1184: Intracellular Calcium Signals Regulate the Pro-fibrotic Phenotype of Scleroderma Fibroblasts via Calcium/calmodulin-dependent Kinase II

Sirapa Vichaikul¹, William Brodie², Megan Mattichak², Qi Wu², Dinesh Khanna² and Eliza Pei-Suen Tsou², ¹Michigan Medicine, Howell, MI, ²University of Michigan, Ann Arbor, MI

Background/Purpose: We performed an in-depth analysis of the involvement of histone reader bromodomain extra-terminal proteins (BETs) in scleroderma (SSc) fibrosis and showed that JQ1, a pan-BET inhibitor, halted fibrosis in both dermal fibroblasts from SSc patients and in vivo models. Transcriptomic analysis of JQ1-treated SSc fibroblasts revealed the involvement of calcium signaling pathway as a novel anti-fibrotic mechanism. Calcium signaling has been implicated in various fibrotic conditions. Studies show that interference of intracellular Ca²⁺ levels through blockade of calcium channels or receptors has potential anti-fibrotic effects in various models. In addition, inhibiting downstream calcium-binding proteins, such as calcium/calmodulin-dependent kinase II (CaMKII), blocked fibrosis in various models. In addition to calcium, CaMKII can also be activated by mitochondrial-mediated oxidative stress. Based on the preliminary results we generated and the literature suggesting the involvement of Ca²⁺ in fibrosis, we hypothesize that Ca²⁺- mediated signaling events through CaMKII contribute to SSc fibrosis.

Methods: Dermal fibroblasts were isolated from biopsies from patients with diffuse cutaneous SSc (dcSSc). To examine the effect of Ca²⁺ in culture, fibroblasts were cultured in Ca²⁺-free media or in full media with BAPTA-AM, an intracellular Ca²⁺ chelator. In separate experiments, fibroblasts were treated with CaMKII inhibitor kn93 or mitoQ, a specific mitochondrial antioxidant. Overexpression of CAMK2A was achieved by transfecting SSc fibroblasts with expression vectors. A scratch wound assay and gel contraction assay were used to evaluate fibroblast function. Cell proliferation was assessed by IncuCyte imaging and BrdU incorporation. Intracellular Ca²⁺ was measured using Fluo-4 by flow cytometry. A p-value of < 0.05 was considered significant.

Results: Dermal SSc fibroblasts cultured in Ca²⁺-free media showed significantly lower levels of fibrotic genes, including ACTA2 and COL1A1 (p< 0.05), compared to ones cultured in full media. Similar results were found when intracellular Ca²⁺ was depleted by treating the cells with intracellular Ca²⁺ chelator BAPTA-AM (both p< 0.05). Overexpression of CAMK2A in dcSSc fibroblasts resulted in increased cell proliferation as well as pro-fibrotic gene expression. In contrast, inhibition of CaMKII in dcSSc fibroblasts by kn93 decreased cell proliferation, migration, and gel contraction. It also downregulated profibrotic gene expression including smooth muscle actin and collagen 1. Removal of mitochondrial reactive oxygen species significantly lowered intracellular Ca²⁺ levels in dcSSc fibroblasts.

Conclusion: We uncovered a novel mechanism by which intracellular Ca²⁺ and CaMKII promotes a pro-fibrotic phenotype in SSc fibroblasts. Our data suggest that activation of CaMKII, possibly via both Ca²⁺/calmodulin and oxidation, contributes to the intracellular Ca²⁺ levels in these cells, thereby promotes the fibrotic phenotype. These findings expanded the understanding for Ca²⁺-related events in SSc pathogenesis, which have been largely unexplored.

Disclosures: S. Vichaikul, None; W. Brodie, None; M. Mattichak, None; Q. Wu, None; D. Khanna, None; E. Tsou, None.