The molecular function of the Popeye domain containing genes in the heart

The electrical impulse, which stimulates the beating of our heart, is generated in an oscillatory fashion in a small cluster of cardiac cells, which is called the sinuatrial node. The node cells will pace the heart at different frequencies, depending on the physiological demands. An increase in beating frequency is mediated by the release of adrenergic hormones, which stimulates pacemaker activity in sinuatrial node cells. We have identified a gene family called the Popeye domain containing genes, which have an important but not yet fully understood role in regulating pacemaker activity. We have observed in mouse models lacking single Popdc genes an inability of the sinuatrial node to adapt to physical activity. Hearts of these mutant animals were unable to increase heart frequency in response to stress, but rather the stressed heart was beating at a lower frequency or was not beating at all for short periods of time. Mutations in one member of this gene family were also identified in patients with abnormally slow heart frequency, strongly suggesting that these proteins are an essential component for proper functioning of the cardiac pacemaker. In this grant proposal we will perform further genetic experiments in mice to fully understand the role of the different family members in cardiac pacemaker function. Moreover the precise mechanisms by which these proteins are accomplishing their tasks within sinuatrial node cells will be further analyzed by making specific mutations in essential parts of the protein. We will also generate a mouse model for the human point mutation in order to further learn how these molecular alterations affect cardiac pacemaking and analyze the biophysical and biochemical mechanisms by which the Popeye domain containing proteins stimulate pacemaker activity in sinuatrial node cells. We are convinced that the proposed experiments will help to define the role of this gene family in the heart, which is vital to our survival and wellbeing.

The Popeye domain containing (Popdc) genes are involved in adrenergic signaling in the heart. In order to overcome genetic redundancy, mice will be generated that lack the entire gene family and will be phenotypically assessed by ECG analysis. In order to assess the phenotypic consequences, a point mutation will be introduced into mice, which interferes with cAMP binding, and a nonsense mutation, which is present in patients with severe early onset bradycardia. In order to identify ion channels with which Popdc proteins might interact, we will analyze the action potentials of cardiac myocytes isolated from different null mutants. Alterations in ion channel anatomy might lead to the identification of malfunctioning currents and ultimately to ion channels, which directly interact with Popdc proteins. Candidates will be further assessed by co-localization and immunoprecipitation analysis. This study is likely to provide novel opportunities for rational drug design.

Impact Summary
This grant proposal aims at defining the function of the Popeye domain containing genes. The nature of this grant application is to perform basic research, which will therefore be primarily of interest to other academic researchers worldwide, which are interested in the membrane biology of cardiac myocytes, in cyclic AMP binding proteins, or working on cardiac pacemaker function. With this research we will provide novel insight into cardiac pacemaker function, which is a scientific area that still lacks a complete understanding of how the cardiac pacemaker works and in particular how adrenergic signaling might have its impact on cardiac pacemaking. The animal models that we have already generated and will be generating during the course of this grant might be of interest to research labs interested in developing biological pacemakers, since we have observed gradual loss of pacemaker cells in the null mutant animals. Since we have identified Popdc proteins as novel cAMP binding proteins we feel that, in the longer term , these proteins might also become a target for drug development in order to modulate cardiac pacemaker function. The mouse models might also be of use to study therapeutic interventions to prevent the development of sick sinus syndrome, a condition which develops in the elderly patient and for which currently the only therapeutic option is the implantation of a pacemaker devices. Since a significant fraction of the total pacemaker implants are due to sinus dysfunction, the development of novel options for therapeutic intervention would result in a significant cost reduction for the health sector. However, this will require further research into this subject beyond the current grant application. The mice will however also be further exploited by collaborating with labs interested in organ systems which also show strong and specific gene expression of Popdc genes, but which are outside the primary interest of the applicants. The use of tissue-specific Cre lines will allow the assessment of organ-specific functions of the Popdc genes. In this regard the recent observation that Popdc genes are associated with the development of colon cancer is an example of possible future collaborations. We are also proposing to generate an animal model for a human mutation we have found in one of the Popdc genes in patients having a severe and early onset bradycardia. We are particularly interested in studying the phenotype of this mutant and whether this acts as a dominant negative mutation but this research most likely will foster further search into mutations in Popdc genes associated with various forms of cardiac arrhythmia.