Name: contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice
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This work was supported by the Deutsche Forschungsgemeinschaft (KFO 196 &#8220;Signaltransduktion bei adaptativen und maladaptiven kardialen Remodelling-Prozessen&#8221;, FR 1638/1-2) and by the DZHK (German Centre for Cardiovascular Research, a part of the German Centres of Health Research, which is a BMBF (German Ministry of Education and Research) initiative).

NOTE: The basic steps of the isolation procedure are shown in Figure&#160;1. All steps are described in detail in the following protocol. Papillary muscle isolation, mounting in organ bath chamber, acquisition and analysis is performed in a consecutive and compulsory timescale.
All animal experiments were performed in accordance with German legislation on protection of animals and were approved by the Ethics Review Board of University of Heidelberg.
1. Preparation of Instrumentation
<ol>
	<li>For contractility measurements use a multichambered organ bath setup. Components of an ex vivo contractility organ bath setup are shown in Figure 2. 
	NOTE: To minimize the amount of time between the preparation of the papillary muscle and the beginning of the experimental protocol, set up equipment and prepare buffers prior to collecting experimental tissue.This provides the greatest amount of time that the tissue remains viable to conduct experiments.</li>
	<li>Start up computer and data acquisition equipment. Turn on organ bath heat (preset to 32 &#176;C) and allow units to preset temperature.</li>
	<li>Prepare the acquisition equipment. Start recording data as mentioned in the software manual.</li>
	<li>Check and conduct calibration (if necessary) on each channel and zero all channels to standardize the electric signal supplied from the respective force transducer (associated with that channel) to a 2-g force supplied by a certified weight.</li>
	<li>Turn on gas supply (95% O2/5% CO2) to organ bath chambers. Continuously gas all chambers during the entire protocol measurements.</li>
</ol>
2. Preparation of Buffers and Physiological Solutions
<ol>
	<li>Prepare a Krebs-Henseleit buffer solution to achieve final concentrations of 119 mM NaCl; 25 mM NaHCO3; 4.6 mM KCl; 11mM glucose; 1 mM Na-pyruvate; 1.5 mM CaCl2; 1.64 mM MgSO4; 1.18 mM KH2PO4 and adjust pH to 7.4 (see Table 1 Physiological solution).</li>
	<li>Prepare separate Krebs-Henseleit buffer solution to use during isolation as described in step 2.1 with additional 30mM 2,3-Butanedione monoxime (BDM) and 50 IE Heparin/ml. Cool solution to 4&#176;C (see Table 1 Preparation solution). For ischemia simulation, prepare ischemia-solution as described in Table 1.</li>
	<li>For the protocol of the Ca2+-dependent contractility, add Ca2+ to the Krebs-Henseleit buffer solution to requested final concentration. 
	NOTE: prepare these solutions directly before its usage and gass with carbogen to avoid precipitation of Calciumcarbonate. Prewarm solution to 32&#176;C before using.</li>
</ol>
3. Preparation of the Gassing Tube and Dissection Dish used during the Papillary Excision Procedure
<ol>
	<li>Connect a soft gassing tube (external diameter 2-4 mm) to the gas connection (100% oxygen, carbogen alternatively) with expert working.</li>
	<li>Create a closed ring of the gassing tube fitting inside the dissection dish. Puncture the gassing tube several times that a continuous bubbling is assured. This gassing tube can be used several times.</li>
	<li>Prepare dissection dish with a silicon elastomer at the bottom (0.5 cm thick), so that pins can be stuck into it. This dish can be used several times.</li>
</ol>
4. Isolation of Left Anterior Papillary Muscle from Mice
NOTE: Before starting the isolation of the papillary muscle, check that the gas lines are clear of blockages.
<ol>
	<li>Prepare organ bath setup as mentioned in the protocol section 1-3 before starting the preparation of the papillary muscles. Prepare all solutions at the day of the experiment.</li>
	<li>Sacrifice the mouse (approximately 8-12 weeks old) by cervical dislocation according to expert working and institutional guidelines.</li>
	<li>Fix the animal in dorsal position by fixing forepaws and hind paws on a dissection board, open the thorax lateral on both sides by cutting through the ribs with sharp bone scissors, then cut the diaphragm with a transversal cut. Remove the pericardium. Now the heart and the aorta are accessible.</li>
	<li>Fix the heart with blunt forceps on the vascular truncus close to the heart and separate the heart quickly with scissors from the lungs and the surrounding tissue.</li>
	<li>Transfer the beating heart to a Petri dish filled with cooled preparation solution gassed with oxygen (step 2.2). Allow the heart to beat and stimulate heart contractions softly via touching the cardiac apex with forceps.</li>
	<li>As soon the heart is completely exsanguinous, transfer it to the dissection dish filled with cooled preparation solution (step 2.2) and gassed with O2. From this step on use a stereomicroscope.</li>
	<li>Fix the heart with a small pin through the right ventricle so that the heart is seen from dorsal (right ventricle is situated on the right side from the operator&#8217;s view).</li>
	<li>Using microsurgical scissors separate both atria on the atrioventricular level, cut connecting tissue from the ventricles and discard. Perform a cut through the ventricular wall from the AV-valve-level to the apex of the heart avoiding any mechanical pressure.</li>
	<li>Open the ventricle and fix the left-sided free wall with forceps, thus having a look at both left ventricular papillary muscles.</li>
	<li>Slightly cut away the ventricular tissue lateral on both sides of the left anterior papillary muscle preserving a part of the valvular sail on the papillary muscle preparation and avoid touching or stretching the papillary muscle as much as possible. Dissect the remaining ventricular wall tissue from the papillary muscle.</li>
	<li>Attach silk threads (7/0, metric 0.5) on both sides of the papillary muscle preparation, one on the valvular sail and one on the muscular part. Fix the preparation in the organ bath chamber filled with organ bath solution and gassed with carbogen, as temperature 32 &#176;C is suggested.</li>
</ol>
5. Equilibration and Stimulation of the Papillary Muscle
<ol>
	<li>Stimulate the papillary muscle preparation with rectangular pulses (duration of 2 ms and current of 100 mA) at stimulation frequency of 1 Hz immediately after the fixation in the organ bath.</li>
	<li>Ensure a frequent change of the organ bath solution, either by continuous change or by frequent manual change (every 5 min) according to the setup of the organ bath type used.</li>
	<li>Gradually increase the pretension until maximal twitch force in reached. Maximal twitch force is reached when the increase of the pretension is not followed by a further augmentation in the twitch force.</li>
	<li>After 45-60 min of equilibration, start the experimental protocol.</li>
</ol>
6. Suggested Experimental Protocols for Contractility Measurements
NOTE: The experimental protocol outlined below comprises standard maneuvers to characterize cardiac contractility under physiological and pathophysiological conditions. In the representative section we describe these protocols in detail also showing representative results (see also Table 2).
<ol>
	<li>Recording and analysis of the experimental protocols
	<ol>
		<li>For recording, use a suitable software with adequate temporal resolution (acquisition rate &#8805; 1kHz).</li>
		<li>Analyze parameters including twitch force amplitude (height), time to peak tension (TTP) and half maximal relaxation time (R50/TFall, respectively) as mentioned in the software program (as example Chart 5.5 of ADInstruments, see Figure 4).</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

