Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Summary

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Abstract

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca²⁺ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca²⁺ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Protocol

1. Animal Anesthesia and Organ Harvesting

1. Anaesthetize the mouse by intraperitoneal (i.p.) injection of Ketamine (200 mg/kg BW) and Xylazine (20 mg/kg BW).
2. To anticoagulate inject 250 IU Heparin i.p. to avoid blood clotting and thrombus formation.
3. Wait until deep narcosis is reached, which is characterized by areflexia. To check for areflexia, test corneal reflex by gently touching the cornea or test flight reflex by tail pinching.
4. Transfer the mouse onto operating table and fix it in supine position.
5. Incise the skin and the abdominal wall below the xiphoid and perform clamshell thoracotomy: Extend the cut to both si....

Results

Isolation of adult murine cardiomyocytes from genetically engineered mice to study the function of specific genes of interest in vitro has become a powerful tool to further understand cardiac pathophysiology. This method is currently used by only a small but increasing number of basic science laboratories worldwide. However, isolation of adult ventricular murine cardiomyocytes can be tricky and needs to be done thoroughly and repetitively by experienced hands. Figure 1 shows freshly isolated exe.......

Discussion

With the growing development of genetically engineered mouse strains to study cardiac function and cardiac pathology related to gene deletion there is also an increasing interest in specialized methods to study effects of the specific gene deletion in vitro. In our laboratory we study the roles of a family of Kv channel ancillary subunits on cardiac repolarization. The KCNE genes comprise a family of 5 genes (KCNE1-5) that play important roles in human ventricular and atrial repolarization

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tetrodotoxin	Alomone
4-aminopyridine	ICN Biomedicals
Heteropodatoxin 2	Alomone
Tetraethylammonium	Sigma Chemicals
Collagenase Type 2	Worthington