The role of Ca2+ influx mechanisms in the regulation of human cardiac fibroblasts in 2D- and 3D- culture models

Cardiac fibrosis is a serious health problem commonly associated with cardiovascular diseases and remains an increasing health burden around the globe. The development of cardiac fibrosis involves the activation and differentiation of mainly resident cardiac fibroblasts (CF) by biochemical factors including angiotensin II (Ang II) and transforming growth factor β (TGF-β). As both factors were shown to interfere with the Ca2+ handling in CF, the role of Ca2+ influx mechanisms were studied in 2D and 3D cultures of CF. In a first step, the influence of the inhibition of the store-operated calcium entry (SOCE) and the transient receptor potential channel canonical 3 (TRPC3) by BTP2 (YM-58483) and Pyr3 (Pyrazole 3), respectively, on the Ca2+ handling were determined in 2D cultured normal human ventricular cardiac fibroblasts (NHCF-V). Both inhibitors were shown to reduce the basal and Ang II-dependent Ca2+ oscillations, as well as the Ang II-induced Ca2+ transients. Moreover, BTP2 was demonstrated to reduce the TGF-β-dependent Ca2+ transient and the ER calcium content under basal condition. Long-term treatment indicated that BTP2 and Pyr3 inhibited the proliferation of NHCF-V and induced cytotoxic effects in 2D cultured cells. An important difference between both inhibitors were identified for their effects on the pro-fibrotic gene expression. BTP2 blunted the expression of the major cardiac collagen isoform Col1a1, but Pyr3 was without effect. BTP2 also down-regulated the matricellular protein connective tissue growth factor (CTGF). Importantly, BTP2 increased the expression of important effectors of the unfolded protein response (UPR). In the next step, the NHCF-V were used to generate human engineered connective tissues (hECT). To induce cell activation a co-treatment with Ang II and TGF-β (AT) was applied. This treatment was shown to increase hECT compaction, contraction, stiffness, and strength, and to decrease extensibility. In the following, the anti-fibrotic potential of BTP2 was shown by a significant reduction of the AT-induced compaction, contraction, stiffness, and strength, and by an increase in elasticity and extensibility of the fibrotic hECT. Molecular analysis revealed that BTP2 treatment resulted in enhanced cell loss within the initial culture period and a downregulation of Col1a1. Mechanistically, BTP2 induced mild ER-stress indicated by an up-regulation of the UPR mediator DDIT3. Finally, Pyr3 also significantly reduce the AT-induced contraction and stiffness but surprisingly, none of the other biomechanical parameters. In summary, the BTP2-dependent Ca2+ influx inhibition induced ER-stress which interfered with ECM protein expression and fibrotic processes in hECT, whereas, Pyr3 interfered only with the contractile behavior of NHCF-V.2021-12-1