In this manuscript we describe a method to investigate contractility of murine papillary muscle in vitro which can be used to answer several scientific questions related to heart physiology and pathology in mice as well as to support the analysis of transgenic lines and the discovery of new pharmaceutical approaches to treat heart dysfunctions. We illustrate the use of this method to assess physiological, pathological and pharmacological properties of cardiac muscle contractility (see Fig 3). Additional applications not illustrated here include evaluation of intracellular pathways using different pharmacological agents (see also 12).
Cardiac diseases such as ischemic heart disease and heart failure display an eminent clinical problem and remain the leading cause for mortality and morbidity worldwide22. For many proteins expressed in the heart, their role for cardiac contractility is still poorly understood or not studied until now. The contractility measurement on isolated papillary muscles demonstrates a meaningful and valid approach to study cardiac contractility in vitro in mouse models.
Although the method is technically feasible and shows a good reproducibility, there are several critical steps necessary to ensure its success: First, after isolating the heart, ensure a blood free solution during the preparation procedure to be able to work properly (visualization). Another critical step is that tissue preparation is performed carefully to ensure viability by avoiding any touching or stretching of the papillary muscle. As described in the method section try to keep the valvular sail connected to the papillary muscle to ensure a trouble-free attachment of the silk thread. After fixation of the preparation in the organ bath chamber, change the organ bath solution several times during the first minutes to flush out the BDM. Increase the pretension of the preparation slightly and gradually until no more increase of twitch force occurs. There are some exclusion criteria for the evaluation of the single protocols such as arrhythmic beats and spontaneous increase of the pretension as a sign for ischemic conditions. Several experimental protocols such as postrest potentiation and force-frequency relation can be performed with the same preparation sample, whereas a new preparation sample should be used for the analysis of drug-mediated effects on contractility. The preparation protocol is optimized to isolate the left anterior papillary muscle. Adapting the protocol, also the posterior muscle can be used for this method of contractility measurements.
This method of isolated papillary muscle measurement provides various information about the calcium-, temperature- and frequency-dependent contractility as well as about the contractile response after applying pharmacological agents or the simulation of pathological conditions. However, the interpretation and extrapolation of these results needs to be handled with scientific care. The described method is an in vitro model of cardiac muscle, disconnected from its normal environment and neural innervation. Therefore, the experimental conditions are not physiological and the results received in this protocol cannot be transferred without in-depth interpretation to the situation in vivo. For example, the method does not take into account changes in blood pressure, hormones or extrinsic neural control. The supply of oxygen and metabolic supplements for the preparation in the organ bath is limited by diffusion. An insufficient supply of oxygen and metabolic supplements would lead to ischemic conditions and subsequently invalid experimental results. To avoid this, the experimental conditions should be optimal, especially concerning oxygenation, concentration of calcium and temperature of the organ bath solution. With the experimental conditions of the protocol described here, valid results can be afforded.
In this manuscript a method of simulating ischemia-like conditions is described. Additionally to the simulation of hypoxia, all energetic supplements of the organ bath solution were depleted to mimic the ischemic state in vivo as good as possible. In contrast to hypoxia, it was shown that the simultaneous depletion of oxygen and energy supply leads to changes in calcium transients comparable to alterations seen in vivo23.
The procedure described for the isolation of mouse papillary muscle can be adapted to other species, e.g., rat. However the optimal conditions of the organ bath setup could vary between different animal models and may be adapted. Parameters that may need adaptation are the temperature and the calcium concentration of the physiological solution.
In summary, this contractility method provides a very powerful approach to assess heart physiology, pathophysiology and pharmacology. When used properly, it provides the ability to study cardiac contractility in an isolated but well controlled environment.

