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

Summary

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.

Abstract

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.

Introduction

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 contractility^1,3,4. 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 species^5-8. 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 conditions^9-12.

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.

Protocol

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

1. 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.
2. Start up computer and data acquisition equipment. Turn on organ bath heat (preset to 32 °C) and allow units to preset temperature.
3. Prepare the acquisition equipment. Start recording data as mentioned in the software manual.
4. 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.
5. Turn on gas supply (95% O₂/5% CO₂) to organ bath chambers. Continuously gas all chambers during the entire protocol measurements.

2. Preparation of Buffers and Physiological Solutions

1. 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).
2. 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°C (see Table 1 Preparation solution). For ischemia simulation, prepare ischemia-solution as described in Table 1.
3. For the protocol of the Ca^2+-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°C before using.

3. Preparation of the Gassing Tube and Dissection Dish used during the Papillary Excision Procedure

1. Connect a soft gassing tube (external diameter 2-4 mm) to the gas connection (100% oxygen, carbogen alternatively) with expert working.
2. 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.
3. 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.

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.

1. 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.
2. Sacrifice the mouse (approximately 8-12 weeks old) by cervical dislocation according to expert working and institutional guidelines.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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’s view).
8. 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.
9. Open the ventricle and fix the left-sided free wall with forceps, thus having a look at both left ventricular papillary muscles.
10. 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.
11. 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 °C is suggested.

5. Equilibration and Stimulation of the Papillary Muscle

1. 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.
2. 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.
3. 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.
4. After 45-60 min of equilibration, start the experimental protocol.

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).

1. Recording and analysis of the experimental protocols
   1. For recording, use a suitable software with adequate temporal resolution (acquisition rate ≥ 1kHz).
   2. 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).

Results

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.

Discussion

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). Ad...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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/10 ml	ratiopharm	PZN 3874685
Histamine dihydrochloride	Sigma-Aldrich	H7250	Hazard statement H 315-H 317-H319- H334-H335