Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062586-08
Application #
7355982
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Buxton, Denis B
Project Start
2000-07-01
Project End
2011-01-31
Budget Start
2008-02-01
Budget End
2011-01-31
Support Year
8
Fiscal Year
2008
Total Cost
$322,145
Indirect Cost

  Institution

Name
Wayne State University
Department
Physiology
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202

  Publications

Sleiman, Naama H; McFarland, Timothy P; Jones, Larry R et al. (2015) Transitions of protein traffic from cardiac ER to junctional SR. J Mol Cell Cardiol 81:34-45
Jacob, Sony; Sleiman, Naama H; Kern, Stephanie et al. (2013) Altered calsequestrin glycan processing is common to diverse models of canine heart failure. Mol Cell Biochem 377:11-21
Guo, Ang; Cala, Steven E; Song, Long-Sheng (2012) Calsequestrin accumulation in rough endoplasmic reticulum promotes perinuclear Ca2+ release. J Biol Chem 287:16670-80
McFarland, Timothy P; Sleiman, Naama H; Yaeger, Daniel B et al. (2011) The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells. Mol Cell Biochem 353:81-91
McFarland, Timothy P; Milstein, Michelle L; Cala, Steven E (2010) Rough endoplasmic reticulum to junctional sarcoplasmic reticulum trafficking of calsequestrin in adult cardiomyocytes. J Mol Cell Cardiol 49:556-64
Milstein, Michelle L; Houle, Timothy D; Cala, Steven E (2009) Calsequestrin isoforms localize to different ER subcompartments: evidence for polymer and heteropolymer-dependent localization. Exp Cell Res 315:523-34
Milstein, Michelle L; McFarland, Timothy P; Marsh, James D et al. (2008) Inefficient glycosylation leads to high steady-state levels of actively degrading cardiac triadin-1. J Biol Chem 283:1929-35
Houle, Timothy D; Ram, Michal L; McMurray, Walter J et al. (2006) Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ. Exp Cell Res 312:4150-61
Kiarash, Arash; Kelly, Carmen E; Phinney, Brett S et al. (2004) Defective glycosylation of calsequestrin in heart failure. Cardiovasc Res 63:264-72
Ram, Michal L; Kiarash, Arash; Marsh, James D et al. (2004) Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart. Mol Cell Biochem 266:209-17

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