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 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 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 β-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 β-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.
NOTE: The basic steps of the isolation procedure are shown in Figure 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
2. Preparation of Buffers and Physiological Solutions
3. Preparation of the Gassing Tube and Dissection Dish used during the Papillary Excision Procedure
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.
5. Equilibration and Stimulation of the Papillary Muscle
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).
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 °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 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 °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 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).
β-adrenergic stimulation
Catecholamines like adrenaline and noradrenaline are hormones which are released during (patho-) physiological stress conditions modulating cardiac contractile force by activation of β-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 β-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 3F). The stimulation of β-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 3H).
Table 1: Components and concentrations of the used buffer solutions.
Table 2: List of suggested experimental protocols to investigate cardiac contractility in isolated papillary muscle preparations.
Figure 1. Key steps of the papillary muscle preparation. (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. Please click here to view a larger version of this figure.
Figure 2. Organ bath setup. (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). Please click here to view a larger version of this figure.
Figure 3. Representative results of contractility measurements of murine left anterior papillary muscles. (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). Please click here to view a larger version of this figure.
Figure 4. Analysis of contractile parameters. 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). Please click here to view a larger version of this figure.
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 authors have nothing to disclose.
This work was supported by the Deutsche Forschungsgemeinschaft (KFO 196 “Signaltransduktion bei adaptativen und maladaptiven kardialen Remodelling-Prozessen”, 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).
Sodium chloride | Sigma-Aldrich | S7653 | |
Sodium bicarbonate | Sigma-Aldrich | S5761 | |
Potassium chloride | Sigma-Aldrich | P9333 | |
Glucose | Sigma-Aldrich | D9434 | |
Sodium pyruvate | Sigma-Aldrich | P5280 | |
Calcium chloride dihydrate | Sigma-Aldrich | 223506 | |
Magnesium sulfate heptahydrate | Sigma-Aldrich | 230391 | |
Potassium phosphate monobasic | Sigma-Aldrich | P 5655 | |
2,3-Butanedione monoxime | Sigma-Aldrich | B0753 | |
Forskolin | Sigma-Aldrich | F3917 | Hazard statement H312, solve in DMSO |
3-?Isobutyl-?1-?methylxanthine | Sigma-Aldrich | I5879 | Hazard statement H 302, solve in DMSO |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
Isoprenaline hydrochloride | Sigma-Aldrich | I5627 | Hazard statement H 315-H319-H335 |
Sodium Heparine 250.000 IE/10ml | ratiopharm | PZN 3874685 | |
Histamine dihydrochloride | Sigma-Aldrich | H7250 | Hazard statement H 315-H 317-H319- H334-H335 |