STIM1 Enhances SR Ca2+ Content through Inhibiting Phospholamban in Rat Ventricular Myocytes

Abstract

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an ER/SR Ca2+ sensor, is unknown with respect to its cellular localization, its Ca2+-dependent mobilization, and its action on Ca2+ signaling regulation. This study is seeking to determine the function of STIM1 in Ca2+ signaling in adult ventricular myocytes. Using freshly isolated adult rat ventricular myocytes and those in short-term primary culture and confocal microscopy, we measured real-time Ca2+ signaling and tracked the cellular movement of STIM1 with m-cherry and immunofluorescence. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional sarcoplasmic reticulum (jSR). Depleting SR Ca2+ using the SERCA inhibitor thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated with fluorescence recovery after photobleaching (FRAP). Consistent with this result, native protein gel electrophoresis showed that STIM1 in heart exists mainly as a multimer, which is not altered by SR Ca2+ depletion. Additionally, we found no store-operated Ca2+ entry (SOCE) in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpression in these cells has a surprising phenotype with increased SR Ca2+ content and with increased SR Ca2+ leak. These changes in Ca2+ signaling in the SR appear to be due to STIM1-phospholamban (PLN) binding and thereby through indirect activation of SERCA2a. We therefore conclude that STIM1 contributes to the regulation of SERCA2a activity, the rate of SR Ca2+ leak and that these actions are independent of SOCE, a process that is absent in normal heart cells.