Calsequestrin (CSQ) concentrates in lumens of junctional SR and contributes to effectual delivery of SR Ca2+. CSQ is comprised of numerous glycoforms and phosphoforms reflecting actions of diverse cellular enzymes and trafficking molecules. Recently, we found in heart failure a striking change in the distribution of CSQ molecular forms, with no change in total levels. Three major changes in CSQ were: 1) a 4-fold decrease in Man3-4 glycoforms, indicating loss of a post-ER mannosidase step; 2) a doubling of CSQ with glycans of Man8-9, indicating greater ER retention; and 3) a roughly two-fold increase in CSQ phosphorylation on CK2-sensitive sites. To understand normal and defective CSQ processing in heart, we propose the following specific aims: Aim 1 is to characterize post-ER trafficking in heart and heart failure. Overexpression of dominant negative Sar1p inhibits CSQ ER exit, and will be used to differentiate ER and post-ER components of CSQ glycosylation and phosphorylation in cell types that differ in ER/post-ER processing of CSQ, by indirect immunofluorescence and analysis of glycan structure. Cells will include COS, C2C12, primary rat heart cells, and cells from failing rat heart Classic density-gradient purified junctional SR, from normal and failed heart tissue, will be analyzed for markers of other COPII pathway components, and for the mannosidase(s) that generates the low mannose forms (Man3,4), an atypical mannosidase that may be defective in heart failure. Aim 2 is to characterize cellular effects of CSQ phosphorylation using new CSQ vectors that overexpress and tag normal or unphosphorylatable CSQ. Effects of phosphorylation will be tested by fluorescence and electron microscopy, and measures of heart cell physiology. Aim 3 is to further explore glycosylation in terminal cisternae of normal and failing heart. Comparing frozen tissue samples of control and heart failure, we will determine whether triadin-1 glycans are a so defective, and whether differences exist in ER/Golgi mannosidases.