Cardiovascular disease is the number one killer in the United States. Toll-like receptor (TLR)-mediated innate immune and inflammatory responses play a central role in the pathophysiologic mechanisms of myocardial ischemia/reperfusion (I/R) injury. It is well known that activation of TLR4-mediated NF-kB signaling plays a deleterious role in myocardial I/R injury, while activation of the PI3K/Akt signaling pathway protects against myocardial I/R injury. We have reported that either TLR4 deficiency or TLR2 modulation protects the myocardium from I/R injury via a PI3K/Akt dependent mechanism. We have discovered that TLR4 deficiency increases the expression of microRNA-146a (miR-146a) and miR-486 in the myocardium. We have also found that TLR2 modulation increases the amount of miR-146a and miR-486 in the myocardium, in circulating exosomes, and in exosomes secreted by bone marrow mesenchymal stem cells. We have demonstrated that increased expression of miR-146a in the myocardium decreases myocardial infarct size and improves cardiac function following myocardial I/R. MiR-146a targets IRAK1 and TRAF6 which are the important intermediates in TLR-mediated NF-kB activation, thus, attenuating I/R-induced NF-kB activation. In addition, we have found that miR-486 inhibits PTEN expression, leading to activation of PI3K/Akt signaling. Our findings suggest that either TLR4 deficiency or TLR2 modulation induce cardioprotection via up-regulation of microRNA expression that inhibits NF-kB activation and activates PI3K/Akt signaling. When considered together, our data and the literature clearly show that TLR4-mediated NF-kB activation is closely linked to disease progression in myocardial I/R injury, while activation of PI3K/Akt signaling pathways is closely associated with attenuation of myocardial injury. At present, we do not fully understand the mechanisms of differential regulation of NF-kB and PI3K/Akt in I/R injury. This application is directed at deciphering the cellular and molecular mechanisms that differentially regulate these signaling pathways in I/R induced myocardial injury. We will then apply that mechanistic knowledge in a practical fashion to develop new and novel therapies for heart attack. Specifically, we will define the role of microRNA in differential regulation of NF-kB and PI3K/Akt in myocardial I/R injury. We will also attempt to apply this knowledge for the development of new and novel miR based therapies for heart attack. We hypothesize that i) miRNAs differentially activate PI3K/Akt signaling and inhibit NF-kB activation in myocardial I/R injury and ii) miRNAs are carried in exosomes which are efficiently uptaken by cardiac myocytes, resulting in cardioprotection via differential activation f protective versus deleterious intracellular signaling. Specific Aim 1 will elucidate the mechanisms by which modulation of NF-kB and PI3K/Akt regulates the expression of miR-146a and miR-486 in cardiac myocytes. Specific Aim 2 will define the effect of miR-146a and miR-486 on myocardial injury and functional recovery. Specific Aim 3 will investigate the role of synthetic exosomes carrying miR-146a and miR-486 as therapeutics in myocardial I/R injury.