Calreticulin localized to the myoendothelial junction mediates heterocellular communication in resistance arteries

Abstract

Regulated heterocellular communication depends on bidirectional signaling that occurs at myoendothelial junctions (MEJs), which are composed of endothelial cell (EC) extensions that make direct cytoplasmic contact with smooth muscle cells (SMC). The MEJ is the anatomical hallmark of resistance arteries and is a key signaling microdomain with endoplasmic reticulum (ER) as we demonstrate via expression of the ER retention signal –KDEL, and ER tracker in a live artery, both of which localize to holes in the internal elastic lamina. Presumably, the expression of ER in the MEJ could facilitate localized Ca2+ events at MEJs generated in response to second messengers from EC agonists or smooth muscle (SMC) agonists (by movement of IP3 or Ca2+ through gap junctions to the EC extensions composing MEJs). To determine what proteins may regulate the spatially localized Ca2+ events at the MEJ, we used an iTRAQ proteomic screen on in vitro MEJs and found significant enrichment of calreticulin. Calreticulin is a calcium binding protein that has been shown to bind ~50% of ER calcium. We confirmed the presence of calreticulin at MEJs using third‐order mesenteric arterioles. Thus, we hypothesized that deletion of EC calreticulin could alter Ca2+ events at the MEJ that were generated after application of EC or SMC agonists. For this reason we created an EC specific calreticulin conditional knockout mouse (Calreticulinfl/fl/CDH5‐CreERT2+). Initial experiments were performed in mice between 10–20 weeks old. In these experiments, carbachol (CCH; EC specific) or phenylephrine (PE; SMC specific) was added to third order mesenteric arteries and the Ca2+ events were quantified. The control mice had a robust increase in Ca2+ events at MEJs after PE stimulation, but Calreticulinfl/fl/CDH5‐CreERT2+ mice had a significantly reduced number of Ca2+ events. Furthermore, Calreticulinfl/fl/CDH5‐CreERT2+ mice had significantly enhanced constriction to PE, demonstrating the importance of calreticulin at the MEJ in the PE response. Conversely, CCH stimulation showed no differences in Ca2+ events and no difference in dilation to CCH in the Calreticulinfl/fl/CDH5‐CreERT2+ mouse. This polarized response in Ca2+ events at MEJs in Calreticulinfl/fl/CDH5‐CreERT2+ endothelium was lost in mice aged >75 weeks. These results demonstrate: 1) the enrichment of calreticulin to MEJs, 2) polarized activation of EC Ca2+ events is based upon whether the agonist is EC (apical) or SMC (MEJ) in origin and 3) aging alters how calreticulin polarizes Ca2+ events after agonist stimulation. Overall, we believe EC calreticulin can regulate Ca2+ events generated in responses to heterocellular communication.Support or Funding InformationNational Institutes of Health HL088554, HL120840, T32 HL007284, and American HeartAssociation 14PRE20420024