BS38 Cyclic tensile strain protects human cardiac fibroblasts from acidic pH-induced activation

Cardiac fibrosis is associated with heart disease (HD) and kills many people each year. Despite the clinical need, there remains limited therapeutic options, with drugs targeting growth factor signalling only (e.g., TGFβ). The microenvironment of the cardiac stromal tissue is complex, with mechanical and chemical factors. Cardiac Fibroblasts (CFs) sense damaged tissue during HD/ischemia by detecting and responding to changes in chemical and mechanical signals. This response results in the activation of CFs into highly contractile, collagen-secreting myoblasts. These myoblasts seek to restore mechanical integrity to damaged tissues, but failure to resolve their activation state results in continued collagen secretion, a pathological scarring process termed fibrosis. To date, CF activation and deactivation has largely been studied in the context of growth factor signalling. Although acidic pH is known to play a prominent role in myocyte injury, the role of acidic pH on CFs is not well understood. Furthermore, CFs are known to be mechanoresponsive, but the combination of mechano-chemical signalling has not been well studied. The aim here was to assess the fibrotic response (activation state) of CFs to changes in mechanical strain and pH, mimicking the beating heart, at pH values recorded in healthy (pH 7.4 and 7.1) and diseased (pH 6.8) cardiac stromal tissue. This was achieved using an immortalised human CF cell line stimulated with cyclic tensile strain (CTS) (10% strain, 1.0 Hz, 4 hours) using a Flex Jr.™ Tension System. CTS was administered in medium at pH 7.4, 7.1 and 6.8, and then cells were either analysed immediately, or following 24 hours rest. The response of the CFs to TGFβ1 was also evaluated. qRT-PCR was used to assess the gene expression of fibrotic markers (including collagens, aSMA, FN1, MMPs, TIMPs and CTGF) normalised to GAPDH. Immunofluorescence imaging was used to assess the expression levels of aSMA protein. The CFs responded to TGFβ1 by increasing fibrotic markers, like that reported in primary human CFs. The CFs responded to acidic pH by increasing expression of anabolic fibrotic markers (figure 1), and decreasing expression of catabolic markers, suggesting a shift towards a fibrotic phenotype. aSMA was upregulated, suggesting a switch towards myoblastic phenotype. Interestingly, the addition of CTS prevented the pH-induced fibrotic phenotype (figure 1), suggesting that only statically cultured CFs were pH-responsive. Importantly, when the CTS-treated cells were rested for 24 hours, their phenotype returned to fibrotic. This data suggests an interaction between pH and mechano-signalling pathways. CFs may, therefore, respond differently to ischemia-induced acidity depending on whether they are in dynamic (beating) or static (infarcted) tissue. Ongoing efforts will identify the mechano-chemical signalling pathways operating in this system, with potential for novel therapeutic targets to be identified for treatment of cardiac fibrosis.