The investigation of proteins like ion channels referring their role for cardiac contractility is essential to discover different pathomechanisms and to establish new therapeutic strategies for cardiac diseases such as ischemia and heart failure.
Contractile function of mammalian cardiomyocytes is known to be modulated by various ion channels, transporters and other proteins. Action potential evoked activation of voltage dependent sarcolemmal L-type Ca2+ channels leads to Ca2+ influx from extracellular space and subsequently to Ca2+-induced Ca2+ release (CICR)1, which triggers cellular contraction2. Ca2+-signaling plays a central role in cardiac contractility and adaptation to physiological or pathological stress. Catecholamines activate cardiac &#946;-adrenergic receptors, thus stimulating adenylyl cyclase (AC) which synthesizes cAMP. Being activated, protein kinase A (PKA) phosphorylates different intracellular and membrane associated proteins like L-type Ca2+ channels, phospholamban and ryanodine receptors resulting in modification of Ca2+ transients and cardiac contractility1,3,4. cAMP is degraded by phosphodiesterase (PDE). Activation of Gs-coupled receptors other than &#946;-adrenoceptors also leads to accumulation of cAMP.
The technique of contractility measurements in isolated ventricular muscle strips is well established for larger mammalian species5-8. Based on the possibility of gene targeting in mice it is important to establish methods to analyze murine cardiac physiology. However, existing data about the physiological properties of isolated muscle preparations in mice differ depending on experimental conditions9-12.
The described method is used to analyze cardiac contractility of left ventricular papillary muscle preparations in vitro. Investigation of cardiac contractility is performed in the absence of influences modifying cardiac contractility in vivo, like blood pressure, neurohumoral stimulation and physical or metabolic stress. The beating rate of the contracting muscle preparation can be rigorously defined and changed arbitrarily. Twitch force can be analyzed in the context of specific stimuli such as calcium concentration, beating frequency or temperature. In addition, this method can be used to investigate different signaling pathway components and to compare cardiac performance of genetically modified mouse models by controlling experimental conditions mentioned above.

