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Browsing by Subject "toxicity screening"

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  • Loppi, Tiia (2024)
    Ischemic heart disease, which often progresses to heart failure, is one of the leading causes of death worldwide. Ischemic conditions result in the death of heart muscle cells i.e. cardiomyocytes. Due to their poor regenerative ability, lost cardiomyocytes are replaced with a fibrotic scar. The loss of cardiomyocytes further leads to compensatory mechanisms, including cardiomyocyte hypertrophy and fibrosis. When prolonged, these responses turn maladaptive leading to pathological cardiac remodeling and alterations in cardiac function. In order to achieve better clinical results, discovery of new drug treatments that promote cardiomyocyte regeneration and decrease pathological cardiac remodeling would be invaluable. One potential target is serine/threonine protein kinase AKT (also known as protein kinase B), a key component of the PI3K/AKT signal pathway, which has been shown to be one of the mechanisms regulating heart regeneration and remodeling post-ischemia through its several downstream targets. The aim of this study was to investigate the effects of AKT-targeted compounds with and without endothelin-1-induced hypertrophy on the phenotype of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The compounds were either commercially available substances linked to AKT regulation, or new experimental compounds synthesized at the Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki. Prior to the hiPSC-CM phenotypic studies, the toxicity of the compounds was investigated using the lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in three different cardiac cell models: human cardiac fibroblasts (HCFs), H9c2 cells derived from embryonic rat myocardium, and hiPSC-CMs. Compound-induced proliferative and hypertrophic responses of hiPSC-CMs were studied using immunofluorescence staining and high-content analysis. Toxicity screening of the compounds showed that only AKT045 was concentration-dependently toxic in all three cell types after 24-hour treatment. Based on the toxicity screening, several compounds caused more pronounced concentration-dependent effects in H9c2 cells as compared to hiPSC-CMs and HCFs. The most considerable effects were observed with AKT042 and AKT048, as they decreased the viability of H9c2 myoblasts 46% and 55% at 30 μM concentration, respectively. In phenotypic studies, AKT050 decreased hiPSC-CM proliferation significantly. This result indicated inhibition of AKT and was consistent with previous studies. Commercially available AKT activator SC79 did not induce expected effects, as it tended to attenuate both proliferative and hypertrophic response in hiPSC-CMs. However, AKT activation has been shown to increase both proliferation and hypertrophy in previous studies. Other compounds induced a prohypertrophic rather than an antihypertrophic effect in hiPSC-CMs. Although proliferative responses to other compounds varied slightly, AKT042 and AKT043 seemed to increase the proliferation of hiPSC- CMs. However, the AKT activation or inhibition could not be confirmed in this study and therefore additional studies are needed to assess the full extent of effects and mechanisms of these compounds.