Browsing by Subject "High-content analysis"

Cardiomyocyte oxygen deprivation followed by apoptosis and cardiomyocytes being replaced with fibrotic tissue can lead to heart failure. Cardiovascular diseases are the most common cause of death world-wide, contributing to 17.8 million deaths in 2017. Treatments currently available aim to maintain cardiac function but are unable to repair the damage, resulting in a poor prognosis for heart failure. Cardiomyocytes are able to proliferate but the endogenous mechanisms of cardiac repair are insufficient to replace the damaged cardiomyocytes, as only an estimated 0.3-1 % of adult cardiomyocytes are regenerated annually. It is known that before birth and up to seven days after birth mice can maintain ability to regenerate cardiomyocytes even after large damage, but this capability is lost within seven days following birth. After this, cardiomyocytes exit cell cycle and will not re-enter it sufficiently to enable cardiac repair. In adults the growth of heart muscle results mainly from hypertrophic growth meaning that the cells grow in width and length. Cardiomyocyte regeneration is an important therapeutic target to which there are no effective pharmacological therapies available yet. The aim of this study was to investigate the effect of 14 novel compounds on cardiomyocyte viability, phenotype and cell cycle activation. Novel compounds were synthesized at the Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, University of Helsinki in Finland. Initial toxicity and cell viability screening was conducted with lactate dehydrogenase assay (LDH assay) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) using COS-1 cells. Based on these assays tolerable concentrations of compounds were determined. Activity analysis was conducted using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and immunocytochemistry staining in conjunction with high-content analysis (HCA). Stress response was measured by imaging and analyzing expression of pro-B-type natriuretic peptide (proBNP) and cell cycle activation was imaged and analyzed by using 5- bromo-2’-deoxyuridine (BrdU) as a marker of active cell cycle. In addition, the DNA content of the cardiomyocytes was measured using 4’,6-diamidino-2-phenylindole (DAPI) staining as well as cardiomyocyte morphology investigated with cardiac troponin T (cTnT) staining. One of the compounds, K6, decreased proBNP expression, which can be considered as a sign of decreased stress response. However, K6 also decreased the number of BrdU positive cardiomyocytes that can be considered as a sign of decreased cell cycle activity. Together these markers indicate that the decreased activity may not be due to a stress response caused by the compound. Another compound, K12, increased proBNP expression in all tested concentrations and it also decreased the number of BrdU positive cardiomyocytes. Together these could be considered as an indication of cardiotoxicity. The rest of the compounds did not exhibit remarkable biological activity or there was too great variance between the results of the independent experiments (n=3) to draw definite conclusions. Compounds for this study were chosen for the sole reason of not been tested for biological effects before. Using a defined compound library or screening a larger number of compounds could deliver more predictable results. Early toxicity and viability screenings were a good approach allowing to define toxic compounds and concentrations before continuing with further studies. Pharmacological therapies to induce cardiac regeneration will continue to be an important area of interest in cardiovascular drug research. Phenotypic screening in conjunction with high-content analysis offers variable and statistically significant data on cardiomyocyte proliferation and stress response. The results of the screening could be improved with careful selections of test molecules based on their structure and biological activity. Early toxicity and viability screening further improve the predictability of results. As a result of this study a compound that would induce cardiomyocyte proliferation was not found, however, one compound that seemed to decrease cardiomyocyte stress response was detected and this compound could be of interest for further studies.