Diseases of the cardiac pacemaking and conduction system account for a large portion of the overall cardiovascular disease burden. Despite this, relatively little is known about the molecular pathways that control the development and maintenance of this highly specialized network of cells. In recent years, the development of molecular markers of the conduction system has opened a pathway for exploration of transcriptional control of conduction system development. We have learned that the dosages of particular transcription factors during precise spatial and temporal windows during cardiogenesis are critical for normal development and maintenance of the cardiac electrical system. The question of how these dosages are regulated has not yet been explored. MicroRNAs (miRNAs) are genomically-encoded small interfering RNAs that fine-tune target gene dosage by binding to specific mRNA transcripts and inhibiting translation. The discovery that miRNAs regulate the pool of proliferating ventricular cardiomyocytes during cardiogenesis by silencing a critical transcription factor raises the intriguing possibility that such translational control is equally important in conduction system development. In this proposal, previously described techniques to identify and characterize miRNAs will be employed to achieve three specific aims.
Specific Aim 1. To determine if miRNAs are necessary for normal conduction system development. miRNAs will be eliminated from the developing conduction system using tissue-specific deletion of Dicer.
Specific Aim 2. To identify miRNAs expressed in the developing conduction system. Conduction system cells will be isolated to identify candidate miRNAs with microarrays. Promising candidates will be validated in vivo with in situ hybridization.
Specific Aim 3. To identify and validate downstream targets of conduction system-specific miRNAs. An in- silico approach will be used to generate a list of candidate miRNA-mRNA pairs. MiRNA targets will be validated in culture and in vivo with transgenic mice overexpressing conduction system-specific miRNAs. This work seeks to unveil novel mechanisms of the molecular regulation of growth, patterning, and maintenance of phenotype in the mammalian cardiac conduction system. Understanding how this complex and elegant system is controlled at the molecular level may eventually permit targeted therapies for the millions of patients suffering from disorders of the cardiac electrical system. ? ? ?
