Stromal Interaction Molecule and Gelsolin Mediate Pathological Cardiac Remodeling

Abstract

Pathological cardiac remodeling is characterized by a series of poorly understood changes in myocardial microstructure involving changes in the cardiomyocytes cytoskeleton. Stromal interaction molecule 1 (STIM1) resides mostly in the sarco/endoplasmic reticulum where is senses intraluminal Ca2+ levels and activates the Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 leading to cytoskeletal remodeling are not known. Proximity‐labeling technique (BioID) can capture weak and transient protein‐protein interactions. Hence, in the present study we investigated the STIM1 interactome used the BioID technique. A promiscuous biotin ligase BirA* was fused to the cytoplasmic C‐terminous of STIM1 and generated MEF stable cell line expressing STIM1‐BirA*. Biotin was added to the culture medium of MEF expressing STIM1‐BirA as well as in control cells not expressing STIM1‐BirA. Cells were harvested after twenty‐four hours and total lysates were incubated with streptavidin magnetic beads to pull down the biotinylated proteins which were subsequently identified by mass spectrometry. STIM1 interactors were validated in neonatal rat cardiac myocytes (NRCM) coupled with biochemical, immuno‐staining techniques and confocal microscopy. Screening of biotinylated proteins identified several high confidence known and previously unknown interactors of STIM1. One of the novel interactors was Gelsolin (GNS), a Ca2+‐dependent actin‐severing protein that is a key regulator of actin filament assembly and disassembly. STIM1 and GSN relationship was further investigated using NRCM. We show that STIM1 upregulation in NRCM was enough to induce upregulation of GNS protein levels. Immunostaining confirms colocalization between STIM1 and GSM. Furthermore, STIM1 overexpression in NVCM show altered actin dynamic and tubulin overexpression probably in response to cytoskeletal instability. We report the first use of BioID to characterize the STIM1 interactome. Our work unravels an unprecedented role of GSN as a part of the STIM1 interactome and its role in the modulation of the actin cytoskeleton.Support or Funding InformationWe gratefully acknowledge funding from the the National Heart, Lung and Blood Institute (award HL114869, to Dr. S.M.), and from UTHSC seed funding.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.