Cardiomyocyte excitation-contraction coupling and normal cardiac function are controlled by the coordinated activities of integral membrane proteins, signaling molecules, and structural proteins. We recently demonstrated that human cardiac arrhythmias can arise from mutations to proteins that are important for the proper targeting and retention of ion channels and transporters to specialized cardiac membrane domains. Specifically, mutations to the adaptor protein ankyrin-B have been linked to a variety of cardiac disorders that are collectively referred to as ankyrin-B syndrome and include catecholamine polymorphic ventricular tachycardia, sinus arrhythmia, atrial fibrillation, and delayed conduction/conduction block. The objective of this grant is to examine the molecular basis for the multifaceted capabilities of ankyrin-B in heart. Our recent findings demonstrate that ANK2, which encodes ankyrin-B, is comprised of 53 exons that are alternatively spliced to yield a diverse population of ankyrin-B polypeptides. Similar findings of tissue-specific alternative isoforms for the related ankyrins (-G and -R) provide support for our central hypothesis that the human heart maintains a previously unidentified complement of ankyrin-B isoforms with unique and critical functions for normal cardiac function. In support of this hypothesis, we identified three novel alternative ANK2 splice variants that remove key exons known to encode binding sites for the sodium/calcium exchanger, inositol (1,4,5) triphosphate receptor, and the large structural and resident M-line protein obscurin. Moreover, we demonstrated that the subcellular targeting of an ankyrin-B isoform is regulated by the alternative splicing of an ANK2 exon that encodes an obscurin binding site. We hypothesize that some specific ankyrin isoforms play critical roles in ion channel and transporter targeting to ttie junctions of transverse-tubules and the sarcoplasmic reticulum in myocytes, while other ankyrin isoforms are critical for the proper organization of protein complexes associated with myofibrillar proteins. We propose the following aims: Specific Aim 1. Identify and characterize novel ankyrin-B cardiac isoforms. Specific Aim 2. Define the role of these novel ankyrin-B isoforms for protein targeting, stability, and function in cardiomyocytes.