Inhibition of Apoptosis Signal-regulating Kinase 1 (ASK1) Attenuates Angiotensin II- and TGFβ-stimulated Cardiac Myocyte Hypertrophy and Fibroblast Collagen Synthesis: Therapeutic Potential of ASK1 Inhibitors in Myocardial Remodelling

Introduction: Myocyte hypertrophy and fibroblast collagen synthesis contribute to cardiac remodelling post myocardial infarction (MI). Whilst molecular mechanisms underlying this process remain to be fully elucidated, apoptosis signal-regulating kinase 1 (ASK1), which plays an important role in stress-induced apoptosis, has been implicated as playing a critical role. The aim of this study was to determine the effectiveness of small-molecule ASK1 inhibitors on these processes. Methods: Selective ASK1 inhibitors, G2261818A (G226) and G2358939A (G235), were co-cultured at increasing doses with isolated neonatal rat cardiac fibroblasts (NCF) or myocytes (NCM) and stimulated with 100 nM angiotensin II (AngII), or TGFβ1 (NCF only). 3H-proline and 3H-leucine incorporation was used to assess collagen turnover and hypertrophy, respectively. Collagen I (Coll I), tissue growth factor β (TGFβ) and connective tissue growth factor (cTGF) gene expression were determined by real time PCR. Human monocytic cell line, THP-1cell, was also treated with ASK1 inhibitors to determine inflammatory cytokine gene expression in response to LPS stimulation. IL-1β, IL-6 and TNFα gene expression was determined by rt-PCR. Results: G226 and G235 dose-dependently inhibited AngII stimulated NCM hypertrophy (G226 at 0.1 μM: 82.4 ± 3.8%, p < 0.01; G235 at 0.1 μM: 69.1 ± 9.5%, p < 0.05, vs AngII stimulated control) and NCF collagen synthesis (G226 at 0.1 μM: 82.6 ± 5.1%, p < 0.001, G235 at 0.1 μM: 91.1 ± 2.7%, p < 0.05, vs AngII stimulated control). Both G226 and G235 also inhibited NCF Coll I, TGFβ and cTGF gene expression. However, only G226 inhibited TGFβ stimulated collagen synthesis in a dose-dependent manner. LPS stimulated IL-1β, IL-6 and TNFα gene expression were inhibited by both G226 and G235 dose-dependently. Conclusions: These results demonstrated that inhibition of ASK1 activity with selective inhibitors has anti-hypertrophic and anti-fibrotic effects in cardiac cells as well as anti-inflammatory effects in monocytic cells. Thus, ASK1 inhibitors may represent a novel therapeutic approach to the attenuation of cardiac remodelling post MI.