Abstract 339: DWORF: a Novel Cardiac Micropeptide That Enhances SERCA Activity and Cardiomyocyte Contractility

Background: Our lab has discovered a novel conserved micropeptide of 34 amino acids encoded by a muscle-specific RNA that was previously annotated as a putative long non-coding RNA (lncRNA). This micropeptide, which we named DWORF (DWarf Open Reading Frame), shares structural similarity with the known SERCA modulators phospholamban (PLN), sarcolipin (SLN) and myoregulin (MLN). Objective: To define the functional role of DWORF in the heart and elucidate its mechanism of action. Methods and Results: Immunofluorescence staining and imaging in isolated adult mouse cardiac myocytes indicates that DWORF specifically localizes to sarcoplasmic reticulum (SR) membranes where it co-localizes with SERCA. Co-IP experiments performed in COS or HEK cells co-transfected with DWORF and SERCA show that DWORF forms a stable complex with various SERCA isoforms. Further biochemical analysis indicates that DWORF binds to the same site on SERCA as PLN, and overexpression of DWORF is capable of competing with PLN for SERCA binding. Co-expression of GFP-tagged DWORF and PLN in the presence of SERCA followed by SERCA pull-down and GFP Western indicates that PLN and DWORF have similar affinities for SERCA binding. Transgenic mice with cardiac specific over-expression of DWORF show enhanced cellular contractility as evidenced by increased peak Ca2+ transient amplitude and faster cytosolic Ca2+ decay rates (reduced Tau values). Furthermore, Ca2+-dependent Ca2+-uptake assays performed in homogenates from DWORF transgenic and littermate control hearts show a statistically significant leftward shift of the Ca2+-dependence curve for SERCA indicating a higher affinity of SERCA for Ca2+. Intriguingly, DWORF expression is silenced in the heart in response to pathological calcineurin signaling, implicating this micropeptide in the cellular pathway for calcineurin action. Conclusions: We have identified a novel muscle-specific micropeptide, DWORF, which is expressed in the heart at high levels and has the capacity to bind SERCA and modulate its function to increase contractility in transgenic mice. Our findings provide exciting data in support of DWORF as a previously unrecognized endogenous SERCA activator that plays a role in cardiac contractility.