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.

Trial registration: ClinicalTrials.gov NCT02362425.

Keywords: Bromocriptine; Exodosis; MANF; Myopathy; Ryanodine receptor isoform-1; SEPN1; SIL1; Skeletal muscle.

Conflict of interest statement

The authors declare they have no competing interests.

© 2022. The Author(s).

Figures

Fig. 1

Targeted validation of exodosis-related proteins. A MANF HTRF assay of media collected from T0034 skeletal muscle cell line treated with 100 nM Tg for 8 h. Extracellular MANF is increased following Tg treatment, mean ± SEM, n = 6 wells/treatment group, ***p = 0.0008, unpaired two-tailed t-test. B Carboxylesterase 1/2 fluorescence activity measured in media collected from T0034 skeletal muscle cell line treated with 100 nM Tg for 8 and 24 h, mean ± SEM, n = 10/treatment group, ****p < 0.0001, 2-way ANOVA, Sidak’s multiple comparison test. C PDIA1/P4HB IP of media collected from T0034 skeletal muscle cell line treated with 100 nM Tg for 8 h, mean ± SEM, n = 4/treatment group, *p = 0.022, unpaired two-tailed t-test. Of note, bands at approximate 28 kDa and 51 kDa are IgG light and heavy chains from IP, respectively

Fig. 2

Calcium depletion elicits exodosis in skeletal muscle cell line. Mass spectrometry analysis of concentrated media from T0034 skeletal muscle cell line treated with 100 nM Tg for 8 h identified 4465 extracellular proteins. Red circles indicate the 54 ERS proteins that were identified. Biological processes of all identified extracellular proteins highlighted by pie chart inset

Fig. 3

Bromocriptine attenuates exodosis in skeletal muscle cell line. A Mass spectrometry analysis of media from T0034 skeletal muscle cell line pre-treated with vehicle, 20 µM bromocriptine, or 50 µM dantrolene 30 min prior to 100 nM Tg for 8 h. Solid line represents mean fold change B Extracellular ERS proteins identified from mass spectrometry analysis pre-treated with vehicle, 20 µM bromocriptine, or 50 µM dantrolene 30 min prior to 100 nM Tg for 8 h; solid line represents mean fold change, ****p < 0.0001, 1-way ANOVA, Dunnett’s multiple comparison test, Tg vs bromocriptine or dantrolene. C Extracellular MANF identified by mass spectrometry in media from skeletal muscle cells pre-treated with vehicle, 20 µM bromocriptine, or 50 µM dantrolene 30 min prior to 100 nM Tg for 8 h; mean ± SEM, *p < 0.05, 1-way ANOVA, Dunnett’s multiple comparison test. D Extracellular MANF identified by MANF HTRF assay of media collected from T0034 skeletal muscle cell line pre-treated with vehicle, 3 µM, 10 µM, 20 µM bromocriptine, or 10 µM, 30 µM, 50 µM dantrolene 30 min prior to 100 nM Tg for 8 h; mean ± SEM, n = 6–12 wells/treatment group, *p < 0.05, ***p < 0.001, ****p < 0.0001, 1-way ANOVA, Dunnett’s multiple comparison test, Tg vs other treatment groups. E) Densitometry analysis of western blot of concentrated media collected from T0034 skeletal muscle cell line pre-treated with vehicle, 20 µM bromocriptine, or 50 µM dantrolene, mean ± SEM, n = 2/treatment groups, *p < 0.05, **p < 0.01, 1-way ANOVA, Dunnett’s multiple comparison test vehicle/Tg vs vehicle/vehicle, bromocriptine/Tg, or dantrolene/Tg

Fig. 4

MANF is marginally elevated in individuals with RYR1 mutations. A Extracellular human MANF in plasma obtained from healthy volunteers or individuals with RYR1 mutations, mean ± SEM, n = 7 healthy volunteers, n = 9 RYR1 mutations, participant sex indicated next to confirmed genotype, p = 0.1665, two-tailed t-test. B, C Circulating levels of MANF is significantly correlated with age in individuals with RYR1 mutations, but not healthy volunteers, n = 7 healthy volunteers, r = -0.5122, p = 0.2398; n = 9 RYR1 mutations, r = 0.7511 *p = 0.0196