DWORF Directly Regulates Cardiac SERCA Function

Abstract

The cardiac sarco/endoplasmic reticulum Ca 2þ -ATPase (SERCA2a) plays a key role in intracellular Ca 2þ regulation in the heart. Impaired SERCA2a function has been reported in a number of pathological conditions. Therefore, understanding mechanisms of SERCA2a regulation is of clinical importance. It has been recently discovered that SERCA2a can be regulated by the peptide dwarf open reading frame (DWORF). However, the specific mechanism of this regulation remains unclear. It has been suggested that DWORF enhances SERCA2a function through the displacement of inhibitory peptide phospholamban (PLB). We tested the alternative hypothesis that DWORF can regulate SERCA2a by directly interacting with the pump independently of PLB. Confocal imaging of HEK293 cells transfected with fluorescently labeled DWORF and the ER Ca 2þ sensor R-CEPIA1er showed that DWORF localizes within the ER network. Fluorescent resonance energy transfer (FRET) experiments in cells transfected with fluorescently labeled SERCA2a and DWORF revealed that the two proteins directly interact with a 1:1 stoichiometry. ER luminal [Ca 2þ ] ([Ca 2þ ] ER ) was measured with R-CEPIA1er in SERCA2a stable line cells transfected either with DWORF or PLB. For each individual cell, the maximum ER Ca 2þ uptake rate and the maximum ER Ca 2þ load were analyzed. This analysis revealed that expression of DWORF increased ER Ca 2þ uptake rate and load, whereas PLB had an opposite effect on SERCA function. The effects of DWORF were associated with an increase in SERCA-ATPase activity. Analysis of calcium-induced calcium release events revealed that DWORF expression increased ER Ca 2þ release amplitude and frequency. These results suggest that DWORF can directly regulate ER Ca 2þ transport by improving both the SERCA2a kinetic and catalytic efficacy. This new mechanism of SERCA2a regulation might play an important role in regular Ca 2þ cycling in the heart and malfunction of this mechanism may lead to contractile dysfunction in the diseased heart.