FUNCTIONAL CHARACTERIZATION OF SCN5A, THE CARDIAC SODIUMCHANNEL GENE ASSOCIATED WITH CARDIAC ARRHYTHMIAS
AND SUDDEN DEATH
LING WU
ABSTRACT
The cardiac sodium channel α subunit Nav1.5 (encoded by the SCN5A gene) plays an important role in the generation and propagation of electrical signals in the heart, and can cause cardiac arrhythmias, heart failure and sudden death when mutated or dysregulated. However, the precise composition of the multi-protein complex for the sodium channel has not been completely defined. The molecular mechanisms by which Nav1.5 mutations cause cardiac arrhythmias have not yet been well-studied in vivo. It remains to be explored whether Nav1.5 is expressed in other tissues and plays novel roles in other tissues or organs. This dissertation addresses these aspects of Nav1.5 regulation.
I found that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and distinctly modulates the physiological function of Nav1.5. A yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domain DII and III) of Nav1.5. The interaction was further demonstrated by both in vitro GST pull-down and in vivo co-immunoprecipitation assays. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities, whereas two siRNAs that knocked down expression of MOG1 decreased current densities. In neonatal myocytes, over-expression of MOG1 increased current densities nearly two-fold, and MOG1 siRNAs down-regulated the sodium currents. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues and was highly localized in the intercalated discs. These results suggest that MOG1 may be a critical regulator of sodium channel function in the heart and reveal a new function for MOG1. Furthermore, I showed that MOG1 increased sodium current density by increasing cell membrane localization of Nav1.5. This study further demonstrates the functional diversity of Nav1.5-binding proteins, which may be important for the function of Nav1.5 under different cellular conditions.
The second major project I worked on was identification by microarray analysis of genes differentially expressed in transgenic mice with cardiac expression of LQTS mutation N1325S of SCN5A. I identified 33 genes in five different functional groups that showed differential expression. STAT1, which encodes a transcription factor involved in apoptosis and interferon response, showed the most significant difference of expression between TG-NS and control mice (a nearly 10-fold increase in expression, P = 4 x 10-6). The results were further confirmed by quantitative real-time PCR and Western blot analyses. Accordingly, many interferon response genes also showed differential expression in TG-NS hearts. This study represents the first microarray analysis for LQTS and implicates STAT1 in the pathogenesis and progression of LQTS and heart failure developed in the transgenic mice.
The third project focused on the distribution of Nav1.5 protein in the mouse brain which was investigated using immunohistochemistry. Immuno-staining with a Nav1.5-specific antibody revealed that Nav1.5 protein was localized in certain distinct regions of brain including the cerebral cortex, thalamus, hypothalamus, basal ganglia, cerebellum and brainstem. Notably, I found that Nav1.5 protein co-localized with neurofilaments and clustered at a high density in the neuronal processes, mainly axons. These results suggest that Nav1.5 protein may play a role in the physiology of the central nervous system (generation and propagation of electrical signals by axons).
These studies on Nav1.5 provide new insights into the regulation of the function of Nav1.5, and shed light on the possible pathogenetic mechanism of cardiac arrhythmias at the molecular level.