Isolation and Physiological Analysis of Mouse Cardiomyocytes

Summary

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.

Abstract

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.

Introduction

Cardiomyocytes provide the contractile force for the heart. Each myocyte contains organized cytoskeletal and contractile elements essential to contraction. The heart not only contains cardiomyocytes but also fibroblasts, connective tissue, and modified myocytes such as Purkinje fibers. Changes in any or all of these cell types can be seen in heart failure, making determination of the ultimate cause of heart dysfunction difficult. Decreased ventricular contractility of a failing heart is preceded by varying degrees of fibrous tissue build-up and hypertrophy and dysfunction of the cardiomyocytes¹. Arrhythmias can be caused by dysfunction in the conducting cel....

Protocol

Ensure all procedures involving animals are approved by the appropriate animal use and care body.

1. Prepare Stock Buffers in Advance

1. Prepare 2 L stock Ca²⁺ free Tyrode’s (Table 1). Adjust the pH to 7.4 with NaOH.
2. Prepare 2 L stock 1.2 mM Ca²⁺ Tyrode’s (Table 2). Adjust the pH to 7.4 with NaOH.

2. On the Day of the Experiment, Prepare Perfusion, Transfer, and Digestion Buffers

1. Prepare 150 ml perfusion buffer in Ca²⁺ free Tyrode’s (Table 3) and fil....

Results

Once the contractility and transient tracings are collected (Figure 2), data are easily analyzed with the appropriate software. The whole tracing, or portions thereof can be averaged (Figure 3). Contractility can be analyzed in a variety of ways. For systolic function, one can assess the magnitude of contraction with fractional shortening, or speed of contraction with time to peak shortening, and contraction velocity. Diastolic function can be analyzed similarly with time to 50% shorteni.......

Discussion

Isolation of quality cardiomyocytes requires some level of practice and optimization. This protocol contains key steps that can greatly affect the outcome of the digestion. These should be considered carefully when performing and troubleshooting the protocol. The time from removal of the heart to cannulation and perfusion should be minimized, ideally less than five minutes. Rinsing the heart immediately in ice-cold perfusion buffer after removing from the mouse and dissecting and cannulating the heart in ice-cold buffer .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	RPI	S23020
KCl	Sigma	P-3911
MgCl2	Sigma	M-8266
HEPES	EM Science	S320
NaH2PO4	Sigma	S5011
CaCl2·2H2O	Fisher	C79
D-(+)-Glucose	Sigma	G7528
2,3-Butanedione-monoxime (BDM)	Sigma	B-0753
Taurine	Sigma	T-0625
Bovine Serum Albumin (BSA)	Sigma	A-7030
Collagenase B	Roche Diagnostics	1-088-823
Collagenase D	Roche Diagnostics	1-088-882
Protease XIV	Sigma	P-5147
Heparin	APP Pharmaceuticals, LLC	401586D
Isofluorane	Butler Schein	11695-6776-2
Fura-2 AM	Teflabs	103
DMSO – anhydrous	Sigma	276855
IonOptix cell pacing and fluorescent analysis system	IonOptix
250 μm nylon mesh filter	Sefar America	Lab Pak 03-250/50
23 G Luer-stub adaptor	Becton Dickinson	1482619E
PE-50 tubing	Becton Dickinson	427410