Atomic Force and Fluorescence Microscopy Imaging of Cardiomyocyte Plasma Membrane, Insulin Receptor and Caveolin‐3

Impaired insulin signaling is a component of diabetic cardiomyopathy. Our hypothesis is that myocardial insulin resistance involves a dissociation of insulin receptors (IR) from caveolae and its intracellular targets. Our aim was to develop techniques to image the spatial relationship between IR and caveolae using atomic force microscopy (AFM) and immunohistochemistry (IHC). We used an embryonic rat cardiac cell line (H9c2) to evaluate various fixation protocols and determined that 4% formaldehyde (rather than glutaldehyde, methanol or combinations), maintained the integrity of the plasma membrane. We tested a variety of AFM tips (standard, tungsten, carbon coated) in both contact and tapping modes on hydrated and dehydrated cells. We now can scan cells in solution, with a resolution of up to 10 nm. To validate our methods and reagents we conducted IHC and immunoblotting. IHC images included fixed and living cells. Future studies will use AFM techniques with gold conjugated antibodies for the IR and caveolin 3, in cells cultured in normal and high extracellular glucose. Ultimately, we intend to evaluate cell surface receptor distributions on adult rat ventricular myocytes. Resolving the localization of IR and caveolae in intact cells and comparing normal and diabetic‐like groups could further our understanding of the pathogenesis of insulin resistance in diabetic cardiomyopathy. APS Undergraduate Summer Research Fellowship (EGM)