Aging-related increase in store-operated Ca²⁺ influx in human ventricular fibroblasts

Abstract

Senescence-related fibrosis contributes to cardiac dysfunction. Profibrotic processes are Ca dependent. The effect of aging on the Ca mobilization processes of human ventricular fibroblasts (hVFs) is unclear. Therefore, we tested whether aging altered intracellular Ca release and store-operated Ca entry (SOCE). Disease-free hVFs from 2- to 63-yr-old trauma victims were assessed for cytosolic Ca dynamics with fluo 3/confocal imaging. Angiotensin II or thapsigargin was used to release endoplasmic reticulum Ca in Ca-free solution; CaCl (2 mM) was then added to assess SOCE, which was normalized to ionomycin-induced maximal Ca. The angiotensin II experiments were repeated after phosphoenolpyruvate pretreatment to determine the role of energy status. The expression of genes encoding SOCE-related ion channel subunits was assessed by quantitative PCR, and protein expression was assessed by immunoblot analysis. Age groups of <50 and ≥50 yr were compared using unpaired t-test or regression analysis. Ca release by angiotensin II or thapsigargin was not different between the groups, but SOCE was significantly elevated in the ≥50-yr group. Regression analysis showed an age-dependent phosphoenolpyruvate-sensitive increase in SOCE of hVFs. Aging did not alter the mRNA expression of SOCE-related genes. The profibrotic phenotype of hVFs was evident by sprouty1 downregulation with age. Thus, an age-associated increase in angiotensin II- and thapsigargin-induced SOCE occurs in hVFs, independent of receptor mechanisms or alterations of mRNA expression level of SOCE-related ion channel subunits but related to the cellular bioenergetics status. Elucidation of mechanisms underlying enhanced hVF SOCE with aging may refine SOCE targets to limit aging-related progression of Ca-dependent cardiac fibrosis. NEW & NOTEWORTHY Human ventricular fibroblasts exhibit an age-related increase in store-operated Ca influx induced by angiotensin II, an endogenous vasoactive hormone, or thapsigargin, an inhibitor of endoplasmic reticulum Ca-ATPase, independent of receptor mechanisms or genes encoding store-operated Ca entry-related ion channel subunits. Selective inhibition of this augmented store-operated Ca entry could therapeutically limit aging-related cardiac fibrosis.

Ca2+ Signaling in Cardiac Fibroblasts and Fibrosis-Associated Heart Diseases

“…Ca 2+ influx, and the expression of Orai1 but not STIM1, have been shown to be much larger in ventricular fibroblasts derived from failing hearts compared to those of non-failing hearts [68]. In aged ventricular fibroblasts, while store-operated Ca 2+ release and entry were shown to increase, the expressions of Orai1 and STIM1 remained unchanged [67]. These results suggest that Orai/STIM channels play a role in fibroblast Ca 2+ signaling, and may therefore contribute to cardiac remodeling under pathological conditions.…”

“…In fibroblasts, although the Orai and STIM subunits have been detected by qPCR or RT-PCR in mouse and human cardiac fibroblasts [37,46], CRAC currents have not been recorded by patch-clamp [37]. Recently, the functional Orai1/STIM1 channels have been demonstrated by Ca 2+ imaging measurements in human ventricular fibroblasts [67,68,125]. Ca 2+ influx, and the expression of Orai1 but not STIM1, have been shown to be much larger in ventricular fibroblasts derived from failing hearts compared to those of non-failing hearts [68].…”

STIM and Orai Mediated Regulation of Calcium Signaling in Age-Related Diseases

“…This study suggests that STIM1/Orai1 may regulate cardiac fibroblast activity and activation. It has been shown that SOCE may increase in aged human cardiac fibroblasts which was associated with a reduction in the expression of pro-fibrotic sprouty homologue 1 (Spry1) possibly contributing to senescence-mediated fibrosis in the heart; however, in these studies expression levels of STIM/Orai were not changed ( Mohis et al, 2018 ) so the catalyst driving the increase in SOCE in this study remains unclear. Moving forward, it will be important better understand the different functions and roles of STIM and Orai isoforms in the various cell types of the heart and whether these STIM/Orai isoforms contribute to crosstalk between these cell types during physiological and pathophysiological instances.…”

Resident macrophages as potential therapeutic targets for cardiac ageing and injury

“…87 Some studies have suggested that age-related fibrosis may be due to decreased matrix degradation rather than increased collagen synthesis. 88,89 Macrophages secrete stromal cell proteins and are directly involved in ECM remodelling by producing MMPs and their inhibitors.…”