Questo lavoro è stato sostenuto dalla Deutsche Forschungsgemeinschaft (KFO 196 &quot;Signaltransduktion bei adaptativen und maladaptiven kardialen rimodellante-Prozessen&quot;, FR 1638 / 1-2) e dalla DZHK (Centro tedesco per la ricerca cardiovascolare, una parte dei Centri tedeschi of Health Research , che è un BMBF (Ministero tedesco dell&#39;Istruzione e della Ricerca), iniziativa).

NOTA: I passaggi fondamentali della procedura di isolamento sono mostrati in Figura 1 Tutti i passaggi sono descritti in dettaglio nel seguente protocollo.. Isolamento del muscolo papillare, il montaggio in organo da camera bagno, acquisizione e l&#39;analisi viene effettuata in un lasso di tempo consecutivo e obbligatoria. Tutti gli esperimenti sugli animali sono stati eseguiti in conformità con la legislazione tedesca in materia di protezione degli animali e sono state approvate dal Consiglio di esame etico dell&#39;Università di Heidelberg. 1. Preparazione di Instrumentation <ol><li> Per contrattilità misurazioni utilizzare un setup vasca organo multichambered. Componenti di un ex vivo contrattilità configurazione bagno organo sono mostrati nella Figura 2. NOTA: Per minimizzare la quantità di tempo tra la preparazione del muscolo papillare e l&#39;inizio del protocollo sperimentale, impostare attrezzature e preparare buffer prima di raccogliere tissue.This sperimentali provides la maggior quantità di tempo che il tessuto rimane vitale per condurre esperimenti. </li><li> Avviare computer e apparecchiature di acquisizione dati. Attivare bagno organo di calore (preimpostato a 32 ° C) e lasciare unità alla temperatura preimpostata. </li><li> Preparare l&#39;apparecchiatura di acquisizione. Iniziare la registrazione dei dati come indicato nel manuale del software. </li><li> Controllare e condurre calibrazione (se necessario) su ciascun canale e azzerare tutti i canali di standardizzare il segnale elettrico fornito dal rispettivo trasduttore di forza (associato a quel canale) a 2 g forza fornita da un peso certificata. </li><li> Accendere approvvigionamento di gas (95% O 2/5% di CO 2) alle camere bagno organo. Continuamente gas tutte le camere durante l&#39;intero misure di protocollo. </li></ol> 2. Preparazione di Tamponi e soluzioni fisiologiche <ol><li> Preparare una soluzione tampone Krebs-Henseleit per raggiungere concentrazioni finali di 119 mM NaCl; 25 NaHCO MM 3; 4.6 mM KCl; 11MM glucosio; 1 mM Na-pyruvate; 1.5 mM CaCl 2; 1.64 mM MgSO 4; 1.18 mM KH 2 PO 4 e regolare il pH a 7,4 (vedi Tabella 1 soluzione fisiologica). </li><li> Preparare una soluzione tampone a parte Krebs-Henseleit da utilizzare durante l&#39;isolamento come descritto al punto 2.1 con l&#39;aggiunta di monoxime 30MM 2,3-butanedione (BDM) e 50 IE Eparina / ml. Raffreddare la soluzione a 4 ° C (vedere Tabella 1 Preparazione Soluzione). Per la simulazione ischemia, ischemia preparare soluzione come descritto nella Tabella 1. </li><li> Per il protocollo del Ca 2 + - contrattilità dipendente, aggiungere Ca 2+ alla soluzione tampone Krebs-Henseleit a concentrazione finale richiesta. NOTA: preparare queste soluzioni direttamente prima del suo utilizzo e Gass con CarboGen per evitare la precipitazione di Carbonato di Calcio. Soluzione Preriscaldare a 32 ° C prima dell&#39;uso. </li></ol> 3. Preparazione del Gassing Tube e dissezione del piatto utilizzata durante il Excision Procedure papillare <ol><li> Collegare un gas morbidocantare tubo (diametro esterno 2-4 mm) per il collegamento del gas (ossigeno al 100%, CarboGen alternativamente) con l&#39;esperto di lavoro. </li><li> Creare un anello chiuso del tubo gassazione raccordo all&#39;interno del piatto dissezione. Forare il tubo di gassificazione più volte che una ebollizione continua è assicurata. Questo tubo gassazione può essere utilizzato più volte. </li><li> Preparare piatto dissezione con un elastomero di silicone nella parte inferiore (0,5 cm), in modo che i perni possono essere bloccati in esso. Questo piatto può essere utilizzato più volte. </li></ol> 4. Isolamento dei anteriore sinistra Muscolo papillare da topi NOTA: Prima di iniziare l&#39;isolamento del muscolo papillare, controllare che le linee di gas sono chiare di blocchi. <ol><li> Preparare configurazione vasca organo come menzionato nella sezione del protocollo 