Este trabajo fue apoyado por AHA SDG 10SDG2630086 a Zhao X, RO1-AR061385 a Ma J y GO subvención RC2AR05896 a Brotto M.

<ol>
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Medición de la fuerza contráctil y la tendencia a la fatiga es importante para la evaluación global de la función del músculo esquelético. El objetivo principal de este ensayo es determinar los cambios en la fuerza muscular y las propiedades fatigantes bajo ciertas condiciones patológicas, como la sarcopenia y la fatiga muscular, y para evaluar el efecto de las drogas / reactivos sobre la contractilidad muscular. Puesto que la fuerza muscular está muy relacionada con la concentración de Ca 2 + libera...

Los músculos del esqueleto unen a los huesos del esqueleto y generar fuerzas contráctiles bajo el control del sistema nervioso central. Acoplamiento excitación-contracción (ECC) se refiere al proceso de convertir un estímulo eléctrico a una respuesta mecánica. Ca 2 + de señalización es un componente esencial de la función contráctil en el músculo esquelético. Efectiva movilización de Ca 2 + del retículo sarcoplásmico (SR) es un componente importante para la ECC en células musculares 1, 2, y los cambios en la concentración intracelular de Ca 2 + señalización subyacen a la disfunción contráctil correspondiente en un número de 3-5 enfermedades musculares. La adecuada valoración de la contractilidad del músculo es esencial y complementaria a Ca 2 + de imágenes y otros ensayos para obtener información sobre la función del músculo esquelético, no sólo a nivel contráctil, sino también a nivel cinética. Fuerza y ​​la velocidad también se puede obtener a informar a la importante propiedad de losla fuerza muscular y el estado del proceso de ECC bajo diferentes condiciones fisiológicas y fisiopatológicas. Este campo fecundo de investigación tiene una historia muy rica y muchas teorías de la contracción muscular apareció más de dos milenios 6. Investigación músculo moderno probablemente se inicia en 1674-1682 con la observación microscópica de estrías cruzadas y miofibrillas en las fibras musculares por Leeuwenhoek 6. Casi un siglo más tarde, Luigi Galvani observó que los contratos de rana musculares vigorosamente cuando el nervio se toca con bisturí durante una descarga de chispa de una máquina distante eléctrico 7-9. Contracción también se podría producir mediante la conexión del nervio pierna en el músculo a través de un conductor de metal. Los detalles del mecanismo de señalización eléctrica complejo preconizadas por Galvani se formularon eventualmente por Hodgkin, Huxley y Katz en su famosa ecuación 10, 11 que se convirtió en la base de la electrofisiología. Las observaciones notables del Ringer a los efectos de la Ca 2 + extracelular sobre la contractilidad del corazón y los músculos esqueléticos rana 12-15 representan el primer paso importante en el reconocimiento de Ca 2 + como un regulador clave de la contractilidad del músculo 16, 17. Desde la década de 1980 hasta la actualidad una explosión de descubrimientos en el campo de la contractilidad muscular se realizó debido a la introducción de la contractilidad del músculo y protocolos de fatiga del músculo esquelético murino 18. Jones y Edwards fueron los primeros en sugerir que la fatiga de baja frecuencia intermitente (inducida por el ejercicio en la reducción de fuerza) 19 se asoció con cambios en la maquinaria ECC y no el aparato contráctil. A finales de la década de 1980 y principios de 1990, Kolkeck et al 20, Kolbeck y Nosek 21, y Reid 22 estaban usando los músculos del diafragma de modelos de roedores para estudiar los efectos de las teofilinas, cortiosterone y radicales libres sobre la contractilidad del músculo esquelético, mientras que Brooks y Faulkner fueron los primeros en informar sobre las medidas de fuerza de repetirse y las mediciones de consumo en rápido y lento-los músculos de los ratones 22. Además, Lannegren, Westerblad, Cordero y Westerblad fueron los primeros en vincular directamente la contractilidad ex vivo con la concentración de Ca 2 + regulación y comenzó a cuestionar el papel de la acidosis en la fatiga muscular 23, 24. Nuestros laboratorios han contribuido de manera significativa desde la década de 2000 hacia la comprensión de nuevos genes con modulador y funciones de regulación en el músculo ECC con papeles críticos en la contractilidad muscular, la fatiga y el envejecimiento mediante el uso de una combinación de estudios intactos de ratón contractilidad del músculo, la concentración de Ca 2 Control + en intactos y sin piel las fibras musculares y las manipulaciones genéticas moleculares 3-5, 25-29. Aquí se detalla el protocolo experimental para la medición de la contractilidad murino soleus aisladas y longus extensor común de los dedos (EDL) músculos, que corresponden a una mayoría de lento oxidativo (tipo I y IIa fibras musculares) y un músculo en su mayoría rápido glyocolytic (tipo IIb y fibras musculares