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 ....

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....

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