It is established that stress can promote addictive drug use and relapse in humans with substance use disorders, thus understanding the stress mechanisms responsible is likely important in developing effective treatments for drug addiction. One effect of stress is the stress-induced release of endogenous dynorphin neuropeptide systems in the brain which activate kappa opioid receptors, that in turn stimulate p38 mitogen-activated protein kinase pathway (MAPK). P38 MAPK activation results in the dysphoria experienced during a stress response. We want to learn more about how p38 affects mood and develop efficient CRISPR techniques to manipulate p38 activation. Other techniques of silencing p38 have been used but come with disadvantages. With a CRISPR approach, virus expressing excision sequences for gene editing can be injected into transgenic mice encoding Cre recombinase in a cell-type specific manner. In this study, I tested a CRISPR/Cas9 virus (AAV1-Flex-SaCas9-sqMapk14), designed to excise the p38 gene. As expected, wild type male mice developed significant conditioned place aversion to the KOR agonist, U50,488. In contrast, mice injected with virus bilaterally in the VTA did not acquire aversion. This finding suggested that successful excision of p38 in VTA had occurred. To confirm with immunohistochemistry, I stained CRISPR injected and U50,488 activated brain slices with p38 and phospho-p38 selective antibodies. We expect slices from CRISPR treated mice to show fewer positive cells in the VTA as compared to controls. My characterization of these antibodies is on-going, but preliminary results suggest differences in CRISPR injected and wild type mice. With the development of a technology allowing for efficient manipulation of p38 MAPK within cell site and brain regional specificity, we hope to provide further insight to the stress response and to better understand its role in addiction.

We can use human pluripotent stem cells to derive cardiomyocytes (hPSC-CMs) in vitro, with the goal of transplanting them into hearts of heart attack survivors to restore contractile function. Studies using animal models have shown that transplanted hPSC-CMs can improve heart function, however they result in arrhythmias, which may not be well-tolerated. These arrhythmias are likely caused by the cells' immature electrical properties (pacemaker-like activity) and their lack of resemblance to adult cardiomyocytes. We aim to mature hPSC-CMs in vitro by controlling gene expression to engineer adult-like cells. To do this, I am studying whether DNA methylation (modification associated with transcriptional repression) plays a role in regulating hPSC-CM maturation. To investigate this, I have cultured hPSC-CMs with the methylation-inhibiting drug 5-azacytidine and used quantitative real-time PCR to measure gene expression. 5-azacytidine treatment increased expression for ATP2A2, MYL2, and SCN5A. Next, I treated hPSC-CM DNA with bisulfite reagent and used PCR to determine whether cardiac gene promoters are methylated. I found evidence of methylation at the MYL2 promoter, a maturation gene that encodes for regulatory myosin light chain and triggers contraction. To understand how methylation regulates MYL2 expression, I used a stem cell line where MYL2 is tagged with GFP. After 5-azacytidine treatment, MYL2 RNA expression increased four-fold compared to control hPSC-CMs. Next, I tested whether MYL2 expression changes after treatment due to the number of hPSC-CMs expressing MYL2 and/or the intensity of MYL2 expression in individual cells, using flow cytometry to measure GFP intensity. My data suggests the latter, and supports a role for DNA methylation in restricting hPSC-CM maturation. By regulating DNA methylation, we can engineer cells in vitro to match expression patterns measured in adult cardiomyocytes, leading to enhanced contractile function. Ultimately, this will contribute to improved hPSC-CM therapies to restore cardiac function after a heart attack.

In response to various forms of stress such as heat shock and oxidative stress, cells produce aggregates of mRNA and proteins called stress granules. These granules sequester mRNA and signaling proteins to promote cell survival. Stress granules are beneficial in the short term, but the chronic presence of stress granules can be cytotoxic and cause hyperaggregation of misfolded proteins. After a heart attack, the heart experiences a lack of oxygen, which creates free radicals and metabolic stress. Whether the stress response is involved in this process is unknown, as most research on stress granules and their role in disease comes from work in neuronal and cancer cells. To test whether the stress granule response is conserved across cell types, I cultured cancer cells, pluripotent stem cells, and stem cell-derived cardiomyocytes (heart muscle cells) and subjected these cells to various stresses, including sodium arsenate poisoning, heat shock, and oxidative stress. I imaged each treatment using immunofluorescence 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 and oxidative stress treatments had cell type-specific stress granule responses. It is widely believed the stress response is conserved across a range of cell types, but these results indicate some stress pathways differ between cardiomyocytes, cancer cells, and stem cells. To test how stress granules impact cardiomyocyte function, I generated stem cells with knockouts of the two genes required for stress granule assembly: G3BP1 and G3BP2. In future studies, I will differentiate these cells into cardiomyocytes and test whether the inability to form stress granules affects their ability to survive in response to stress. This work is important in understanding the impact stress granules have on the regeneration of heart cells in damaged heart tissue.

Since the beginning of the Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) pandemic, a large number of COVID-19 patients have suffered a variety of cardiovascular complications. In a previous study from the Murry Lab, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) were infected with live SARS-CoV-2 alpha variant, resulting in increased cell death and functional abnormalities. Once infected with SARS-CoV-2 delta variant, hPSC-CMs showed reduced cell death, compared with the ones infected with alpha. The SARS-CoV-2 genome has at least 14 open reading frames (ORFs), which is the transcribed part of a gene. A difference in expressed ORFs can create new variants. The delta variant has 60 amino acid deletions in the ORF7a sequence, resulting in the absence of the ORF7a protein. Thus, I hypothesized that this protein might play a role in cardiomyocytes’ cell death. To demonstrate that this protein has a direct effect on hPSC-CMs cell death, I have created a lentivirus plasmid construct that expresses only ORF7a protein. To assemble the virus, I transfected HEK-293 cells with the ORF7a plasmid with two other plasmids encoding for the packaging and the replication of the lentivirus. Once the virus is ready, I will then infect hPSC-CMs with the lentivirus. If the ORF7a protein is responsible for cell death, we expect to see increased cell death in the hPSC-CMs infected with the ORF7a lentivirus. Showing support of the ORF7a protein inducing harmful effects on cardiomyocytes has the potential to provide valuable insight for scientists to target this particular protein for drug development to combat cardiovascular complications in COVID-19 patients.