1-3 prima di iniziare la preparazione dei muscoli papillari. Preparare tutte le soluzioni al giorno dell&#39;esperimento. </li><li> Sacrifica il mouse (circa 8-12 settimane) da cervicale dislocazione According per linee guida istituzionali di lavoro di esperti e. </li><li> Fissare l&#39;animale in posizione supina fissando zampe anteriori e posteriori zampe su una tavola di dissezione, aprire il torace laterali su entrambi i lati tagliando attraverso le costole con forbici ossee affilate, poi tagliare la membrana con un taglio trasversale. Rimuovere il pericardio. Ora cuore e l&#39;aorta sono accessibili. </li><li> Fissare il cuore con una pinza smussato sul tronco vascolare vicino al cuore e separare il cuore rapidamente con le forbici dai polmoni e il tessuto circostante. </li><li> Trasferire il cuore pulsante di una capsula di Petri riempite con una soluzione preparazione raffreddata gasati con l&#39;ossigeno (punto 2.2). Lasciare che il cuore a battere e stimolare le contrazioni del cuore dolcemente via toccando l&#39;apice cardiaco con una pinza. </li><li> Non appena il cuore è completamente exsanguinous, trasferirlo al piatto dissezione riempito con soluzione di preparazione raffreddata (passo 2.2) e gasati con O 2. Da questo punto in uso uno stereomicroscopio. </li><li> Fissare il heaRT con un piccolo perno attraverso il ventricolo destro in modo che il cuore è visto dalla dorsale (ventricolo destro si trova sul lato destro dalla vista dell&#39;operatore). </li><li> Utilizzando le forbici microchirurgiche separano entrambi gli atri a livello atrioventricolare, taglio tessuto connettivo dai ventricoli e scartare. Effettuare un taglio attraverso la parete ventricolare dalla valvola livello AV all&#39;apice del cuore evitando ogni pressione meccanica. </li><li> Aprire il ventricolo e fissare la parete libera lato sinistro con una pinza, avendo così uno sguardo a due ventricolo muscoli papillari sinistro. </li><li> Leggermente tagliare il tessuto ventricolare laterali su entrambi i lati del muscolo papillare anteriore sinistra conservando una parte della vela valvolare sulla preparazione muscolo papillare ed evitare di toccare o allungamento del muscolo papillare quanto possibile. Sezionare il restante tessuto della parete ventricolare dal muscolo papillare. </li><li> Fissare fili di seta (7/0, metrico 0,5) su entrambi i lati della preparazione muscolo papillarearazione, uno sulla vela valvolare ed una sulla parte muscolare. Fissare la preparazione nella camera bagno di organo pieno di soluzione del bagno organo e gasati con CarboGen, la temperatura a 32 ° C viene consigliata. </li></ol> 5. equilibrazione e stimolazione del muscolo papillare <ol><li> Stimolare la preparazione muscolo papillare con impulsi rettangolari (durata di 2 ms e corrente di 100 mA) a frequenza di 1 Hz stimolazione immediatamente dopo il fissaggio nel bagno organo. </li><li> Assicurare un frequente ricambio della soluzione del bagno di organo, sia dal cambiamento continuo o da frequenti modifica manuale (ogni 5 min) a seconda dell&#39;impostazione del tipo a bagno d&#39;organo utilizzato. </li><li> Aumentare gradualmente la pretesa fino a forza contrazione massima a raggiunto. Forza contrazione massima viene raggiunta quando l&#39;aumento del precarico non è seguito da un ulteriore aumento nella forza contrazione. </li><li> Dopo 45-60 minuti di equilibrio, avviare il protocollo sperimentale. </li></ol> NOTA: Il protocollo sperimentale descritto di seguito comprende manovre standard per caratterizzare la contrattilità cardiaca in condizioni fisiologiche e fisiopatologiche. Nella sezione rappresentante descriviamo questi protocolli in dettaglio anche mostrando i risultati di rappresentanza (vedi anche tabella 2). <ol><li> Registrazione ed analisi dei protocolli sperimentali <ol><li> Per la registrazione, utilizzare un software adatto con adeguata risoluzione temporale (velocità di acquisizione ≥ 1kHz). </li><li> Analizzare parametri tra cui la forza contrazione ampiezza (altezza), il tempo di picco di tensione (TTP) e per metà tempo di rilassamento massimo (R 50 / tfall, rispettivamente) come indicato nel programma di software (come ad esempio Grafico 5.5 di ADInstruments, vedi Figura 4). </li></ol></li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

