Calcium release-activated channel regulator 2 A (CRACR2A), a novel player in the pathophysiology of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare and devastating disease resulting from the obstruction of pulmonary arteries (PA). PAH is multifactorial, involving all cell types of human pulmonary circulation, including PA smooth muscle cells (hPASMCs), pulmonary endothelial cells (hPECs), and PA adventitial fibroblasts (hPAAFs). A missense variant in the CRACR2A ( Calcium release-activated channel regulator 2 A ) gene was identified in patients with PAH, but its role in PAH pathogenesis remains unknown. CRACR2A was described as a regulatory protein of Orai1, the main calcium (Ca 2+ ) channel involved in Store-Operated Ca 2+ Entry (SOCE) and PAH pathogenesis. Exome sequencing identified 9 variants in the CRACR2A gene in both the French and US cohorts. Using immunofluorescence staining, we found that CRACR2A is expressed in hPASMCs and hPAAFs from both control and PAH patients. By immunoblot, we showed that CRACR2A protein expression is similar in hPECs and hPAAFs from PAH patients and controls, whereas expression is reduced in hPASMCs from PAH patients (PAH-hPASMCs) compared to controls. CRACR2A knockdown decreases SOCE and cell proliferation in hPASMCs from control patients. In PAH-hPASMCs, CRACR2A knockdown increased SOCE and Orai1 expression. By a liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach, we found that CRACR2A is involved in vesicle transport and protein trafficking. In vivo, CRACR2A knockdown accelerates the development of experimental PAH in the monocrotaline-exposed rats. The identification of 9 variants in CRACR2A , along with our in vitro studies, indicates that CRACR2A plays an essential role in pulmonary vascular cell homeostasis. In addition to its role in regulating SOCE, proliferation, and migration, in vivo downregulation of CRACR2A exacerbates experimental PAH, suggesting that CRACR2A dysfunction contributes to PAH pathogenesis.