Chemical Biology Tools for Biochemical and Functional Analysis of Myosin Phosphatase

Atrial fibrillation (AF) is a common cardiac arrhythmia characterized by irregular electrical impulses and impaired atrial contraction. This mechanical dysfunction contributes to blood stasis and increases the risk of thrombus formation, even after restoration of normal rhythm. Emerging evidence links atrial hypocontractility in AF to dysregulation of myosin light chain phosphatase (MLCP), a holoenzyme composed of a catalytic subunit (PP1cβ) and a regulatory subunit that targets the phosphatase to sarcomeric substrates. PPP1R12C is an atrial-enriched MLCP regulatory subunit that directs PP1cβ to the myosin regulatory light chain (MLC2a), modulating contractile force. In AF, PPP1R12C is upregulated, leading to excessive dephosphorylation of MLC2a and weakening of atrial contraction. This dissertation addresses the structural and functional analysis of the PPP1R12C–PP1cβ holoenzyme using a suite of chemical biology tools. To overcome challenges posed by PPP1R12C’s intrinsic disorder and the dynamic assembly of the complex, we employed mammalian expression systems and AlphaFold modeling to predict structure and optimize expression. A ligand-inducible degron system was developed by fusing PPP1R12C to a destabilized eDHFR domain, allowing rapid, reversible stabilization of the protein with trimethoprim (TMP). This approach enabled tight temporal control of PPP1R12C levels in HL-1 atrial cardiomyocytes. Functional assays demonstrated that stabilization of PPP1R12C reduced MLC2a phosphorylation and contractile amplitude in atrial cells, mimicking the hypocontractile phenotype observed in AF. Conversely, PPP1R12C depletion restored phosphorylation levels and improved contractile function. These findings establish a direct causal link between PPP1R12C abundance and atrial contractility and underscore the critical role of myosin phosphatase regulation in sarcomeric signaling. In summary, this work provides new biochemical and functional insights into the PPP1R12C– PP1cβ holoenzyme and introduces versatile experimental tools to dissect its regulation. These findings support the therapeutic potential of targeting myosin phosphatase in atrial-specific cardiac disorders such as atrial fibrillation.