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Ischemic cardiac preconditioning protects the heart during myocardial infarction by activating critical cardioprotective genes such as eNOS, SOD, and HO-1. Clinical trials only show marginal effects of conventional preconditioning strategies, however, in part due to transient activation of cardioprotective genes. Recent studies have shown that N6-methyladenosine (m6A) mRNA methylation is the most abundant RNA modification in eukaryotes, and governs mRNA stability and, in turn, the level of protein expression. We hypothesize that regulation of m6A mRNA methylation levels of cardioprotective mRNAs will result in stable expression of the cardioprotective proteins, rendering ischemic cardiac preconditioning more robust and reducing infarct size. To test this hypothesis, we will test the effects of introducing m6A methylases/demethylases into ischemic preconditioned/post conditioned hearts and subjecting them to myocardial infarction. We will assess the half-life of key cardioprotective mRNAs (e.g., eNOS, SOD, and HO-1) and cardiac apoptosis to determine which m6A methylases/demethylases have a synergistic effect on cardiac preconditioning.

Although stem cell therapy is promising for repairing damaged cardiac tissue and improving heart function, there are safety concerns, especially regarding the risk of arrhythmias, which can be life threatening. To address this issue, we propose to develop a novel screening system to evaluate arrhythmic risk associated with stem cell therapy using a high-throughput multielectrode array system that can measure conduction velocity and action potential duration in cardiomyocytes co-cultured with different types of stem cells, such as mesenchymal stem cells, skeletal myoblasts, and resident cardiac stem cells. We will assess the arrhythmic potential of each of these types of stem cells under normoxic and hypoxic conditions, with/without application of oxidative stress or catecholamines. We hypothesize that these methods will prove to be an effective way to screen for arrhythmic risk of cardiac stem cell therapy. Ultimately, our approach can potentially be personalized to develop a robust screening protocol in order to identify which stem cell type carries the least amount of risk for arrhythmia. This system will have great clinical benefit to improve the risk/benefit ratio of human stem cell therapy for heart disease.

Ischemic heart disease has remained the number one killer around the world for over the past twenty years. While stem cell therapy has become a promising new frontier to repair the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Strategies to genetically modify stem cells to activate pro-survival and anti-apoptotic and anti-inflammatory pathways, such as Akt and heme oxygenase-1, have been shown to improve the lifespan of transplanted stem cells within the ischemic myocardium, but constitutive overexpression of these pathways at high levels has been shown to have side effects. Therefore, more specific and controlled gene activation would be necessary. Current techniques used for gene regulation include zinc finger and TALE proteins, but there are still disadvantages to each of these methods, such as ease and cost of use. Also, those methods use synthesized promoters to express synthesized cDNA, which lack regulatory elements, including introns and 3′ untranslated regions for microRNA mediated post-transcriptional regulation. A new novel technique, the CRISPR/dCas9 system, was recently developed as a simple and efficient method for endogenous gene regulation. With its use of single guide chimeric RNA’s (sgRNA’s), this system has been shown to provide a high level of specificity and efficiency. When targeting different loci, past studies have found that the CRISPR/dCas9 system can activate gene expression at varying levels. In addition, this system makes use of the genome’s endogenous regulatory elements, such as the aforementioned introns and 3′ UTR’s, which can help provide a safer method of gene activation. If targeted to a gene promoting cellular survival or decreasing cell death, it could potentially improve stem cell longevity in a more efficient and controllable manner. As a result, our hypothesis is to use the CRISPR/dCas9 system to activate expression of an anti-inflammatory and anti-apoptotic gene, such as heme oxygenase-1 (HO-1), to an optimal level to increase transplanted stem cell survival while also mitigating its cytotoxic effects due to lack of internal regulation, thus prolonging its effects within the ischemic myocardium leading to greater therapeutic benefit.

The miR-17-92 cluster is an important microRNA cluster in the animals. It was mainly investigated as an oncogene in tumors but never studied in cardiovascular disease. On one aspect, miR-17-92 cluster is documented to play anti-apoptotic roles in tumor cells, including hypoxia-induced apoptosis. The families of miR-17, miR-19, and miR-92 promote resistance to apoptosis by directly inhibiting pro-apoptotic protein, and by the two major cell survival signaling pathways—PI3K/AKT, and MAPK/ERK. On the other aspect, the component members of miR-17-92 cluster are high expressed in the hearts of canine and mice, suggesting that there are effect targets of the cluster in the myocardium. So that, we hypothesized that the miR-17-92 cluster may protect the heart by diminishing the apoptosis and alleviating ischemia/reperfusion injury. This may be a new regulating target for patients with myocardial infarction and undergoing cardiac surgery.