<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

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.

The protocol of this manuscript for contractility measurements of isolated murine papillary muscle preparations is tuned to optimal conditions to achieve reproducible experimental results under physiological conditions. To define optimal experimental conditions we performed pilot experiments varying organ bath temperature and extracellular calcium concentration (see also12). The protocol described here was performed with an extracellular calcium concentration of 1.5 mM and a temperature of 32 &#176;C.
Basal contractility properties
To characterize basal contractility properties, parameters for twitch force amplitude, twitch rate, time to peak (defined as the time required to reach maximal contraction force starting from 5% basal) and relaxation 50 time (half maximal relaxation time; defined as time required to reach half of relaxation force) are used in this protocol. A representative trace of twitches and the analysis of these parameters are illustrated in Figure 3A-B.
Calcium-dependent contractility
Extracellular calcium concentration crucially determines cardiac excitation-contraction coupling and by varying this parameter, the calcium sensitivity of the contractile apparatus in cardiomyocytes can be evaluated. As shown in Figure 3C the augmentation of the extracellular calcium concentration results in altered calcium transients and subsequently in altered twitch force generation. The calcium-concentration of the extracellular solution is increased step-by-step (i.e., between 1.5 to 7mM). By applying this protocol, the calcium homeostasis regarding the calcium-dependent contractility of the contractile apparatus can be evaluated. In the organ bath setup used in this manuscript, the change of the extracellular calcium concentration was performed by changing the organ bath solution manually leading to a short interruption of the pacing and subsequently to an artificial postrest potentiation (see Force potentiation after non-pacing interval).
Force potentiation after non-pacing interval (PRP)
At the end of a contraction cycle calcium is sequestered into sarcoplasmatic reticulum (SR) predominantly by an ATP-dependent calcium pump (SERCA, sarco-endoplasmic reticulum calcium-ATPase), thus lowering the cytosolic calcium concentration during the diastole. If the time interval between two contractions is enlarged, more calcium can be pumped back into the SR and subsequently more calcium can be released during the next excitation. The postrest-potentiation protocol (PRP) describes alterations of the twitch force amplitude after a previous stimulation with rest period of a defined duration. In human, rat as well as in murine cardiac tissue, an augmentation of the twitch force was shown10,12-14,16(Figure&#160;3D). It is known that a positive post rest potentiation is diminished or even adverse in failing cardiac tissue of human and rat13,14,16,17. This protocol is used to evaluate the function of the diastolic calcium accumulation in intracellular stores4,15.
Frequency-dependent contractility (FFR)
Force-frequency relation describes the correlation between beating rate and contractile twitch force (Bowditch effect)18. The force- frequency relation significantly differs between mammalian species and experimental conditions19. In mice, a specific correlation between beating rate and contraction strength was shown. At 32 &#176;C, an initial negative FFR between stimulation rates of 0.1-1 Hz was shown, whereas the FFR was shown to be positive in the range of 1-4 Hz of stimulation9-12 (Figure&#160;3E).
In failing myocardium a negative correlation between beating rate and contractility is described20,21. In this manuscript, the standard pacing-frequency (1 Hz) is reduced to 0.1 Hz and starting from there, the pacing-frequency is incrementally raised to 5 Hz (0.2; 0.5; 1; 2; 3; 4; 5 Hz).
&#946;-adrenergic stimulation
Catecholamines like adrenaline and noradrenaline are hormones which are released during (patho-) physiological stress conditions modulating cardiac contractile force by activation of &#946;-adrenoceptors. Iatrogenic application of catecholamines plays a prominent role in clinical treatment of patients with acute heart failure to enhance cardiac inotropy temporarily. This effect can also be seen and investigated in in vitro studies. As an agonist of &#946;-adrenoceptors, Isoprenaline increases contractile twitch force. In vivo, an increase in beating frequency also occurs (positive inotropic effect), which additionally modulates the contractile response (Bowditch effect, see also FFR protocol described above). Using the method described here, the inotropic effect of Isoprenaline can be investigated at a fixed beating rate without that chronotropic effect (Figure&#160;3F). The stimulation of &#946;-adrenoceptors can also be achieved by other hormones such as histamine. In murine ventricular tissue, application of histamine results in a biphasic response which contains an initial positive inotropic effect, but with a transient decline in twitch force as shown in Figure 3G. For that reason an interpretation of data measured by application of histamine seems to be difficult; especially the underlying receptor-activation in mice is not entirely solved.
