Altered Expression of ORAI and STIM Isoforms in Activated Human Cardiac Fibroblasts

Abstract

Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR – STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.

Histamine activates an intracellular Ca2+ signal in normal human lung fibroblast WI-38 cells

“…These data are therefore consistent with the results provided by the pharmacological manipulation of the Ca 2+ response. Conversely, all Orai and STIM isoforms were detected in human cardiac fibroblasts (Cendula et al, 2021), in which they support spontaneous Ca 2+ oscillations (Chen et al, 2010). Intriguingly, Orai3 can be directly activated by 50 µM 2-APB independent from ER Ca 2+ store depletion (Zhang et al, 2020), which might explain the immediate rise in [Ca 2+ ] i that occurs upon 2-APB application in the presence (see Figure 6F) but not in the absence (not shown) of extracellular Ca 2+ .…”

Progress on role of ion channels of cardiac fibroblasts in fibrosis

“…As the secondary messenger in cells, Ca 2+ plays an essential role in intracellular functions, including cell signal transduction, gene expression, cell proliferation, differentiation, migration, growth and death ( Berridge, 2002 ). Although it is clear that Ca 2+ signaling plays a vital role in the pathological activation of fibroblasts, due to the large and diverse number of receptors, transporters, and ion channels that affect intracellular Ca 2+ kinetics, the key elements of fibroblast transdifferentiation to myofibroblasts have not been fully elucidated ( Čendula et al, 2021 ). Decreased calcium influx into cells reduces fibroblast differentiation, collagen release, and reduce TGF-β-induced fibrotic remodeling ( Ramires et al, 1998 ; Teng et al, 2015 ).…”

“…FGF23 (25 ng/mL)-treated cardiac fibroblasts had higher expression levels of Orai1 ( Li et al, 2021 ). A regulatory subunit within the endoplasmic reticulum (ER), STIM1 is partially located inside the ER and able to sense the Ca 2+ concentration within ( Parks et al, 2016 ; Čendula et al, 2021 ). STIM1 activates Orai1 to allow Ca 2+ entry into the cells ( Liou et al, 2005 ; Roos et al, 2005 ; Čendula et al, 2021 ).…”

“…A regulatory subunit within the endoplasmic reticulum (ER), STIM1 is partially located inside the ER and able to sense the Ca 2+ concentration within ( Parks et al, 2016 ; Čendula et al, 2021 ). STIM1 activates Orai1 to allow Ca 2+ entry into the cells ( Liou et al, 2005 ; Roos et al, 2005 ; Čendula et al, 2021 ). Depletion of Ca 2+ stores induces a conformational change, which allows STIM after its oligomerization to interact with Orai ( Liou et al, 2005 ; Muik et al, 2011 ; Trebak et al, 2013 ).…”

“…Orai proteins show four transmembrane domains and form hexameric channels in the plasma membrane, which is highly selective for Ca 2+ ( Covington et al, 2010 ; Muik et al, 2011 ). These channels are opened after interaction with active STIM proteins, which allows the inflow of Ca 2+ from the extracellular environment ( Čendula et al, 2021 ). Three genes that encode Orai proteins (Orai1–3) and two encoding STIM proteins (STIM1-2) are currently known ( Ruhle and Trebak, 2013 ; Wen et al, 2016 ).…”