects were not accompanied by a significant modification of either maximal activated force at pCa 4.5 solution or pCa50 at 1.9 microm sarcomere length. This indicates that there was no major functional alteration of the contractile machinery during the protocol as also suggested by contractile and regulatory protein electrophoresis on 2.5-12% gradient and 15% SDS-PAGE gels. Thus, besides modulation induced by the reduced lattice spacing during enhanced heart refilling, Ca2+ sensitivity of the cardiac contractile machinery may be controlled at least partially by internal passive load, which is known to be largely attributable to titin.