A feature of heart failure is the global reversion from adult to fetal patterns of metabolism and misregulation of alternative splicing programs. Furthermore, the physiological significance of thousands of splice forms has yet to be elucidated. This indicates that there is a considerable need to investigate the factors that govern alternative splicing decisions and their physiological significance in the heart. Exon 31 of the clathrin heavy chain (Cltc) pre-mRNA is highly included in the heart and skeletal muscle, and slightly in brain while it is skipped in other tissues. Thus, two tissue specific Cltc isoforms are generated differing only in the presence or absence of exon 31. The inclusion of Cltc exon 31 is reduced in the heart during heart failure conditions. The functional importance of these splice forms is highlighted by the fact that CRISPR engineered mice expressing only the short Cltc splice form lacking exon 31 are protected from pressure-overload induced hypertrophy and heart failure and have reduced AMPK phosphorylation, compared to mice expressing both Cltc splice forms. AMPK is an intracellular energy sensor and its phosphorylation promotes glucose transporter (GLUT) translocation to the plasma membrane and prevents GLUT endocytosis to promote glucose influx. GLUTs are endocytosed in a clathrin-dependent manner. Therefore, the main research goals of this proposal are: (i) to identify the RNA-binding proteins (RBP) that regulate alternative splicing of Cltc pre-mRNA (Aim 1), and (ii) to determine the impact of Cltc splicing regulation on cardiomyocyte metabolism in basal and heart failure conditions (Aim 2).
Aim 2 will further elucidate the energy substrate utilization of cardiomyocytes expressing Cltc alternative splice forms in basal and heart failure conditions (Aim 2a) and determine the impact of Cltc splicing in GLUT dynamics in cardiomyocytes (Aim 2b). Targeting metabolic pathways, GLUT expression, and AMPK are protective in models of heart failure, therefore data from this proposal will identify novel targetable proteins and pathways to treat or prevent cardiac hypertrophy and failure. The world-renowned scientific environment of UNC-Chapel Hill and the surrounding `Research Triangle' area will expose the applicant to experts in the fields of cardiac biology, alternative splicing, cell biology, RNA biology, and metabolism. Furthermore, the training, scientific mentorship, and experimental design outlined in this F32 postdoctoral fellowship proposal have been tailored specifically to the applicant's long-term goal: to build a career focused on the intersection between muscle and RNA biology, nutrient signaling, and alternative splicing function and regulation.
The proposed research is relevant to public health because heart failure has been described as a global pandemic and the prevalence and public health burdens are expected to increase in the coming years. Heart failure is associated with a global reversion from adult to fetal patterns of metabolism and alternative splicing; therefore, fundamental discoveries filling the gap in knowledge on how this process is regulated and the specific function of alternative splice forms are essential to developing novel therapies to treat and prevent heart failure. The proposed research is also relevant to the goals of the NIH that pertain specifically to the cause, diagnosis, prevention, and cure of human disease, and the process of human growth and development.
