Hardin Lab's Current and Future Research
Our research program has focused on the role of macromolecular interactions in the cytoplasm. The primary research focus has been on the spatial and functional organization of carbohydrate metabolism in smooth muscle. We have demonstrated a functional compartmentation of carbohydrate metabolism using 13C-NMR spectroscopy and have elucidated some aspects of the molecular basis for this compartmentation including specific associations with scaffolding proteins including tubulin, caveolin-1, and actin. We are continuing to probe the molecular basis for this compartmented metabolism by use of siRNAs to “knock down” scaffolding protein levels, and by use of overexpression of caveolin-1 in smooth muscle cells, astrocytes, and lymphocytes. In addition we have been utilizing vascular tissue from a genetically type 2 diabetic swine model which have ~80% reduction in caveolin-1 levels. Our plans include the use constructs for scaffolding proteins and glycolytic enzymes expressed with different fluorescent proteins (GFP and variants) to perform FRET measures to better characterize the dynamic associations of these enzymes with structural proteins in cells. In addition we plan to use atomic force microscopy to determine the specific metabolic pathways responsible for cytoskeletal force maintenance. Our overall goal is to provide a model for enzyme localization and control of localized pathway function to understand the role of glycolytically produced ATP in the localized support of cell kinases. Our work has traditionally focused on ATPases such as the calcium pump. We are beginning to move towards studies of smooth muscle signaling and the potential role of glycolysis in the support of cell signaling. Such studies may include Rho kinase and a variety of signaling cascades associated with arteriogenesis. These investigations will focus initially on the role of scaffolding proteins in the regulation of cell signaling. In the future, the coupling of localized energy supply to these signaling kinases will be examined.
Another aspect of our work has been the examination of mitochondrial metabolism using 13C-isotopomer analysis of glutamate as our measure of the pattern of mitochondrial substrate preference. Since the mitochondria is a key control point for apoptosis and hence cell fate (and hence tissue phenotype), we have focused on the possible role of mitochondrial dysfunction in smooth muscle apoptosis and cell phenotype transition. In urinary bladder from diabetic/dyslipidemic swine we have found an altered pattern of substrate utilization with is qualitatively mimicked by growth in organ culture and by provision of the pro-apoptotic long-chain fatty acid palmitate. We are currently making TUNEL measures of apoptosis and real-time PCR measures of PPAR a, d and g mRNA. We believe that smooth muscle apoptosis induced by lipotoxicity may result in alterations in smooth muscle phenotype in smooth muscle ranging from urinary bladder to vascular smooth muscle. We have focused on PPAR d since this isoform has the highest expression levels in smooth muscle and its function is largely unknown.
We have also actively worked on some novel metabolic interventions for hypoxic and ischemic tissue. We have demonstrated that the glycolytic intermediate fructose 1,6-bisphosphate (FBP) is capable of crossing the plasma membrane by use of a dicarboxylate transporter and providing substantial anaerobic ATP production. Indeed in Langendorff perfused rat heart, we achieved near maximal glycolytic rate with FBP as the sole exogenous substrate under normoxic conditions. Our results are consistent with a linear form of FBP being transported and future goals include finding a suitable FBP chelator to stabilize the linear form.
Finally, a recent interest of the lab has been in metabolomics/metabonomics and the application of NMR-based metabonomics to develop a marker for muscle toxicity induced by HMG-CoA reductase inhibitors (“statins”).