Browsing by Subject "high-content analysis"

Cardiac fibrosis (CF) is a physiological response to various stress factors encountered by the heart, with the aim of maintaining proper functioning of this vital pump in an altered situation such as increased mechanical stress or sudden injury in heart muscle. CF is characterized by excessive production of extracellular matrix (ECM) components and stress fibers in cardiac tissue, accompanied by morphological changes of the heart muscle. The responsible cells behind these changes are fibroblasts (FBs) that undergo phenotypic change by transdifferentiating into myofibroblasts (myoFBs). Although being initially a supportive response, CF can lead to deterioration of the heart performance and even heart failure, if prolonged. Given the lack of effective enough therapies against CF, and the strong involvement of CF in cardiovascular diseases (CVDs) that are associated with high mortality rate, the need for new effective therapies is urgent. Indeed, a diversity of approaches to fight CF have been proposed, among them protein kinase C (PKC) and its signaling cascades. PKC has been shown to play a role in fibrosis and many studies suggest antifibrotic properties of PKC, yet the results are challenged by the opposite findings. Despite the dichotomous results, new small molecules that function as partial agonist of PKC seemed to be a promising strategy for the treatment of fibrosis. To further explore the role of PKC activation in CF, the aim of this study was to first develop and characterize a human cardiac fibroblast (HCF)-based CF model, in which the effects of seven new PKC modulators on HCFs could then be evaluated. To create the CF model and provoke a fibrotic response, HCFs were treated with either transforming growth factor β1 (TGF-β1), Angiotensin II (Ang-II), endothelin-1 (ET-1), or combination of treatments, followed by determination of HCF proliferation activity and α-smooth muscle actin expression (α-SMA), a marker of myoFBs. After the treatments, the original goal was to continue in compound testing phase by exposing the HCFs to the PKC-modulators to see whether any differences could be determined in α-SMA expression or proliferation activity. However, no considerable effects of fibrosis-inducing treatments on the activation of HCFs were observed, thus preventing this progression. Nevertheless, toxicity tests were performed on the compounds and the results indicated relatively low overall toxicity for the lower concentration: six out of seven compounds yielded over 70% HCF viability at 3 μM concentration with three of them reaching even over 80% viability, while the corresponding value for the previously published PKC agonist HMI-1a3 was 54%. Although these results are promising for the lower concentrations of PKC-modulators, it is obvious that more in-depth studies are required prior to drawing any unambiguous conclusions.

Dilated cardiomyopathy is a non-ischemic cardiac disorder predisposing to heart failure, and the characteristics of dilated cardiomyopathy emerge under normal loading conditions. Dilated cardiomyopathy can be consequence of various conditions e.g. genetic mutations, virus infection or toxin exposures. One of the significant causes of familial dilated cardiomyopathy in Finland is mutation S143P in LMNA-gene, coding for A type lamins. Current drug therapy for dilated cardiomyopathy aims to alleviation of symptoms, prevention of complications and progression of the disease, however, efficacy of current therapy is insufficient, and novel therapy strategies are urgently required. Transcription factors are fundamental regulators of gene expression, and GATA4 is a crucial transcription factor both in embryonic and in adult heart and thus an intriguing target for therapeutic manipulation. Compounds targeting GATA4 have shown anti-hypertrophic and cardioprotective effects. Here, effects of two different hypertrophic stimuli, endothelin-1 and mechanical stretch, on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were examined with high-content analysis and quantitative reverse transcription PCR (qRT-PCR), respectively. One hiPSC-CM line was used as a healthy control, whereas the other carried the S143P mutation in LMNA-gene (DCM-CMs). Additionally, effects of GATA4-targeting compound C-2021 on cardiomyocytes were investigated. In summary, according to proBNP staining, DCM-CMs are more hypertrophied at baseline. DCM-CMs seemed to be less susceptible to mechanical stretch-induced enhancement in BNP gene expression. In addition, compound C 2021 may have anti-hypertrophic properties suggesting it to be a potential drug candidate in cardiac diseases. Finally, lamin A seemed to mislocalize to nucleoplasm instead of nuclear lamina in DCM-CMs.

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.