STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca²⁺ entry and mitochondrial Ca²⁺ homeostasis in cardiomyocytes

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca²⁺ sensors that trigger store-operated Ca²⁺ 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 Ca²⁺ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the I_SOC 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 Ca²⁺ 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_SOC 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 Ca²⁺ cycling, we observed, using Rhod-2/AM Ca²⁺ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca²⁺ (mCa²⁺) 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 (IP₃Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca²⁺ 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 mCa²⁺ uptake capacity possibly via the STIM2-IP₃Rs-VDAC-MCU and MNF2 complex.