Ischemic conditions
Acute cardiac ischemia is defined as a critical limitation of blood flow leading to undersupply of the cardiac tissue with oxygen and nutrients resulting in loss of contractility, development of ischemic contracture and cell death. To simulate ischemia in vitro, muscles are exposed for 30 min to glucose- and pyruvate-free extracellular solution bubbled with 95 %N2/5% CO2. With that approach a depletion of ATP in the cardiomyocytes should be induced. Within few minutes after starting the ischemia-simulation, a decline of the twitch force occurs with appearance of an ischemic contracture pictured as a spontaneous increase in the preload tension (Figure&#160;3H).
<img alt="Table 1" src="/files/ftp_upload/53076/53076table1.jpg" />
Table 1: Components and concentrations of the used buffer solutions.
<img alt="Table 2" src="/files/ftp_upload/53076/53076table2.jpg" />
Table 2: List of suggested experimental protocols to investigate cardiac contractility in isolated papillary muscle preparations.
<img alt="Figure 1" src="/files/ftp_upload/53076/53076fig1.jpg" /> 
Figure 1.&#160;Key steps of the papillary muscle preparation.&#160;(A) Dissection of both atria. (B) View on the left ventricular anterior papillary muscle. (C) Dissection of the papillary muscle from the ventricular wall. (D) Attachment of two silk threads before the fixation in the organ bath chamber. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53076/53076fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53076/53076fig2.jpg" /> 
Figure 2.&#160;Organ bath setup.&#160;(A) Schematic setting of the organ bath setup. Water jacket organ bath chambers equipped with aeration glass frits provide stable temperature control. To generate square-wave pulses field electrodes are attached to the tissue support. A force transducer senses the contraction of the muscle thereby amplifying the generated signal. This signal is filtered and transferred back to the computer by an A/D converter. The signal is then digitized and can be saved for later analyses. (B) Picture of a single papillary muscle preparation fixed in the organ bath chamber from the organ bath setup (C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53076/53076fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53076/53076fig3.jpg" /> 
Figure 3.&#160;Representative results of contractility measurements of murine left anterior papillary muscles.&#160;(A) Representative analysis of twitch force amplitude, Time to peak tension (TTP) and half maximal relaxation time (R50) of a single twitch. Representative recordings of stimulated twitches of murine papillary muscle preparations under basal stimulation with a beating rate of 1 Hz (B), during increasing extracellular calcium concentrations (C), during defined resting intervals (post rest potentiation, PRP) (D), or different stimulation rates (force-frequency relation, FFR) (E) and after application of increasing concentrations of Isoprenaline- (F) or Histamine-application (G). Representative recording of stimulated twitches and preload during ischemia-stimulation (H). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53076/53076fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53076/53076fig4.jpg" /> 
Figure 4.&#160;Analysis of contractile parameters.&#160;To characterize contractile function, parameters including twitch force amplitude (height), time to peak tension (TTP) and half maximal relaxation time (R50/TFall, respectively) are analyzed in this protocol using the software program Chart 5.5 (ADInstruments). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53076/53076fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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

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14.9K Views.  Universität Heidelberg. 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.

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.

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

Medicine

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.

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.

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.

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

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.

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.

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.

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.

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.

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

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

Summary

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Chapters in this video

In vitro Measurements of Tracheal Constriction Using Mice

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

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

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Isolation and Physiological Analysis of Mouse Cardiomyocytes

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

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

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

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

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes