STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca entry and mitochondrial Ca homeostasis in cardiomyocytes.

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca sensors that trigger store-operated Ca entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the I current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the I current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca cycling, we observed, using Rhod-2/AM Ca imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca (mCa) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IPRs), voltage-dependent anion channel (VDAC), mitochondrial Ca uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa uptake capacity possibly via the STIM2-IPRs-VDAC-MCU and MNF2 complex.