IIx) con diferentes propiedades contráctiles. En este protocolo, intactos músculo-tendón complejos se aislaron y se bañó en una IDA sistema PowerLab Radnotti cámara suministrada con oxígeno puro o una mezcla de oxígeno (95%) y CO 2 (5%). Fuerzas contráctiles fueron generados por estímulos eléctricos de un estimulador Grass y detectado usando un transductor de fuerza que se ha integrado con un sistema de ADI PowerLab/400, permitiendo la personalización de las rutinas de macro para controlar la adquisición, la recogida, la digitalización y el almacenamiento de datos. Esta configuración puede medir la fuerza muscular, fuerza muscular, así como la relación de la fuerza frente a la frecuencia, la fatiga muscular, la recuperación de la fatiga muscular, la velocidad y en general las propiedades cinéticas de la contracción muscular. Además, los efectos de los fármacos sobre la contracción muscular puede controlarse a través de estos experimentos. </p&gt; Las ventajas de este método reside en la eliminación de los componentes neuronales y vasculares lejos del músculo esquelético, lo que permite la evaluación directa de las propiedades intrínsecas de contraer el músculo. Además, los ensayos de contractilidad ex vivo permiten la manipulación del medio extracelular que rodea a los músculos aislados, que permite el uso de manipulaciones farmacológicas de diversos canales iónicos y transportadores de permeación a fin de definir sus papeles fisiológicos para la función del músculo esquelético. Este sistema ex vivo que nos ha permitido descubrir recientemente un comportamiento distinto alternan en ciertas preparaciones de músculo mutantes, que estaban vinculados con alteración intracelular de Ca 2 + 4 propiedades de manejo. Alternancia se definen como episodios de ráfaga fluctuantes de la fuerza contráctil durante la fase de declive del perfil de fatiga. Durante estos eventos fuerzas contráctiles aumentar momentáneamente por encima de su nivel anterior de la fuerza durante la estimulación fatiga, tal vez porque sea más Ca 2 + se publica o la maquinaria contráctil se ha vuelto más sensible a Ca 2 + 30. Tratamiento de ácido ciclopiazónico (CPA), un bloqueador reversible de retículo sarcoplásmico calcio ATPasa (SERCA), la cafeína, un agonista de rianodina canal (RyR) y se repitió fatigoso estímulos pueden inducir alternans mecánicos 4, lo que sugiere que alternans están directamente relacionados con modulación del proceso de acoplamiento CE. Demostración del método para inducir y registrar en alternancia mecánica en la configuración de la contractilidad vitro sirve como ejemplo para mostrar los parámetros experimentales diversificados que se podrían obtener con este sistema o similares, en base a los intereses individuales de investigación. Este método puede ser de interés para los investigadores que estudian la fisiología muscular. Configuración similar también se puede utilizar para aisladas complejos muscle-tendon/ligament esqueléticos de otroubicaciones anatómicas, así como para las fibras individuales y las tiras musculares.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> 2-APB </td><td> Tocris </td><td> 1224 </td><td> Bloqueador de una serie de Ca 2 + canales de entrada incluyen SOC y TRP, etc </td></tr><tr><td> SKF96365 </td><td> Sigma </td><td> SKF-96365 </td><td> Bloqueador de un número de Ca 2 + canales de entrada incluyendo SOC y mediada por el receptor de Ca 2 + entrada etc </td></tr><tr><td> BTP-2 </td><td> Millipore </td><td> 203890-5MG </td><td> Relativamente específico SOC bloqueador </td></tr><tr><td> CPA </td><td> Sigma </td><td> C1530 </td><td> Reversible bloqueador SERCA </td></tr><tr><td> cafeína </td><td> Sigma </td><td> C0750 </td><td> Rápida acción agonista RyR </td></tr><tr><td> Radnoti Cuatro Tissue Unidad de Órganos del Sistema Bath </td><td> Radnoti </td><td> 159920 </td><td></td></tr><tr><td> Combinación de soporte de tejido / electrodo de estimulación </td><td> Radnoti </td><td> 160151 </td><td> Vertical Tipo Zig Zag con soporte de tejido </td></tr><tr><td> Quad Bridge Amp </td><td> ADInstruments </td><td> FE224 </td><td></td></tr><tr><td> PowerLab/400 </td><td> ADInstruments </td><td></td><td> Este producto ya no se encuentra disponible. Elige otra versión del sistema de adquisición de datos. </td></tr><tr><td> Transductores de fuerza (5 mg - 25 g) </td><td> ADInstruments </td><td> MLT0201/RAD </td><td></td></tr><tr><td> Tabla 4.02 </td><td> ADInstruments </td&gt; <td></td><td> LabChart 7.3 es la última versión del software para gráficos. </td></tr><tr><td> S8800 Dual Pulso Digital Estimulador </td><td> TECNOLOGÍAS DE LA HIERBA </td><td></td><td> Este producto ya no se encuentra disponible. S88X de salida dual de la Plaza de pulso estimulador es un estimulador más reciente. </td></tr><tr><td> Transformador RF Unidad de Aislamiento </td><td> TECNOLOGÍAS DE LA HIERBA </td><td> Modelo SIU5 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles

