Zinc Protection Against Ischemia-Reperfusion Injury in Heart

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Project Summary This project is aimed to provide NIH R15 REAP research opportunities for our underrepresented graduate students at UT Arlington, a Hispanic-Serving Institute in North Texas. During acute ischemia-reperfusion (I/R), reactive oxygen species (ROS) is generated at the reperfusion phase and results in catastrophic damage to heart. Thus, the administration of antioxidant agents to prevent or ameliorate ROS detrimental effects is an active research area. The nutrient, zinc, has multifaceted antioxidant effects and has been shown to protect against I/R injury in heart. But a safe formulation, dosage, and delivery mode of zinc have not been established. Previously, zinc dosages yielded toxic effects (e.g., halted growth, cell death), and the delivery mode of zinc as an ionophore across plasma membrane produced many side-effects (e.g., membrane damage, cell death, etc.). Our long-term goal is to establish a therapeutic zinc regimen to protect heart against I/R injury. To progress to such human clinical trials, we must initially address two fundamental questions: What entity besides zinc transporters regulates zinc influx in muscle cells? Can enhancement of such intrinsic entity, without using side effect-prone zinc ionophore, protect the cardiomyocytes from I/R injury? Toward addressing the two questions, we performed unbiased genome-scale CRISPR/Cas9-based screening to search for such new entity. Unexpectedly, a gene on the top validated candidate list was SLC5A3, also called SMIT1, a sodium myo-inositol transporter. SLC5A3 has never been linked to zinc metabolism. Our preliminary data showed that knockdown of SLC5A3 negated zinc-induced protection in HL- 1 cardiomyocytes in hypoxia /reoxygenation (H/R) injury. Interestingly, hypoxic post-conditioning (PostC) enhanced HL-1 cell survival upon H/R injury, and its beneficial effect was diminished in SLC5A3-knockdown cells. Moreover, PostC with zinc perfusion protect the heart (in terms of reduced apoptosis, maintaining left ventricle ejection fraction in echocardiography study) from I/R induced cardiac dysfunction in mice. The protective function of zinc in both cultured cardiomyocytes and whole heart was blunted by a SLC5A3 inhibitor phloridzin. Based on these data, we hypothesize that SLC5A3 mediates zinc influx in cardiomyocytes, which can be enhanced to protect heart from I/R injury. Two specific aims are formulated to test this hypothesis: 1) to define the molecular mechanisms by which SLC5A3 mediates zinc influx in cardiomyocytes; 2) to conduct proof-of-concept study by targeting SLC5A3-mediated zinc influx to protect heart from I/R injury. The innovation includes: (i) the first study to show an unrecognized role of SLC5A3 in regulating zinc homeostasis; (ii) the identification of the therapeutic potential of SLC5A3- mediated zinc influx for heart I/R injury. As one of six founding members in Bone-Muscle Research Center at UTA, the PI’s laboratory has required expertise, excellent resources to train PhD students in cardiovascular physiology.