Mitochondrial dysfunction has been implicated as a contributing factor for heart failure. Genes involved in mitochondrial biogenesis and quality control such as the peroxisome proliferator- activated receptor gamma coactivator 1? (PGC-1?) have been severely downregulated in rodent models of heart failure. Transcription of PGC-1? can be promoted when cAMP Response Element Binding Protein (CREB) is phosphorylated at Ser-133 by PKA upon beta-adrenergic signaling. However, recent evidence shows that CREB itself has numerous factors modulating it, including phosphorylation on other sites (e.g Ser-142), availability of dimerization partners (c- Jun, CREB subtypes) and other co-factors (e.g. TORCs) that may be altered in heart failure. Here we will test whether bypassing the complex regulatory steps involved in PGC-1? activation and directly targeting cAMP Response Elements (CRE) known to regulate PGC-1? gene expression can have beneficial effects on expression of PGC-1?. Importantly, we also propose to test whether mitochondrial biogenesis can be increased via CRE-mediated PGC-1? upregulation. I intend to utilize the newly developed CRISPR/Cas9 technology as a transcriptional activator (instead of editing genes, with inactivated Cas9 fused to transcriptional activation domains) to target CRE sequences to promote transcription of PGC-1?. Use and efficacy of these gene modulating tools has not been evaluated in cardiomyocytes. With this project, I will establish if metabolic reprogramming and enhanced mitochondrial biogenesis can be effected with CRISPR-mediated activation of PGC-1? while providing fundamental knowledge of the role of CRE in PGC-1? activation and mitochondrial function. I would have also developed a streamlined and generalizable method for using gene modulating tools for expression and begun to test its efficacy in preventing or reversing heart failure.
Mitochondrial dysfunction is a precipitating factor for heart failure. A new genome targeting tool called CRISPR-Cas9 shows great promise as a therapeutic by directly targeting a gene for up- regulation. Their mechanism, behavior, ef?ciency, feasibility for improving mitochondrial function in cardiomyocytes have not yet been evaluated and I aim to characterize this.
