Cala Steven E, Carruthers Nicholas J, Stemmer Paul M, Chen Zhenhui, Chen Xuequn
Wayne State University.
Indiana University.
Res Sq. 2023 Feb 8:rs.3.rs-2557992. doi: 10.21203/rs.3.rs-2557992/v1.
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. Analyses of protein enrichments from the 3 membrane preparations (crude microsomes, MedSR, and HighSR), showed that only a third of microsomal proteins in heart, or 354 proteins, were enriched ≥2.0-fold in SR. 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 (ryanodine receptor) 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 from other putative ER/SR subdomains also showed characteristic distributions among SR subpopulations. 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）钙处理在心脏中的重要性已使人们对钙释放和再摄取蛋白复合物有了详细了解，而对心脏中其他内质网（ER）功能的了解则较少。为了更全面地了解心脏SR和ER功能，我们基于心肌细胞中高度活跃的SR钙ATP酶（SERCA）和兰尼碱受体的作用导致的密度增加，对心脏微粒体进行了分析。装载草酸钙的粗制心脏微粒体囊泡产生了两个更高密度的亚组分，即中密度SR（MedSR）和高密度SR（HighSR）。对三种膜制剂（粗微粒体、MedSR和HighSR）的蛋白质富集分析表明，心脏中只有三分之一的微粒体蛋白，即354种蛋白，在SR中富集≥2.0倍。先前研究的SR蛋白均被富集，与经典ER功能相关的蛋白也是如此。收缩蛋白、线粒体蛋白和肌膜蛋白未被富集。比较MedSR与HighSR囊泡中SERCA阳性SR蛋白的水平，产生了一系列SR亚组分富集情况，表明在同一膜片中共同定位的钙泄漏（兰尼碱受体）水平不同。所有已知的连接SR蛋白在MedSR中富集程度更高，而经典ER蛋白在HighSR膜中富集程度更高。来自其他假定的ER/SR亚结构域的蛋白在SR亚群中也表现出特征性分布。我们得出结论，心脏微粒体的活性钙加载反映了SERCA摄取钙和RyR导致钙泄漏的综合活动，允许对多个功能性ER/SR亚结构域进行评估。来自这些亚结构域的蛋白组在跨膜亚组分中表现出相似的富集模式，反映了心脏ER和SR单个膜片中SERCA和RyR的相对水平。
