Disruption of STIM1-mediated Ca2+ sensing and energy metabolism in adult skeletal muscle compromises exercise tolerance, proteostasis, and lean mass

Abstract

Objective: Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated Ca²⁺ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is known to be indispensable during development, its biological and metabolic functions in mature muscles remain unclear.

Methods: Conditional and tamoxifen inducible muscle STIM1 knock-out mouse models were coupled with multi-omics tools and comprehensive physiology to understand the role of STIM1 in regulating SOCE, mitochondrial quality and bioenergetics, and whole-body energy homeostasis.

Results: This study shows that STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and is present at SR-mitochondria interfaces. Inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle led to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in the settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis.

Conclusion: These results show that STIM1 regulates cellular and mitochondrial Ca²⁺ dynamics, energy metabolism and proteostasis in adult skeletal muscles. Furthermore, these findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent Ca²⁺ handling.

Keywords: Calcium signaling; ER stress; Energy metabolism; Exercise tolerance; Mass spectrometry; Mitochondria; Proteomics; Proteostasis; STIM1; Skeletal muscle.

Graphical abstract

Figure 1

Muscle-specific loss of STIM1 affects survival and mitochondrial quality. (A) Representative images and fluorescence intensity profiles of flexor digitorum brevis (FDB) fiber immunostained with STIM1, COXIV, and TOM20. (B) Distribution of STIM1-LacZ to subcellular localization with mitochondria (black arrow) in FDB muscle as determined by transmission electron microscopy (TEM). STIM1-LacZ expression is detected by staining of β-galactosidase activity. Myofibers were isolated from flexor digitorum brevis (FDB) muscles and loaded with Rhod-2 to determine mitochondrial Ca²⁺ content in the (C) basal state (n = 75–128 fibers) and in response to (D) electrical stimulation (n = 12–19). (E) Representative traces of mitochondrial Ca²⁺ transients in response to repeated bursts of electrical activity. (F) Representative transmission electron micrographs show (a) normal mitochondrial ultrastructure of STIM1^fl/fl muscle and (b–f) abnormal mitochondrial size, shape, and cristae of mSTIM1^−/− muscle. (G) Metabolomic analysis of acylcarnitines in gastrocnemius muscles at 2 weeks of age (n = 5–6). (H) STIM1 mRNA expression normalized to 18 S measured by RT-qPCR in tibialis anterior muscle after a 90-minute bout of treadmill running (n = 4). (I) Western blot and (J) quantification of STIM1 normalized total protein in gastrocnemius muscle from PGC1α transgenic (Tg) and non-transgenic (NT) littermates (n = 4). All data represent mean ± SEM. (C, D, G, and J) were analyzed using the Student's 2-tailed t-test. ∗p ≤ 0.05. (H) was analyzed by 1-way ANOVA with Dunnett's multiple comparisons test. ∗∗p≤ 0.01 and ∗∗∗p ≤ 0.001. n represents biological replicates.

Figure 2

Conditional deletion of STIM1 in adult muscle abolishes SOCE. (A) Schematic depicting the breeding strategy to generate mice with inducible muscle-specific deletion of STIM1. Transgenic HSA-MerCreMer mice were crossed with mice in which exon 2 of the STIM1 gene is flanked by loxP sequences (STIM1^fl/fl). Adult mice received tamoxifen (TMX) at ages of 12–13 weeks (B–D) or 8 weeks (E–J). TMX-treated transgenic HSA-MerCreMer:STIM1^fl/fl are denoted as iSTIM1^−/− throughout the manuscript. (B) Western blots and quantification of (C) STIM1S and (D) STIM1L abundance normalized to total protein in gastrocnemius muscles 4, 8, and 18 weeks after TMX (n = 5–8). (E) Representative traces of cytosolic Ca²⁺ in response to a standard SOCE protocol in FDB fibers loaded with Fura-2 (n = 4). (F) Representative traces of cytosolic Ca²⁺ transients generated with a single (2 s pulse [short bars]) or repeated bursts (2 s pulse at 50 HZ every 5 s [long bar]) of electrical activity in STIM1^fl/f (left) and iSTIM1^−/− (right) FDB myofibers loaded with Fura-4 (n = 4). The amplitude of cytosolic Ca²⁺ in Fura-4 loaded FDB fibers (G) during and (H) after each electrical stimulus (2 s at 50 HZ) (n = 17–23 fibers). (I) Representative traces and (J) quantification of mitochondrial Ca²⁺ uptake in response to a standard SOCE protocol in FDB fibers loaded with Rhod-2 (n = 12–13). Data in (C, D, and J) were analyzed using Student's two-tailed t-test. n represents biological replicates.

