Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins

Abstract

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 μg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC–MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.