Browsing by Subject "cardiomyocyte"

Left ventricular hypertrophy (LVH) takes place when cardiomyocytes respond to excessive stress by growing in size. Cardiomyocytes have a very marginal capability to proliferate, which is why hypertrophic growth is almost their only option to meet the requirements of increased workload. In the long run, however, LVH leads to further problems, such as cardiac failure and an increased risk of myocardial infarction. Hypertension is the most prevalent cause of LVH, and its current treatment relies on antihypertensive drugs. They decrease the workload of the heart and therefore alleviate symptoms but have very little effect on the built damage and remodeling. Understanding the details of cellular level signaling pathways and genetic expression in LVH is crucial for future drug development. Regulation of gene expression is a very complex process, which involves more than just DNA being translated into a protein. In this project, two types of factors participating in this regulation were in focus: long non-coding RNAs (lncRNA) and transcription factors GATA4 and FOG2. LncRNAs are RNA sequences of more than 200 nucleotides that do not code for any protein final products themselves but are involved in chromatin remodeling as well as transcriptional and post-transcriptional gene regulation. They are highly organ-selective, which makes them potential targets for drug development. Our group has previously found a selection of cardiomyocyte-selective lncRNAs, which share a similar expression pattern in neonatal mouse hearts. In this project, three of them were silenced in a primary cardiomyocyte culture while simultaneously hormonally inducing hypertrophy. The goal was to see whether these lncRNAs have an effect on the hypertrophic response and apoptosis in the cardiomyocytes. Transcription factors are proteins with partially similar activities to lncRNAs; they regulate, which genes are expressed under certain circumstances. GATA4 is an important transcription factor in the heart as it targets various developmental and functional genes in cardiomyocytes. FOG2 is a cofactor of GATA4; interaction between them regulates the activity of GATA4. Our group has recently developed a selection of compounds that affect protein-protein interaction between GATA4 and NKX2-5, another important transcription factor. The second part of the project was to set up and optimize a compound screening assay for GATA4-FOG2 interaction. The results showed no change in hypertrophic response when the lncRNAs were silenced. Other experimental designs could still reveal if they have effects that could not be seen with these protocols. The silencing had no effect on apoptosis. As for the GATA4-FOG2 interaction experiments, transfecting COS-1 with GATA4 and FOG2 plasmids in a ratio of 10:1 resulted in a signal suitable for compound screening. Initial compound screening results indicated the compounds may have an effect on GATA4-FOG2 interaction, but further studies are needed before drawing conclusions.

This body of research focuses on establishing a drug screening pipeline for discovering drugs which increase the differentiation of pluripotent stem cells into cardiac myocytes, known as cardiogenic molecules. Cardiomyocytes can be utilized in regenerative medicine by offering a platform for testing molecules or drugs which may increase cardiomyocyte proliferation and for using cardiomyocytes produced outside of the body for clinical transplant, in order to heal the damage caused by heart attacks. Building on known models and developmental pathways three assays were designed and implemented for in vitro cardiogenic molecule screening. A pipeline comprised of three primary screening systems; an embryoid body (EB) model, a cardiomyocyte directed differentiation model, and a magnetic activated cell sort (MACS) model. The MACS model uses the cell surface receptors Fetal Liver Kinase 1 (FLK1) and/or Platelet Derived Growth Factor Receptor alpha (PDGFRα) as the most practical platform for screening drugs against an enriched mesodermal population of cells. The MACS system was confirmed with flow cytometry to ensure the enrichment of Myl2-eGFP+ (ventricular cardiomyocytes) cells in the FLK1+ cells. Furthermore unique known molecules help elucidate the molecular mechanisms governing cardiomyocyte differentiation, measured by cardiomyocyte purity in in vitro models. Also demonstrated are assay controls which decrease purity and acts as negative controls for the MACS assay such as a late stage GSK-3 Inhibitor treatment used to constitutively activate the canonical Wnt/β-catenin pathway and effectively reduce the cardiomyocyte proliferation. Additionally, an early stage Wnt Inhibitor compound IWP-4 was used as a potential positive control effectively blocking late stage activation of canonical Wnt/β-catenin pathway and increase the in vitro purity of cardiomyocytes. These controls provide two important reference points for the many molecules screened over the course of these experiments for the 3i Regeneration project. Additional molecular inhibitors are used to elucidate the mechanism of action within the MACS cells; including a Sonic Hedgehog inhibitor (cyclopamine), an NKX2.5 activator (ISX-9) and a novel small molecule (C1). These models act as an effective pipeline bringing a potential drug through first an EB model, followed by a cardiomyocyte enriched model, to finally a MACS model targeting FLK1. This pipeline tests the molecules against conditions of increasing resemblance to the native microenvironment of a cardiomyocyte.

