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To maintain homeostasis, cells continuously evaluate and adjust to their nutrient availability and current energy state. A cell's response to the fed state requires the presence of amino acids and other nutrients, which signal to the mammalian target of rapamycin complex 1 (mTORC1) coordinating cell growth, protein translation, and autophagy. The umami taste receptor, which is a heterodimer between the G protein‐coupled receptors T1R1 and T1R3, is a direct sensor of amino acid availability and signals to mTORC1. Reducing the expression of T1R1/T1R3 in mice and pancreatic beta and cardiomyocyte cell lines diminishes the activation of mTORC1 by amino acids, thereby inducing autophagy. Previous research suggests that elevated autophagic flux reduces the development and progression of atherosclerosis by protecting cells from oxidative stress, reducing apoptosis within atherosclerotic lesions, and promoting lesion stabilization by degrading intracellular components that are harmful to the cell. Other studies suggest that increased autophagic flux decreases the progression of Non‐Alcoholic Fatty Liver Disease (NAFLD) by reducing hepatocyte lipid accumulation and liver inflammation. We hypothesized that the deletion of T1R3 in ApoE−/− mice would decrease aortic atherosclerotic plaque buildup and decrease hepatosteatosis by increasing autophagic flux. Our data suggest that T1R3−/− ApoE−/− mice have less liver lipid accumulation and reduced atherosclerotic plaque area when compared to T1R3+/+ApoE−/− mice. Taken together, these data suggest that modulation of nutrient and amino acid‐sensing GPCRs like T1R1/T1R3 could be used as a potential therapeutic strategy for cardiovascular disease.Support or Funding InformationAmerican Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (E.M.W.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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A small percentage of the 518 kinases in the human genome are known to play important roles in the pathophysiology of ischemia/reperfusion (I/R) injury in cardiomyocytes. However, there are many understudied kinases that may impact the damage to cardiomyocytes caused by I/R. Thus, there are likely unmet therapeutic opportunities in targeting kinases to reduce death caused by myocardial infarction. We utilized an unbiased mass spectrometry affinity chromatography method to investigate how simulated ischemia/reperfusion (sI/R) affected the activity of a large number of kinases in a cardiomyocyte cell line. Our data suggested that the activity of bone morphogenic protein 2‐inducible kinase (BMP2K) was elevated in cardiomyocytes exposed to simulated ischemia/reperfusion (sI/R) injury. BMP2K was cloned from a cell line that was induced to differentiate into an osteoblastic phenotype with BMP2. While no physiological substrates have been identified, BMP2K was observed to phosphorylate myelin basic protein and autophosphorylate in vitro. We determined that a reduction in the expression of BMP2K in cardiomyocytes using siRNA reduced apoptosis and overall cell death caused by sI/R injury. In addition, knocking down BMP2K reduced autophagic flux in cardiomyocytes under all the conditions that we tested. To identify potential BMP2K substrates and interacting proteins, we immunoprecipitated Flag‐BMP2K from HEK‐293 cells, performed SDS‐PAGE, and visualized proteins with Coomassie blue stain. We observed a 75 kD protein that was identified by mass spectrometry analysis as mortalin. Mortalin, which is member of the heat shock protein 70 (Hsp70) family, can function to suppress cell death during glucose deprivation. We validated our mass spectrometry results with co‐immunoprecipitation assays followed by immunoblotting with a mortalin antibody. Our future goals are to determine if mortalin is a direct substrate of BMP2K and to identify the functional significance of the BMP2K/mortalin interaction. In addition, we are currently investigating the mechanism by which BMP2K inhibition regulates both cell death and autophagy using both a cardiomyocyte cell line and primary adult rat ventricular cardiomyoctesSupport or Funding InformationThis research was supported by the American Heart Association Award: 15SDG25090279 (EMW) and by The Iowa Osteopathic Education and Research (IOER) Funds (EMW).

Our previous data suggest that bone morphogenic protein 2‐inducible kinase (BMP2K) plays a significant role in the pathophysiology of hypoxia/reoxygenation injury in cardiomyocytes, and that a reduction in BMP2K expression reduces apoptosis in cardiomyocytes. BMP2K and adaptor‐associated kinase 1 (AAK1) are human homologs of the drosophila Numb‐associated protein kinase (NAK). The function of BMP2K is unknown, and there are no identified physiological substrates. BMP2K shares a 74% sequence homology with the kinase domain of AAK1. Novel dual inhibitors of AAK1 and BMP2K, SGC‐AAK1‐1 and LP935509, have recently been developed. Because knockdown of BMP2K in cardiomyocytes protects against hypoxia‐induced cell death, we hypothesized that SGC‐AAK1‐1 and LP935509 would produce the same effect. To test our hypothesis, we pretreated H9c2 cardiomyocyte cells with SGC‐AAK1‐1, LP935509, or an inactive control compound SGC‐AAK1‐1N before subjecting the cells to hypoxic conditions. By analyzing cleaved caspase‐3 and cleaved PARP levels via immunoblotting, we determined that 3 μM of SGC‐AAK1‐1 or LP935509 reduced apoptosis. Since these compounds inhibit both AAK1 and BMP2K with similar potencies, we wanted to determine which kinase was responsible for reduced apoptosis. We observed that transfection of H9c2 cells with BMP2K siRNA, but not AAK1 siRNA, reduced hypoxia‐induced apoptosis. In addition, BMP2K siRNA increased overall cell viability. Phosphoantibody arrays are currently being done to assess the effect of SCG‐AAK1‐1 on cellular kinase signaling. Ongoing experimental objectives are to identify the mechanism by which these kinase inhibitors protect cardiomyocytes in hypoxic conditions, and characterize the substrates of BMP2K.Support or Funding InformationAmerican Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (E.M.W.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.