In questo manoscritto si descrive un metodo per indagare la contrattilità del muscolo papillare murino in vitro che può essere utilizzato per rispondere ad alcune domande scientifici relativi alla fisiologia del cuore e la patologia nei topi, nonché per sostenere l&#39;analisi delle linee transgeniche e la scoperta di nuovi approcci farmaceutici per il trattamento di disfunzioni cardiache. Illustriamo l&#39;uso di questo metodo per valutare le proprietà fisiologiche, patologiche e farmacologiche di contratt...

L&#39;indagine di proteine ​​come canali ionici che si riferiscono loro ruolo per la contrattilità cardiaca è essenziale per scoprire differenti meccanismi patogenetici e di stabilire nuove strategie terapeutiche per le malattie cardiache come l&#39;ischemia e insufficienza cardiaca. Funzione contrattile dei cardiomiociti mammiferi è noto per essere modulata da vari canali ionici, trasportatori e altre proteine. Potenziale d&#39;azione evocato attivazione di tensione dipendente sarcolemmal tipo L di Ca 2+ canali porta a Ca 2+ afflusso dallo spazio extracellulare e successivamente Ca2 + indotta rilascio di Ca 2+ (CICR) 1, che innesca la contrazione cellulare 2. Ca 2+ -Segnalazione gioca un ruolo centrale nella contrattilità cardiaca e l&#39;adattamento allo stress fisiologico o patologico. Catecolamine attivano i recettori beta-adrenergici cardiaci, stimolando così ciclasi (AC) che sintetizza cAMP. L&#39;attivazione, Protein chinasi A (PKA) fosforila diverse proteine ​​associate intracellulari e di membrana come L-tipo Ca 2+ canali, fosfolambano e recettori rianodinici conseguente modifica di Ca 2+ transitori e cardiaco contrattilità 1,3,4. cAMP è degradata dalla fosfodiesterasi (PDE). L&#39;attivazione dei recettori Gs-accoppiati diversi beta-adrenergici porta anche ad accumulo di cAMP. La tecnica di misurazione contrattilità in isolati strisce muscolari ventricolare è ben definito per le più grandi mammiferi 5-8. Sulla base della possibilità di gene targeting in topi è importante stabilire metodi per analizzare murino fisiologia cardiaca. Tuttavia, i dati esistenti sulle proprietà fisiologiche di preparati muscolari isolate in topi variano a seconda delle condizioni sperimentali 9-12. Il metodo descritto è utilizzato per analizzare contrattilità cardiaca ventricolare sinistra pre muscolo papillareparations in vitro. Investigation della contrattilità cardiaca viene eseguita in assenza di influenze modificanti contrattilità cardiaca in vivo, come la pressione sanguigna, stimolazione neuroumorale e stress fisico o metabolica. Il tasso di battito della preparazione contraente muscolo può essere rigorosamente definito e cambiato arbitrariamente. Twitch forza può essere analizzato nel contesto di stimoli specifici come concentrazione di calcio, battendo frequenza o temperatura. Inoltre, questo metodo può essere usato per studiare diversi componenti della via di segnalazione e di confrontare funzione cardiaca di modelli murini geneticamente modificati controllando condizioni sperimentali sopra menzionati.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761&amp;nbsp;</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>D9434&amp;nbsp;</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280&amp;nbsp;</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P 5655</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich</td><td>B0753</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td>Hazard statement H 302, solve in DMSO</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Isoprenaline hydrochloride</td><td>Sigma-Aldrich</td><td>I5627</td><td>Hazard statement H 315-H319-H335</td></tr><tr><td>Sodium Heparine 250.000 IE/10 ml</td><td>ratiopharm</td><td>PZN 3874685</td><td></td></tr><tr><td>Histamine dihydrochloride</td><td>Sigma-Aldrich</td><td>H7250</td><td>Hazard statement H 315-H 317-H319- H334-H335</td></tr></tbody></table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

