Department of Physiology, Wayne State University, Detroit, MI, 48201, USA.
Institute of Environmental Health Sciences, Wayne State University, Detroit, USA.
Mol Cell Biochem. 2024 Jan;479(1):85-98. doi: 10.1007/s11010-023-04708-0. Epub 2023 Apr 10.
The importance of sarcoplasmic reticulum (SR) Ca-handling in heart has led to detailed understanding of Ca-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 Ca-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 Ca 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 E enrichment values (mean ± S.D.) that spanned the range of possible E values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca loading of cardiac microsomes, reflecting the combined activities of Ca uptake by SERCA, and Ca 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.
肌浆网（SR）Ca 处理在心脏中的重要性导致了对 Ca 释放和再摄取蛋白复合物的详细了解，而对心脏中其他内质网（ER）功能的了解较少。为了更全面地了解心脏 SR 和 ER 的功能，我们分析了基于 SR Ca-ATP 酶（SERCA）和肌浆网 Ca 释放通道（RyR）活性高的心脏微粒体的密度增加。用 SERCA 和 RyR 的作用负载 Ca 草酸盐的粗心脏微粒体囊泡产生了两个更高密度的亚部分，MedSR 和 HighSR。用 SDS-PAGE 对来自 20.0μgMV、MedSR 和 HighSR 蛋白的蛋白进行分级，然后从 20 个单独的凝胶片中用胰蛋白酶消化，并用 LC-MS/MS 分析以确定蛋白含量。从获得的 62000 个单独的肽谱中，我们鉴定了 1105 种不同的蛋白质，其中 354 种在 SR 部分中的丰度比粗膜制剂高≥2.0 倍。所有先前研究过的 SR 蛋白都被富集，与经典 ER 功能相关的蛋白也是如此。收缩、线粒体和肌浆膜蛋白没有被富集。比较 MedSR 与 HighSR 囊泡中 SERCA 阳性 SR 蛋白的水平产生了一系列 SR 亚部分的富集，表明在相同的膜斑中存在不同水平的 Ca 泄漏共定位。所有已知的连接 SR 蛋白在 MedSR 中更丰富，而经典的 ER 蛋白在 HighSR 膜中更丰富。构成其他假定的 ER/SR 亚域的蛋白也表现出平均 E 富集值（平均值±标准差），跨越可能的 E 值范围，这表明功能蛋白组被定位到 ER/SR 膜的相同区域。我们得出结论，心脏微粒体的主动 Ca 负载，反映了 SERCA 的 Ca 摄取和 RyR 的 Ca 泄漏的联合活性，允许评估多个功能 ER/SR 亚域。来自这些亚域的蛋白组在膜亚部分中表现出相似的富集模式，反映了单个心脏 ER 和 SR 斑块中存在的 SERCA 和 RyR 的相对水平。
