Preparation of Acute Myocardial Tissue Slices from Biopsies of Human Neonates and Infants with Congenital Heart Disease for Physiological Measurements

Summary

Cardiac slices are a unique model for cardiovascular research and bridge the gap between single-cell and whole-heart models. This protocol describes the preparation of viable cardiac slices from myocardial tissue samples excised during surgery for congenital heart disease.

Abstract

In cardiovascular research, diverse ex vivo models are used to investigate cardiac function. These models can be categorized according to their complexity, ranging from isolated cardiomyocytes to multicellular 3-dimensional tissue preparations, such as the Langendorff-perfused heart or coronary-perfused wedges. Cardiac tissue slices bridge the gap between these models, as their relatively low thickness overcomes the need for arterial perfusion, while the native cellular alignment and extracellular matrix structure are preserved. This enables the use of tissue when coronary perfusion is not available (e.g., tissue excised during surgery for congenital heart disease). The present protocol describes the preparation of viable cardiac slices from myocardial explants from neonate and infant patients undergoing surgery for congenital heart disease. Upon extraction, the myocardial tissue is transferred to oxygenated, ice-cold, low-calcium solution and transported to the laboratory. Thereafter, the tissue is pre-cut, embedded into low-melting agarose, and sectioned with a vibratome. Tissue recovery is promoted by the stepwise increase of calcium concentration, followed by gradual rewarming to 37 °C for 1 h in the measurement solution. Afterwards, the obtained acute myocardial slices can be used for physiological experiments. Representative results for isometric force measurements and action potential recordings are provided. The importance of the solution and the vibratome parameters to the preparation of viable cardiac slices, as well as limitations regarding the control of the fiber alignment and long-term culture, are discussed.

Introduction

Ex vivo cellular studies on myocardial function rely on a spectrum of models, ranging from isolated single cells to whole-heart preparations such as the Langendorff-perfused heart.

Although isolated cardiomyocytes are the key model for many research questions, they do not entirely reflect the in vivo situation because intercellular interactions and connections to an extracellular matrix (ECM) are missing^1,2. Moreover, enzymatic digestion during the dissociation of myocardial tissue can modify the electrophysiological properties of cardiomyocytes. For instance, Yue ....

Protocol

This study was approved by the local ethics committee of the Medical Faculty of the University of Cologne (reference no. 07-045) and complied with the Word Medical Association Declaration of Helsinki (7th revision, Fortaleza, Brazil, 2013). Written informed consent was given by the parents of each patient.

1. Laboratory Preparation

1. Switch on the water bath that controls the temperature of a custom-made jacketed vessel (Figure 1). Set the temperature to.......

Discussion

Cardiac slices bridge the gap between single-cell and complex multicellular models for physiological research^1,2. Here a protocol has been introduced describing the preparation of viable cardiac tissue slices from human myocardial explants obtained from neonatal and infantile patients undergoing surgery for congenital heart disease. These slices can be used for studying contractile behavior and electrophysiological properties, among other physiological parameters.......

Materials

Name	Company	Catalog Number	Comments
NaCl	Carl-Roth	HN00.2	solution A: 136 mmol/L in aqua dest.
KCl	Merck	1.04936.0500	solution A: 5.4 mmol/L
NaH2PO4 * 2 H2O	Carl-Roth	T879.1	solution A: 0.33 mmol/L
MgCl2 * 6 H2O	Merck	1.05833.0250	solution A: 1 mmol/L
D(+)-glucose	Carl-Roth	HN06.3	solution A: 10 mmol/L
HEPES	Carl-Roth	HN77.3	solution A: 5 mmol/L
2,3-Butanedione monoxime	Sigma-Aldrich	B0753	solution A: 30 mmol/L
NaOH	Carl-Roth	K021.1	solution A: use to adjust pH to 7.4
CaCl2 * 2 H2O	Merck	1.02382.0250	calcium solution: 100 mmol/L in aqua dest.
Agarose Low Melt	Carl-Roth	6351.5	for slicing
Steel blades	Campden Instruments	7550-1-SS	for slicing
Vibratome	Leica	VT1000 S	for slicing
Glass pasteur pipettes	VWR	14673-010	for 'slice collector' tool
Peleus ball	VWR	612-2699	for 'Slice collector' tool
steel chamber	custom-made		for embedding tissue into agarose; cylindric shape (inner: Ø: 1.5 cm, depth 0.9 cm; outer: Ø: 2.0 cm, depth 1.1 cm)
instant adhesive	Henkel AG	PSG2C	Pattex Ultra Gel; for slicing
Jacketed vessel	custom-made		to maintain temperature of agarose in beaker at 37°C; made of steel with a plexiglass bottom
Water bath	Grant	Sub 14	for temperature control of jacketed vessel
Water pump	Aquarium Systems	Mini Jet MN404	for temperature control of jacketed vessel
IMDM	Life Technologies	31980022	for force measurements
DMEM, high glucose	Life Technologies	61965026	for microelectrode recordings
E4031 dihydrochloride	Abcam	ab120158	for microelectrode recordings