Muscoli papillari isolati dai cuori adulte di topo possono essere utilizzati per studiare la contrattilità cardiaca durante le diverse condizioni patologiche / fisiologiche. Le caratteristiche contrattili possono essere valutate in maniera indipendente da influenze esterne, quali tono vascolare o lo stato neuroumorale. Raffigura un approccio scientifico tra le misurazioni di cellule singole con isolate miociti cardiaci e studi in vivo, come l&#39;ecocardiografia. Così, preparazioni muscolo papillare servire come un modello eccellente per studiare la fisiologia cardiaca / patofisiologia e può essere utilizzato per le indagini come la modulazione da agenti farmacologici o l&#39;esplorazione di modelli animali transgenici. Qui, descriviamo un metodo per isolare l&#39;anteriore sinistra muscolo papillare murino di indagare la contrattilità cardiaca in un setup bagno di organo. A differenza di un preparato striscia muscolare isolato dalla parete ventricolare, il muscolo papillare può essere preparato in toto senza danneggiare il muscolo tissuuna e severamente. Il setup bagno organo è composto da diversi elettrodi attrezzate camere a bagno d&#39;organo a temperatura controllata, gasati e. Il muscolo papillare isolato è fissato nella camera bagno di organo e stimolato elettricamente. La forza di contrazione evocata viene registrata utilizzando un trasduttore di pressione e parametri quali contrazione forza ampiezza e contraggono la cinetica vengono analizzati. Diversi protocolli sperimentali possono essere eseguite per indagare la contrattilità e calcio-dipendente dalla frequenza e curve dose-risposta di agenti contrattili come catecolamine o altri farmaci. Inoltre, le condizioni patologiche come l&#39;ischemia acuta possono essere simulati.

Il protocollo di questo manoscritto per misure contrattilità isolati murini preparazioni muscolo papillare è sintonizzato per condizioni ottimali per ottenere risultati sperimentali riproducibili in condizioni fisiologiche. Per definire le condizioni sperimentali ottimali abbiamo eseguito esperimenti pilota variabili temperatura del bagno d&#39;organo e la concentrazione di calcio extracellulare (vedi anche 12). Il protocollo qui descritto è stato eseguito con una concentrazione di calcio extracellulare 1,...

Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

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

Murine sinistra muscolo papillare ventricolare può essere usato per studiare la contrattilità cardiaca in vitro. Questo articolo descrive in dettaglio l&#39;isolamento e protocolli sperimentali per studiare le caratteristiche contrattili cardiaci.

Misure contrattilità isolati su muscoli papillari per le inchieste sui Cardiac inotropo nei topi

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K Views.  Universität Heidelberg.  Murine sinistra muscolo papillare ventricolare può essere usato per studiare la contrattilità cardiaca in vitro. Questo articolo descrive in dettaglio l'isolamento e protocolli sperimentali per studiare le caratteristiche contrattili cardiaci. 

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

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Nelle misure in vitro di costrizione tracheale con Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Ricerca

Medicina

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cateterizzazione cardiaca nei topi per misurare il rapporto pressione del volume: Indagare l&#39;effetto Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Approcci ecocardiografici e protocolli per completa caratterizzazione fenotipica di malattia valvolare cardiaca nei topi

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Isolamento, Cultura e caratterizzazione funzionale di topo adulto Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Biologia

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolamento e fisiologica Analisi di mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Misurazione dinamica e imaging di capillari, arterioli e periciti nel cuore del topo

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Valutazione della myofilament Ca<sup&gt; 2+</sup&gt; Sensibilità Alla base Cardiac eccitazione-contrazione Accoppiamento

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Analisi della disfunzione contrattile cardiaca e dei transitori Ca2+ nei miociti dei roditori

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

Misurazione della contrattilità cardiaca in cardiomiociti primari umani adulti isolati

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

Misure contrattilità isolati su muscoli papillari per le inchieste sui Cardiac inotropo nei topi

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