Project Summary Sudden cardiac death and arrhythmias account for ~15-20% of all deaths worldwide. Voltage gated sodium channels (Nav) in heart are major regulators of myocyte excitability and cardiac function. The Nav channel current (INa) is a large amplitude and short duration inward current that is regulated by rapid channel activation and immediate inactivation. However, a small late component of this current (INa,L) is present at baseline but increases in response to heightened adrenergic challenge and has been correlated with fatal forms of congenital and acquired arrhythmia. The majority of work on Nav1.5 has focused on molecular pathways for positive regulation of INa,L. However, less is known about the pathways for negative regulation. These pathways are critical and can be targeted to alter the pathogenic INa,L in potentially fatal forms of arrhythmia. My previous work identified a key pathway for INa,L regulation via the B56α regulatory subunit of protein phosphatase 2A (PP2A). My previous and current data support that: 1) B56α co-localizes with Nav1.5 in the heart at the intercalated disc, along with ankyrin-G and CaMKIIδ, 2) PP2A activity is regulated by the specific regulatory subunit (B56α) and 3) activation of PP2A through modulation of B56α is sufficient to regulate Nav1.5 activity and resist the increase in INa,L following adrenergic challenge. Our work illustrates a key role of B56α in INa,L regulation as well as the potential for targeting this pathway in arrhythmias. However, the field has been limited by two key issues: 1) the lack of selective and inducible in vivo models to selectively impact key PP2A regulatory proteins, and 2) the lack of molecules that selectively target PP2A activity in vivo. For this proposal, we have generated two novel tools to identify the impact of PP2A-B56α on cardiac action potential and arrhythmias in vivo. First, we have created the first in vivo model to both constitutively as well as inducibly impact local PP2A regulatory subunit (B56α) function, and thus cardiac PP2A activity. This model has the impact in a field where work was limited to in vitro studies or non-selective activation or protein reduction. Second, we have identified a new molecule to directly test the in vivo impact of PP2A activation in disease. Our collaborators at Ohio State have synthesized a new molecule and have shown that this molecule increases PP2A activity. Our preliminary data support our central hypothesis that PP2A, via the B56α regulatory subunit, regulates the intercalated disc Nav1.5 to counteract CaMKII-dependent phosphorylation in disease. Further, we hypothesize that molecules that selectively modulate the PP2A-B56α holoenzyme will impact altered INa,L in heart disease. Combining molecular, biochemical, pharmacological, patch clamping and Ca2+ imaging approaches, we will: 1) define the functional role of PP2A-B56α signaling complex in primary myocytes, 2) determine the effects of PP2A-B56α regulation on ...