This work was funded by grants from Deutsche Forschungsgemeinschaft (DFG), Fritz-Thyssen-Stiftung and Charit&eacute;/MDC.

1. Animal Anesthesia and Organ Harvesting
<ol>
 <li>Anaesthetize the mouse by intraperitoneal (i.p.) injection of Ketamine (200 mg/kg BW) and Xylazine (20 mg/kg BW).</li>
 <li>To anticoagulate inject 250 IU Heparin i.p. to avoid blood clotting and thrombus formation.</li>
 <li>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.</li>
 <li>Transfer the mouse onto operating table and fix ...

<ol>
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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 <sup...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>Tetrodotoxin</td>
 <td>Alomone</td>
 </tr>
 <tr>
 <td>4-aminopyridine</td>
 <td>ICN Biomedicals</td>
 </tr>
 <tr>
 <td>Heteropodatoxin 2</td>
 <td>Alomone</td>
 </tr>
 <tr>
 <td>Tetraethylammonium</td>
 <td>Sigma Chemicals</td>
 </tr>
 <tr>
 <td>Collagenase Type 2</td>
 <td>Worthington</td>
 </tr>
 </tbody>
</table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

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.

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...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

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.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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13.3K ビュー.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). KVチャネル機能不全は、心臓不整脈に関連付けられています。我々はKvをチャネルの補助サブユニットのノックアウトマウスからの心房と心室の心筋細胞を単離するための体系的なプロトコルを利用するような不整脈を引き起こす分子メカニズムを研究するために。隔離された心筋細胞は、その後すぐに携帯電気生理学的研究、生化学的または免疫蛍光法?...