We can use human pluripotent stem cells to derive cardiomyocytes (hPSC-CMs) in vitro, with the goal of transplanting them into the hearts of individuals who have suffered from heart attacks and restore contractile function. After transplantation into animal models, however, hPSC-CMs produce arrhythmias (irregular heartbeats), likely caused by the immature state of hPSC-CMs. This immature state is associated with low expression of cardiac genes regulating heart muscle contraction and electrical properties. We aim to mature hPSC-CMs in vitro by controlling the expression of these genes, so we can engineer them to behave more like adult cardiomyocytes. To do this, I am looking at DNA methylation, a modification occurring at cytosine nucleotides that is associated with transcriptional repression or gene silencing. My project goal is to determine if DNA methylation plays a role in regulating gene expression patterns of cardiac genes in hPSC-CMs. To investigate this, I have treated hPSC-CM genomic DNA with bisulfite reagent, which converts unmethylated cytosine nucleotides to thymine nucleotides. This treatment will allow me to differentiate between unmethylated versus methylated DNA, and determine whether cardiac maturation genes are methylated at their promoters (where gene expression is typically regulated) by running PCR. Additionally, I have cultured hPSC-CMs with the DNA methylation inhibiting drug 5-azacytidine. By blocking DNA methylation, I will be able to determine if methylation has a direct effect on the expression of cardiac genes by measuring gene expression via quantitative real-time PCR. I hypothesize that DNA methylation regulates cardiac gene expression, and inhibiting methylation will cause expression to increase. Thus, if DNA methylation represses cardiac gene expression, we can mature hPSC-CMs by inhibiting methylation. Ultimately, we hope to prevent arrhythmias that occur after hPSC-CM engraftment and develop cell therapies using mature hPSC-CMs to restore heart function after a heart attack.

Many advancements have been made in differentiating human pluripotent stem cells to cardiomyocytes (hPSC-CMs), with respect to obtaining large numbers with high purity. However, a limitation of hPSC-CMs is that they have an immature phenotype and behave like fetal cells. This immaturity limits the application of cardiomyocytes for cell transplantation that would help repair the heart after myocardial infarction. Our lab has identified candidate master transcriptional regulators of cardiac maturation. These regulators have low expression in immature hPSC-CMs and high expression in mature cardiomyocytes. My project goal is to test the role of PPARG coactivator 1 beta (PGC1B), a candidate transcriptional regulator, in regulating hPSC-CM maturation. PGC1B is involved in mitochondrial biogenesis and increases number of mitochondria, which are highly abundant in adult cardiomyocytes. I hypothesize that activating transcription for PGC1B will enhance maturation of hPSC-CMs through mitochondrial biogenesis. To upregulate gene expression, we use a CRISPR activation (CRISPRa) system with a modified version of Cas9 fused to a transcriptional activator VPR (dCas9-VPR) to upregulate transcription of target genes upon introduction of a specific guide RNA (gRNA). I have differentiated WTC11 stem cells into cardiomyocytes, introduced dCas9-VPR and gRNAs for PGC1B via lentivirus, and performed measurements after 2 weeks. I validated the increased expression of PGC1B at the RNA (using quantitative reverse transcriptase PCR) and protein levels (western blot). To assess relative abundance of mitochondria in PGC1B-expressing versus control hPSC-CMs, I will label mitochondria with MitoTracker and quantify using flow cytometry and microscopy. From these experiments, I expect that PGC1B-overexpressed hPSC-CMs would have a higher relative abundance of mitochondria and increased expression of metabolic and maturation genes compared to control hPSC-CMs. My findings will provide insight on the role of PGC1B in mitochondrial biogenesis and stem cell-derived cardiomyocyte maturation.

In response to various forms of intrinsic and extrinsic stresses such as heat shock, electrical stimulation, and viral infection, cells produce non-membrane-bound aggregates of mRNA and proteins called stress granules. These granules sequester mRNA and ribosomal subunits to halt the production of proteins unnecessary for the immediate survival of the cell, thus allowing more energy to be used in combatting the stress. Stress granules are beneficial in the short term, but the chronic presence of stress granules can be cytotoxic. If stress granules are not cleared, hyperaggregation of misfolded proteins, which is thought to play a role in neurological diseases, can occur. After myocardial infarction (heart attack), the heart experiences a lack of oxygen which is known to create free radicals and metabolic stress. Whether the stress response is involved in this process is unknown, as most research on stress granules, especially their role in disease, comes from work in neuronal and cancer cells. To test whether the stress granules response is conserved across cell types and how cardiomyocytes (heart muscle cells) specifically respond to stress, I cultured cancer cells, embryonic stem cells, and embryonic stem cell-derived cardiomyocytes and subjected these cells to various forms of stress, including sodium arsenate poisoning and heat shock. Using fixed immunofluorescence and spinning disk microscopy, I imaged each treatment and quantified the number of stress granules per cell. The sodium arsenate treatment induced stress granule formation in all three cell types, but surprisingly, the heat shock treatment only induced stress granule formation in the stem cells. It is widely believed the stress response is conserved across a wide range of cell types, but these results indicate some stress pathways differ between cardiomyocytes, cancer cells, and stem cells. Future experiments will test additional types of stress and how stress granules contribute to cardiomyocyte function.

COVID-19, the viral disease caused by the novel coronavirus SARS-CoV-2, is associated with cardiovascular complications such as arrhythmias, myocarditis, and even cardiac arrest. There are two possible mechanisms of SARS-CoV-2 entry into human cells; the endosomal-mediated pathway which requires intracellular processing by intracellular proteases, and the membrane fusion pathway mediated by secreted proteases. Importantly, SARS-CoV-2 entry relies on the expression of the transmembrane receptor ACE2, which interacts with the viral spike protein. It’s still unclear if ACE2 is required for both viral entry pathways. Cardiomyocytes express ACE2, thus SARS-CoV-2 can enter heart tissue; however, the mechanism by which this occurs and how it may lead to cardiac dysfunction is unknown. We previously demonstrated that SARS-CoV-2 significantly impairs mechanical and electrical function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our goal is to understand if ACE2 is required for viral entry into the heart, using hiPSC-CMs as a model, in order to better understand COVID-19 pathology affecting the heart. Using a CRISPR/Cas9 system, we targeted the ACE2 gene at three loci to effectively knockout (KO) gene expression from WTC11 iPSCs. Two KO clones were selected and isolated after sequencing. Wild type (WT) and KO iPSCs were directly differentiated into CMs over a 17-day period. Preliminary results confirmed the absence of ACE2 protein expression in both KO clones by western blot. Fluorescent imaging of CMs infected with GFP-tagged SARS-CoV-2 showed severe infection and cell death at varied time points and multiplicities of infection (MOI) in WT WTC-CMs, while ACE2 KO-CMs showed absence of prominent infection and cell death. These data indicate that the lack of ACE2 markedly prevents SARS-CoV-2 entry into CMs, and understanding if blocking viral entry is sufficient to prevent functional impairment will provide key insights into the development of cardiomyopathies in COVID-19 patients.