STIM1 enhances SR Ca ²⁺ content through binding phospholamban in rat ventricular myocytes

Abstract

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca2+ sensor, is unclear with respect to its cellular localization, its Ca2+-dependent mobilization, and its action on Ca2+ signaling. Confocal microscopy was used to measure Ca2+ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. 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 SR (jSR). Depleting SR Ca2+ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca2+ depletion. Additionally, we found no store-operated Ca2+ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca2+ content and increased SR Ca2+ leak. These changes in Ca2+ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca2+ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca2+ leak and that these actions are independent of store-operated Ca2+ entry, a process that is absent in normal heart cells.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling

“…We and others have previously shown that in cardiac myocytes, as well as in smooth muscle cells, STIM1 co-immunoprecipitates with Orai1 and 3 channels under resting conditions 9, 49 . STIM1 might thus represent a large protein complex that can adapt to different stresses to interact with a diversity of complexes 12 . In response to hypertrophic or proliferative stimulus, a large recruitment of Orai3 to STIM1 and Orai1 occurs thus allowing for a store-independent entry 9, 49 .…”

“…A similar phenomenon has been reported in proliferative smooth muscle cells where STIM1 controls heteromultimers of Orai1 and Orai3 that are regulated by leucotrienes C4 9-11 . Interactions with less Ca 2+ selective channels, such as TRPCs, have also been suggested and STIM1 overexpression leads to dysregulated calcium transients 5, 12 . All together, these data indicate that STIM1 activates in response to stress and can interact with a diversity of plasma membrane channels that will however mediate highly localized Ca 2+ signals.…”

High glucose suppresses embryonic stem cell differentiation into cardiomyocytes

“…Differentiated EB in low or high glucose conditions were placed on gelatin-coated chamber slides (Lab-Tek, Thermo Fisher Scientific, Waltham, MA, USA) for Ca 2+ imaging based on a previously described protocol [29]. ES-derived cardiomyocytes at day 9 were loaded with 5 μM fluo 4-AM (Thermo Fisher Scientific, Waltham, MA, USA) for 20 minutes at room temperature and replaced with normal Tyrode’s solution containing (in mM) 140 NaCl, 10 Hepes, 0.5 MgCl 2 , 0.33 NaHPO 4 , 5.5 glucose, 1.8 CaCl 2 , and 5 KCl (pH 7.4).…”

Role of STIM1 (Stromal Interaction Molecule 1) in Hypertrophy-Related Contractile Dysfunction

“…Here, we specifically study the role of the SR protein, Stromal Interaction Molecule 1 (STIM1), and its sarcolemmal partner protein Orai in the development of electrical and contractile remodeling of adult ventricular myocytes after pressure overload. There is some evidence from previous reports that these proteins are present in cardiac myocytes and their abundance increases in disease states 16, 17 . This increase has been linked to maladaptive cardiac hypertrophy 18, 19 .…”

“…It is therefore not surprising that a physiological role for STIM and Orai in cardiac myocytes has not been defined. Yet, many independent groups have found STIM1 to be present in cardiac myocytes 27 , though there are conflicting reports on the function of STIM1 and SOCE in these cells 16, 28, 29 . In the diseased heart, SR Ca 2+ regulation is altered 11 and increased SOCE activity has been observed in rodent cardiac disease models.…”