Abstract WMP39: Protein Phosphatase 1 Regulatory Subunit 12C Contributes to Atrial Myosin Light Chain Dephosphorylation in Atrial Fibrillation

Background: Atrial fibrillation (AF) increases stroke risk five-fold. Atrial hypocontractility from atrial myosin light chain (MLC2a) dephosphorylation contributes to stroke risk in AF. Recent proteomic data has shown increased protein phosphatase 1 subunit 12C (PPP1R12C) targeting to MLC2a in AF. However, it is unclear whether PPP1R12C causes MLC2a dephosphorylation in AF. Objective: Determine whether increased PPP1R12C expression causes MLC2a dephosphorylation and increases AF risk. Methods: Western blots and co-IPs were performed to evaluate the relationship among PPP1R12C, PP1c and MLC2a in human atrial tissues (AF vs SR). Mice with either a knockout (KO) or lentiviral (LV) cardiac overexpression of PPP1R12C were evaluated with invasive EP studies for AF inducibility vs WT controls. Results: In human AF, PPP1R12C was increased 4-fold ( P <0.005, n=6) with an 88% reduction in S-19-MLC2a phosphorylation ( P <0.05, n=4). PPP1R12C-PP1c and PPP1R12C-MLC2a binding was increased 2-fold in AF ( P <0.05, n=6). AF burden in LV-12C mice increased nearly tenfold vs. KO and WT mice ( P <0.05, n=6). Conclusion: In human AF, increased PPP1R12C expression is associated with reduced P-MLC2a through enhanced binding with the PP1c catalytic subunit. This dephosphorylation is a likely contributor to atrial hypocontractility and stroke risk in AF. Additionally, increased PPP1R12C expression in mice increases AF risk. Future studies will examine the effects of increased PPP1R12C expression upon atrial contractile function in mice.

Abstract 283: Nppa Marks A Subset Of Embryonic Cardiomyocytes Predestined To Form Trabeculae In Zebrafish

The development and maintenance of cardiac trabeculae is required for proper ventricular function. Aberrant trabeculation can cause heart failure, arrhythmia, and death. Yet the mechanisms controlling initiation of trabeculation are incompletely understood, partly due to a lack of markers for trabeculation during early embryogenesis. We hypothesized that natriuretic peptide A (nppa) marks trabeculae before cardiomyocytes leave the single-celled ventricular layer. While nppa has been studied in mammals, the external development of the translucent zebrafish embryo offers new insight into the earliest stages of trabeculation. We have found by in situ hybridization, and by creating a fluorescent transgenic zebrafish line, that nppa exclusively marks zebrafish cardiac trabeculae from their onset through adulthood. Live spinning disc confocal video microscopy shows that nppa:GFP-positive cells move into the trabecular layer starting at 60 hours post fertilization (hpf). GFP-positive trabeculae are seen by 5 days post fertilization (dpf) in normal larvae. However, in cardiac troponin T2a mutants and in erbB2 mutants _ both known not to trabeculate _ nppa is still expressed, but the nppa:GFP positive cells fail to form trabecular projections. In embryos injected with a morpholino against atrial myosin heavy chain 6, where weakened atrial contraction leads to weaker blood flow without affecting the ventricle directly, GFP-positive cells form trabeculae similar to uninjected controls. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we created nppa mutants causing frameshift deletions. Mutants have poorly contractile hearts at 3 dpf and have been crossed into transgenic reporter lines in order to specifically assess trabeculation. Together, these data suggest that nppa marks a subset of embryonic cardiomyocytes destined to form trabeculae, and that nppa loss of function causes cardiac defects.