Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Myocardial infarction (MI) initiates a dynamic cellular response almost immediately after reperfusion therapy. Inflammation during the early stages of ischemia reperfusion injury (IRI) initiates a cascade of immune cell signaling networks which contribute to immune cell recruitment and extracellular matrix (ECM) degradation. The paradoxical phenomenon of IRI results in massive cell death and is a primary determinant of adverse ventricular remodeling. Using a novel transcriptome-wide analysis of spatiotemporal gene expression, we determined that the RNA-binding protein, Tristetraprolin (TTP) has regulator effects on the expression of several key pro- inflammatory cytokines and genes related to reactive oxygen species (ROS). However, the role of TTP in early- onset MI is unknown despite its well-established anti-inflammatory properties. The research described in this proposal seeks to elucidate the cardioprotective mechanisms of TTP in early-onset MI. S100A8 and S100A9 act as damage associated molecular patterns (DAMPs) that are highly expressed after 3 days of IRI. Aim 1 investigates the regulatory role of TTP on S100A8/S100A9 expression and functionality. TTP knockout (TTP- KO) and TTP overexpressing (TTP-OE) in myeloid cell lines will be used to determine the effects of TTP on S100A8/S100A9 expression. Mutations to 6 specific AA residues has been shown to abrogate TTP binding of mRNA molecules. TTP mutant cell lines (TTP-Δ1-6) will be generated to determine whether TTP mediates S100A8/S100A9 signaling effects through mRNA destabilization or upstream regulation. The function of TTP and S100A8/S100A9 are closely tied to Ca2+ availability. Therefore, Ca2+ concentration will tested as an environmental variable. TTP has also been shown to diminish SPP1 and Cybb (NOX2) expression, inhibiting ROS production, oxidative stress, and mitochondrial dysfunction following IRI. Therefore, Aim 2 will explore the effects of simulated (sIRI) on ROS production in TTP-KO and TTP-OE myeloid cells in vitro. Mitochondrial function and ATP production will also be measured in cardiomyocytes upon exposure to conditioned media of sIRI-stimulated, TTP-KO and TTP-OE myeloid cells. Lastly Aim 3 will investigate the role of myeloid-specific TTP expression in mitigating adverse ventricular remodeling in vivo. To test this, TTP floxed mice and mice with a 136 bp floxed site upon LysMCre excision generate myeloid-specific TTP-KO and TTP-OE mice respectively. Experimental mice will be exposed to IRI treatment by temporary LAD ligation. Echocardiography will be regularly performed to monitor heart function and ventricular remodeling. Lastly, we will investigate the therapeutic potential of time-dependent TTP delivery by introduction of TTP-expressing adeno-associated viral vector-9 (AAV9) or adoptive transfer with TTP-OE macrophages after 3 days of IRI. The same measurements and assessments will be made to determine the efficacy of each treatment. The rationale behind this research is a sound comprehension of the anti-inflammatory properties and therapeutic potential of TTP in early-onset MI. Specifically, the effects of TTP expression on inflammation, mitochondrial dysfunction, and cardiomyopathy.
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