This project uses a series of complementary approaches including voltage clamp, high resolution confocal imaging, adenoviral gene transfer, and biochemical analyses to critically examine the links between local phosphorylation, cytoskeletal structures, and Ca2+ signaling in heart.
One experimental approach is defining the functional sites to which the phosphatase PP2A is targeted in cardiac myocytes. This series will use a combination of intact cell studies combined with proteomic analyses.
We are also working on identifying the specific amino acid sequence motifs in PP2A B-targeting subunits that determine its segregation to discrete cardiac cell structures. Virus directed gene transfer of GFP fusion proteins is being used in this analysis.
The role of phosphatases and t-tubule cytoskeleton proteins in the regulation of L-type Ca2+ channel activity is also being studied through a combination of voltage clamp and molecular genetics methods (see Figure on right). This study is being performed in normal and heart failure animal models.
We are also examining alterations in PP2A targeting evoked by gene tansfection methods change signal transduction cascades.
Thus this project is defining the links between phosphatase subcellular targeting, cytoskeletal architecture, and Ca2+ signaling in cardiac myocytes.