Contraction of the heart is triggered by an intricate signal transduction pathway known as EC coupling. In this process, depolarization of the cardiac muscle cell activates an intracellular calcium release channel; the ryanodine receptor isoform 2 (RyR2) to release calcium from intracellular stores and this released calcium triggers muscle contraction. Fully understanding this elaborate signaling process is one of the fundamental goals in cardiac muscle research. While, the importance of RyR2 in heart function is well established, as transgenic animals that lack RyR2 suffer embryonic lethality, relatively little is known as to the structural determinants within the RyR2 primary sequence that are involved in EC coupling. Work funded by this proposal will specifically define the regions on RyR2 necessary for EC coupling. Using. molecular biological techniques, we will create a set of chimeric proteins where defined segments of RyR2 are substituted into a backbone made up of the brain ryanodine receptor isoform. RyR3. Although RyR3 is able to support calcium induced calcium release (CICR), it is unable to participate in depolarization induced EC coupling. This makes it the ideal background for these studies. After expressing these chimeric proteins in dyspedic (i.e. RyR-deficient) muscle cell line, we will test the ability of these RyR2-RyR3 chimeras to mediate EC coupling using high-speed fluorescence calcium imaging techniques. When chimeras that support depolarization induced EC coupling are found we will then create additional chimeras containing smaller and smaller amounts of RyR2 until we can define the smallest segment of the RyR2 sequence that can support EC coupling. After completing preliminary studies in our dyspedic skeletal muscle cell line, we will then confirm our findings in embryoid bodies which express cardiac ventricular proteins created from ES cells in which both alleles of RyR2 have been """"""""knocked out"""""""". This will allow us to confirm our data and firmly establish its relevance in the heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32HL067572-03
Application #
6638788
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Commarato, Michael
Project Start
2002-07-01
Project End
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
3
Fiscal Year
2003
Total Cost
$51,904
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Fessenden, James D; Feng, Wei; Pessah, Isaac N et al. (2006) Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol. J Biol Chem 281:21022-31