Figure 3

Conditional STIM1 deficiency in adult mice diminishes lean mass. (A) Body weight, (B) lean mass, and (C) fat mass before and 6 or 12 weeks after TMX administration at 11–13 weeks of age (n = 12). (D) Change in body weight, lean, and fat mass 12 weeks after TMX compared to pre-TMX (n = 12). (E) Tibia length measured 18 weeks after TMX (n = 6–7). (F) Representative images and (G) quantification of the cross-sectional area of succinate dehydrogenase immunostained soleus muscles 8 weeks after TMX administration at 8 weeks of age (n = 5). (H) Representative images of H&E-stained soleus cross-sections 8 weeks after TMX (n = 5). (I) Western blot and (J) quantification of mitochondrial protein expression normalized to total protein in gastrocnemius muscle 4 weeks after TMX (n = 5). All data shown represent mean ± SEM. Data in (A-E, G, and J) were analyzed using Student's two-tailed t-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001. n represents biological replicates.

Figure 4

Conditional STIM1 deficiency alters fuel use and compromises exercise tolerance. (A) Whole-body oxygen consumption, the relationship of energy expenditure to (B) lean mass and (C) whole body weight as well as (D), total activity, (E) food consumption, and (F) respiratory exchange ratio assessed over 48 h in STIM1^−/− v. STIM1^fl/fl fed a chow diet after receiving TMX at 12–15 weeks of age (n = 9–10). (G) Time, and (H) distance to exhaustion, and (I) respiratory exchange ratio in response to a high-intensity exercise test (n = 5–6). (J) Traces of substrate utilization by STIM1^fl/fl (left) and iSTIM1^−/− (right) mice and (K) a bar graph depicting the time to the crossover point where carbohydrate utilization exceeds fat utilization during exercise (n = 5–7). (L) Blood lactate prior to and immediately after 15′ of exercise (n = 5–7). (M) Western blots and (N) quantification of phosphoproteins normalized to total protein in gastrocnemius muscles immediately after 15′ of exercise (n = 5–7). All data represent mean ± SEM. Data in (A, D-I, K, L, and N) were analyzed using Student's two-tailed t-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001. n represents biological replicates.

Figure 5

Deletion of STIM1 alters muscle pyruvate partitioning but not whole-body glucose tolerance. (A) Scheme of isotopologue enrichment depicting labeling of glycolytic intermediates, TCAC intermediates, and TCAC-derived intermediates by [U–¹³C₆]-glucose through the first turn of the TCAC via pyruvate dehydrogenase (black circles) and pyruvate carboxylase (blue circles). Muscles were incubated with 10 mM [U–¹³C]-glucose + 200 μM palmitate with or without 100 nM of insulin. Average ¹³C-labeling of glycolytic and TCAC intermediates in soleus muscles (B) 4 and (C) 8 weeks after TMX administered at 12–15 weeks of age (n = 4–6). (D) Lactate efflux from soleus during 90′ incubation (n = 4–6). (E) Ratio of M5 citrate to M3 pyruvate labeling as an estimation of the contribution of pyruvate to the acetyl-CoA pool through PDH (n = 4–6). (F) Western blots and quantification of phospho to total protein levels in gastrocnemius muscles 4 and 8 weeks after TMX (n = 5–8). (G) Blood glucose and (H) plasma insulin during an oral glucose tolerance test 6 weeks after TMX (n = 6–8). All data represent mean ± SEM. Data in (B-E, G-J) were analyzed using Student's two-tailed t-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001. Outliers identified by Grubb's test were removed to reach statistical significance. n represents biological replicates.

