Identification of ER/SR resident proteins as biomarkers for ER/SR calcium depletion in skeletal muscle cells

Abstract

Background Aberrations to endoplasmic/sarcoplasmic reticulum (ER/SR) calcium concentration can result in the departure of endogenous proteins in a phenomenon termed exodosis. Redistribution of the ER/SR proteome can have deleterious effects to cell function and cell viability, often contributing to disease pathogenesis. Many proteins prone to exodosis reside in the ER/SR via an ER retention/retrieval sequence (ERS) and are involved in protein folding, protein modification, and protein trafficking. While the consequences of their extracellular presence have yet to be fully delineated, the proteins that have undergone exodosis may be useful for biomarker development. Skeletal muscle cells rely upon tightly coordinated ER/SR calcium release for muscle contractions, and perturbations to calcium homeostasis can result in myopathies. Ryanodine receptor type-1 (RYR1) is a calcium release channel located in the SR. Mutations to the RYR1 gene can compromise calcium homeostasis leading to a vast range of clinical phenotypes encompassing hypotonia, myalgia, respiratory insufficiency, ophthalmoplegia, fatigue and malignant hyperthermia (MH). There are currently no FDA approved treatments for RYR1-related myopathies (RYR1-RM). Results Here we examine the exodosis profile of skeletal muscle cells following ER/SR calcium depletion. Proteomic analysis identified 4,465 extracellular proteins following ER/SR calcium depletion with 1,280 proteins significantly different than vehicle. A total of 54 ERS proteins were identified and 33 ERS proteins significantly increased following ER/SR calcium depletion. Specifically, ERS protein, mesencephalic astrocyte-derived neurotrophic factor (MANF), was elevated following calcium depletion, making it a potential biomarker candidate for human samples. Despite no significant elevation of MANF in plasma levels among healthy volunteers and RYR1-RM individuals, MANF plasma levels positively correlated with age in RYR1-RM individuals, presenting a potential biomarker of disease progression. Selenoprotein N (SEPN1) was also detected only in extracellular samples following ER/SR calcium depletion. This protein is integral to calcium handling and SEPN1 variants have a causal role in SEPN1-related myopathies (SEPN1-RM). Extracellular presence of ER/SR membrane proteins may provide new insight into proteomic alterations extending beyond ERS proteins. Pre-treatment of skeletal muscle cells with bromocriptine, an FDA approved drug recently found to have anti-exodosis effects, curbed exodosis of ER/SR resident proteins. Conclusion Changes to the extracellular content caused by intracellular calcium dysregulation presents an opportunity for biomarker development and drug discovery.

Unraveling the Connection: Pain and Endoplasmic Reticulum Stress

“…Under certain stress conditions, such as Ca 2+ depletion in the ER lumen by thapsigargin treatment that inhibits SERCA, these ER proteins can undergo "exodosis", in which they are released from the ER and even from the cells [45]. This protein efflux is a biomarker of ER Ca 2+ depletion and indicates changes in cellular homeostasis [46]. Ca 2+ release from the ER caused by the activation of cell surface receptors through CICR also induces exodosis and the depletion of ER molecular chaperones from the ER, which disturbs the folding environment in the ER and activates the UPR [35] (Figure 2).…”

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase

“…The endoplasmic/sarcoplasmic reticulum (ER/SR) is responsible for a variety of cellular functions, including protein synthesis, protein modification, protein degradation, lipid metabolism, carbohydrate metabolism, and xenobiotic detoxification. 1 Another important role for ER/SR is as a major intracellular reservoir of calcium, maintaining calcium homeostasis and controlling muscle contraction and relaxation. 2 Breaking the intracellular calcium homeostasis, such as excessive Ca 2+ leakage from ER/SR, can cause ER/SR stress, leading to pathological consequences.…”