A continuación se describe un método para medir la contractilidad de los músculos esqueléticos aisladas. Parámetros como la fuerza muscular, la fuerza muscular, la cinética de contracción, fatigabilidad y la recuperación después de la fatiga puede ser obtenida para evaluar aspectos específicos del acoplamiento excitación-contracción (ECC) de proceso, tales como excitabilidad, maquinaria contráctil y Ca 2 + capacidad de manipulación. Este método elimina la inervación y de la sangre y se centra en el músculo esquelético aislado en sí. En forma rutinaria utilizar este método para identificar los componentes genéticos que alteran la propiedad contráctil del músculo esquelético, aunque la modulación de Ca 2 + vías de señalización. A continuación, describimos un fenotipo recién identificado el músculo esquelético, es decir, alternans mecánico, como un ejemplo de las diversas informaciones y rica que se puede obtener utilizando el ensayo in vitro de la contractilidad del músculo. Combinación de este ensayo con ensayos de células individuales, los enfoques genéticos y bioquímicosstrY ensayos pueden proporcionar importantes conocimientos sobre los mecanismos de la ECC en el músculo esquelético.

Watch this Scientific Journal Video about Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles at JoVE.com

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Se describe un método para medir directamente la fuerza muscular, la fuerza muscular, la cinética de contracción y fatigabilidad de los músculos esqueléticos aislados en un<em&gt; In vitro</em&gt; Sistema mediante la estimulación del campo. Información valiosa sobre Ca<sup&gt; 2 +</sup&gt; Propiedades de manipulación y la maquinaria contráctil del músculo se puede obtener utilizando diferentes protocolos de estimulación.

Evaluación ex vivo de la contractilidad, fatigabilidad y alternancia en aislados músculos esqueléticos

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

ex-vivo-assessment-contractility-fatigability-alternans-isolated

Research

JoVE Journal

Biology

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

23.6K vistas.  UMDNJ-Robert Wood Johnson Medical School. Se describe un método para medir directamente la fuerza muscular, la fuerza muscular, la cinética de contracción y fatigabilidad de los músculos esqueléticos aislados en un In vitro Sistema mediante la estimulación del campo. Información valiosa sobre Ca 2 + Propiedades de manipulación y la maquinaria contráctil del músculo se puede obtener utilizando diferentes protocolos de estimulación.

Video: Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

En vivo la micro-circulación de medición en el músculo esquelético de Intra-vital Microscopía

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Investigación

Biología

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

La transfección de ADN de músculos esqueléticos de mamíferos con En Vivo La electroporación

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

La Ex vivo Preparación aislada Microvessel esquelético para la Investigación de la reactividad vascular

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Evaluación de Generación de la Fuerza isométrica y excéntrica de los músculos esqueléticos Aislados de modelos murinos de Distrofias Musculares

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Contractilidad Mediciones en músculos papilares aislados para la Investigación de Cardíaca inotropía en ratones

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

La proteína c-Fos detección inmunohistoquímico: una herramienta útil como marcador de vías centrales implicados en las respuestas fisiológicas específicas<em&gt; En Vivo</em&gt; y<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

La medición de la rigidez de<em&gt; Ex Vivo</em&gt; Ratón aortas Usando microscopía de fuerza atómica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Medición de la absorción de glucosa estimulada por insulina y contracción en músculo esquelético maduro aislado e incubado de ratones

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Ensayo único de cultivo de Myofiber para la evaluación de la funcionalidad de las células madre musculares adultas ex vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...