Molecular and Cellular Studies of Ca2+ Transport ATPase

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DESCRIPTION: (provided by applicant) The Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) is the operator of active Ca2+ transport into intracellular stores. Stored Ca2+ can then be released to trigger cytosolic Ca2+ signaling. Both, Ca2+ transport and Ca2+ signaling play prominent roles in control of relaxation and contraction in cardiac and skeletal muscle. Both mechanisms are altered in cardiac failure. The aims of this project are: (1) clarification of the molecular mechanism whereby ATP is utilized to move Ca2+ against a concentration gradient, and (2) establishment of strategies for SERCA gene transfer, and definition of the consequences of exogenous SERCA expression in cardiac myocytes. The research related to aim (1) will produce specific modifications in native and recombinant ATPase by protein chemistry and site directed mutagenesis, and will define the effects of these modifications on the sequential ATPase reactions that are coupled to Ca2+ transport. The findings will be related to crystallographic data obtained by x-ray diffraction, to indicate how various ATPase protein domains and specific amino acid residues are involved in energy transduction. The research related to aim (2) will optimize conditions for SERCA cDNA delivery to cardiac myocytes by means of recombinant adenovirus vectors. This will include evaluation of several promoters in order to obtain appropriate levels of exogenous SERCA expression only in cardiac muscle, and not in other tissues such as smooth muscle, endothelium, skeletal muscle, and liver. The functional consequences of SERCA overexpression and/or isoform switch on Ca2+ signaling, contraction/relaxation cycle and cellular homeostasis will be defined. Thereby, we will optimize gene transfer strategies for basic studies of cardiac cell physiology in culture, and will evaluate their possible use for amelioration of failing cardiac muscle in situ.