Cardiac Sudden Death Is Death Caused by Nonsequential Or Rapid Electrical Impulses Within

Expression Analysis of a Potassium Channel Regulator and Generation of the Knock-Out Construct

Jennifer Thompson , Tara St. Amand, Ken Chien. Institute of Molecular Medicine, University of California, San Diego. La Jolla, Ca 92092 Introduction The expression of KCR-1, a K+ channel regulator, within the conduction system may play a pivotal role in normal electrophysiological functioning of the heart. Normal electrophysiological functioning of the heart is mandatory in order to prevent cardiac sudden death. Cardiac sudden death is caused by non-sequential or rapid electrical impulses within the heart.1 This irregular heart rhythm (arrhythmia) causes the heart to stop beating suddenly. Normal electrophysiological function of the heart depends on proper development of the cardiac conduction system. The conduction system consists of the Sinoartial (SA) node, Atrioventricular (AV) node, the His-Bundle, and Purkinje fibers. The conduction system is a specialized network of electrical cells in the heart that stimulate the heart to beat. The electrical impulses from which the heart muscle causes the heart to contract initiate at the SA node. Once released from the SA node, the impulse causes the atria to contract. The signal then passes through the AV node, which is located at the junction of the atria and ventricles. The AV node checks the signal, delaying the activating impulse and sends it through the muscle Purkinje fibers of the ventricles, causing them to contract. The AV node serves as a crucial component of the conduction system, as it coordinates the pumping of the atria and ventricles so that they work together to pump blood most efficiently and sequentially. 2 Failures in the conduction system, which can ultimately lead to cardiac sudden death, may result from deficiencies in gene expression within the conduction system. Because KCR-1 is a neuronal gene, being expressed in the brain and the heart, it was of particular interest to us. Previous studies in cardiac sudden death have found that genes found in the brain and the heart, are found in the conduction system within the heart. Currently, there is little known about the development of the conduction system or what makes these cells different from the rest of the myocaridum. Recent studies however have suggested that the cardiac conduction system cells have many of the similar properties as cells in the brain. KCR-1 was initially identified as a K+ channel regulator in the brain. Previous studies involving northern blot analysis revealed that KCR-1’s expression is high in the central nervous system and low in the heart.3 This result was of great significance because it suggests that the low of expression of KCR-1 may be specific to the conduction cells. Conduction cells make up only 1% of the population of heart cells, thus the low expression of KCR-1 in the heart may be specific to these conduction system cells. We have identified KCR-1 as a conduction system specific gene using in situ hybridization of adult mouse hearts. Therefore, our hypothesis states that deficiencies in KCR-1 may result in conduction system defects, such as cardiac sudden death. To test this hypothesis we first analyzed the general expression pattern of KCR-1. Based on KCR-1's expression we then designed the construct for the conditional knockouts of KCR-1. Because we are generating a conditional KO, KCR-1 expression would be restricted to only certain areas of the conduction system. This will provide the data needed to see if requirement for KCR-1 comes from cells found in the brain and heart. Materials and Methods Whole mount insitus were performed on mouse embryos and isolated mouse hearts as described by (St. Amand et al., 1998).4 Construction of the KCR-1 vector to knockout exon 1 was done by PCR amplification and cloning into the pFlox-FRT Neo (courtesy from Chen) vector. PCR amplification was performed on the exon 1 and cloned in between 2 loxP sites. Adjacent 4kb 5` sequence was cloned using Xho1, and 4kb 3` sequence was cloned using Not1/Sac1. Exon 1’s location in between 2 loxP sites will enable it to be taken out when crossed with Cre. Results In situ hybridization was performed to observe KCR-1's expression pattern. In situ hybridization is the use of an RNA probe to detect a specific RNA sequence within certain tissues and/or organs.5 Prior studies indicate KCR-1 is expressed in the conduction system of adult mouse hearts. Therefore, we performed whole mount in situ to analyze KCR-1’s expression during development. Whole mount in situ hybridization of the E8.5 –E11.5 mice embryos and E14.5 isolated hearts revealed that KCR-1 is expressed as early at E8.5 but, expression seemed to be non-specific to a certain area. This non-specific expression pattern continued to be seen through E11.5. We were able to get the 5` sequence cloned into the vector, pFlox-FRT-Neo, however, time constraints prevented us from cloning in the 3` sequence. Discussion As a K+ channel regulator being expressed in the heart and the brain, KCR-1 is thought to play a pivotal role in normal electrophysiological functioning of the heart. Deficiencies in KCR-1 may result in conduction system defects, such as cardiac sudden death. Due to KCR1’s non-specific expression pattern as seen in the mouse embryos, we see that its widespread expression through development makes KCR-1 an undesirable marker for looking at development of the conduction system. Due to KCR-1’s expression in the conduction system as seen in the adult mouse heart, we sought to determine its function in terms of affecting the onset of cardiac sudden death. This required the construction of conditional KCR-1 knockout (KO) mice. We suspect that KO of KCR-1 will lead to conduction system defects, such as sudden death. Because we are generating a conditional KO, KCR-1 expression would be restricted to only certain areas of the conduction system. This will provide the data needed to see if requirement for KCR-1 comes from cells found in the brain and heart. 1 “Sudden Cardiac Death”. http://www.americanheart.org/presenter.jhtml?identifier=4741 Aug. 09, 2002 2 “The Conduction System”. Texas Heart Institute. http://www.tmc.edu/thi/conduct.html March 2002 3 4 St. Amand, Tara et al.,. “Antagonistic Signals between BMP4 and FGF8 Define the Expression of Pitx1and Pitx2 in Mouse Tooth-Forming Anlage”. Developmental Biology 2002 5 “In Situ Hybridization”. Immunohistochemistry-In situ Hybridization. http://home.no.net/immuno/. Jan. 2002