The purpose of this study is to assess if it is possible to reprogram cardiomyocytes from mouse embryonic fibroblasts by using one transgenic factor, Oct4, and a pool of small molecules added to growth medium. The Oct4 gene was conducted in mouse embryonic fibroblasts by lentiviral vectors. Samples were taken for analysis after 15, 20 and 41 days of growing the fibroblasts with small molecules. Compared to control group, no upregulation of cardiac progenitor or cardiomyocyte specific genes were observed. Concentration of Ctgf decreased in treatment cells compared to control cells, which implies that control cells continued functioning as fibroblasts more than treatment cells. The cell morphology of treatment cells changed in the direction of cardiomyocytes compared to control cells; more cylinder-like and branching compared to spindle-like fibroblasts. A different approach was also attempted, the STAP (Stimulus-Triggered Acquisition of Pluripotency) method, in which fibroblasts are exposed to acid treatment while growing. The pluripotent mRNA expression did not rise in this analysis.

Heart failure is a major public health problem and a leading cause of mortality worldwide. The most common cause of heart failure is myocardial infarction. Following a myocardial infarction, a large number of cardiomyocytes die and cardiac muscle is replaced by fibrotic scar tissue. Since the adult heart has inadequate endogenous regenerative capacity, loss of muscle tissue often causes a progressive decrease in cardiac function eventually leading to heart failure. At the moment heart transplantation is the only curative treatment for heart failure, but the low number of donor hearts is limiting the use of this treatment option. As current drugs only slow down the progression of the disease, there is a great need for new regenerative treatments. Direct cardiac reprogramming is a new approach for generating cardiomyocytes for cardiac regeneration. Unlike pluripotent stem cell-based strategies, direct reprogramming enables conversion of a terminally differentiated cell type directly into another cell type without first producing a pluripotent intermediate. Due to their abundancy and role in the repair of myocardial injury, fibroblasts represent an attractive starting cell type for direct cardiac reprogramming. Fibroblasts have been directly reprogrammed to induced cardiomyocytes (iCMs) by overexpression of key cardiac transcription factors, microRNAs (miRNA) or by modulating specific signal transduction pathways with small-molecule compounds. Despite successful reports of direct reprogramming both in vitro and in vivo, the efficiency of direct reprogramming remains, however, too low for potential clinical applications. The aim of this M.Sc. thesis work was to establish direct reprogramming of mouse embryonic fibroblasts (MEFs) to iCMs by viral overexpression of cardiac transcription factors Hand2 (H), Nkx2.5 (N) Gata4 (G), Mef2c (M) and Tbx5 (T) and a small-molecule compound screening platform for identifying small-molecule compounds that could enhance the reprogramming efficiency and potentially replace cardiac transcription factors in direct cardiac reprogramming. In accordance with previous publications MEFs were successfully directly reprogrammed to iCMs using both HGMT and HNGMT cardiac transcription factor combinations. The screening platform was tested using the TGF-β inhibitor SB431542, which has recently been reported to increase the cardiac reprogramming efficiency. In line with previous publications, the reprogramming efficiency was significantly increased by treatment with SB431542. Initial tests with other small-molecule compounds did not have a positive effect on the reprogramming efficiency. The results of this M.Sc. thesis work verify previous publications and demonstrate a method for in vitro small-molecule compound screening, which can be used to identify compounds that increase the reprogramming efficiency in direct cardiac reprogramming. However, the results shown here are only preliminary and more replicates are needed in order to confirm the current results. Nonetheless, the results of this thesis work set a foundation for finding small-molecule compounds that in the future might be used to target direct cardiac reprogramming as a regenerative therapy for myocardial infarction and heart failure.