Figure 6

Metabolomics and proteomics analysis of iSTIM1^−/− muscles reveals adaptive proteostasis. Metabolomic and proteomic analysis in gastrocnemius muscles from iSTIM1^−/− v. STIM1^fl/fl mice a 16 h fast. (A) Volcano plots depicting the relative abundance of organic acids, amino acids, and acylcarnitines identified 4 (left) and 8 weeks (right) after TMX administration at 12–13 weeks of age (n = 8). Gastrocnemius muscles collected 8 weeks after TMX and 16 h of fasting were used to evaluate protein and phospho-protein abundance by MS/MS proteomics and western blotting (n = 5). (B) Volcano plots depicting relative abundance (downregulated in blue and upregulated in red) of proteins (left) and phosphopeptides (right). (C) Principal component analysis of proteins (left) and phosphopeptides (right). (D) Heat map of a subset of differentially regulated proteins involved in proteostasis and contraction identified by pathway analysis. (E) Output of kinase-substrate enrichment analysis (KSEA) wherein at least 2 phospho-peptides with substrate sequence were identified; numbers indicate substrates previously linked to each kinase. (F) Western blot and (G) quantification of phosphoprotein expression normalized to total protein. Data in (A and G) were analyzed using Student's two-tailed t-test ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001. n represents biological replicates.

Figure 7

Chronic deletion of STIM1 alters whole-body fuel selection. (A) Body weight, (B) fat mass (C) percentage body fat, and (D) lean mass of iSTIM1^−/− and STIM1^fl/fl mice fed standard chow (SC) or a high-fat diet (HFD) (n = 6–8). (E) Wet weight of skeletal muscle normalized to tibia length after 20 weeks of diet and 30 weeks after TMX administered at 12–15 weeks of age (n = 6). (F) Respiratory exchange ratio over a 24 h light/dark period with ad libitum access to food after 8 weeks on diet (n = 5–8). Relationship between energy expenditure to (G) lean mass, and (H) body weight (n = 5–8). (I) Respiratory exchange ratio during 16 h of fasting, administration oral glucose bolus (3 g/kg), and a 5 h refeed after 13 weeks on diet, and (J) relative frequency distribution of RER during 16-h fasting period (n = 5–8). (K) Metabolic flexibility defined as the difference in RER in response to the 3 g/kg oral glucose bolus and the last hour of fasting (n = 5–8). Data in (A-F, I, and J) were analyzed using Student's two-tailed t-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001. Outlier identified by Grubb's test was removed to reach statistical significance and is represented by a gray open circle. n represents biological replicates.

Figure 8

Chronic deletion of STIM1 diminishes mitochondrial respiratory efficiency without exacerbating diet-induced glucose intolerance. Respiratory efficiency evaluated by plotting JO₂ vs. ΔΨ_m of mitochondria fueled with pyruvate/malate (PM), α-ketoglutarate (αKG), or palmitoylcarnitine/malate (PcM) isolated from skeletal muscle of mice fed a (A) standard chow (SC) (B) or 20-week high fat (HF) diet 30 weeks after TMX administration at 12–15 weeks of age (n = 4–6). (C) Western blot and (D) quantification of canonical OXPhos subunits normalized total protein (n = 6). (E) Oral glucose tolerance test after 10 weeks on diet (n = 6–8). (J) Western blot and quantification of STIM1 expression normalized to total protein in gastrocnemius muscle after 20 weeks of diet and 30 weeks after TMX (n = 6). All data represent mean ± SEM. Data in (A and B) were analyzed using 2-way ANOVA (S. Figure 6). Data in (D-F and H) were analyzed using Student's two-tailed t-test to test for statistical significance. Outlier identified by Grubb's test was removed to reach statistical significance. n represents biological replicates.