Novel role of the ER/SR Ca²⁺ sensor STIM1 in the regulation of cardiac metabolism

Abstract

The endoplasmic reticulum/sarcoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca²⁺ entry, is expressed in cardiomyocytes and has been implicated in regulating multiple cardiac processes, including hypertrophic signaling. Interestingly, cardiomyocyte-restricted deletion of STIM1 (^crSTIM1-KO) results in age-dependent endoplasmic reticulum stress, altered mitochondrial morphology, and dilated cardiomyopathy in mice. Here, we tested the hypothesis that STIM1 deficiency may also impact cardiac metabolism. Hearts isolated from 20-wk-old ^crSTIM1-KO mice exhibited a significant reduction in both oxidative and nonoxidative glucose utilization. Consistent with the reduction in glucose utilization, expression of glucose transporter 4 and AMP-activated protein kinase phosphorylation were all reduced, whereas pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphorylation were increased, in ^crSTIM1-KO hearts. Despite similar rates of fatty acid oxidation in control and ^crSTIM1-KO hearts ex vivo, ^crSTIM1-KO hearts contained increased lipid/triglyceride content as well as increased fatty acid-binding protein 4, fatty acid synthase, acyl-CoA thioesterase 1, hormone-sensitive lipase, and adipose triglyceride lipase expression compared with control hearts, suggestive of a possible imbalance between fatty acid uptake and oxidation. Insulin-mediated alterations in AKT phosphorylation were observed in ^crSTIM1-KO hearts, consistent with cardiac insulin resistance. Interestingly, we observed abnormal mitochondria and increased lipid accumulation in 12-wk ^crSTIM1-KO hearts, suggesting that these changes may initiate the subsequent metabolic dysfunction. These results demonstrate, for the first time, that cardiomyocyte STIM1 may play a key role in regulating cardiac metabolism.

NEW & NOTEWORTHY Little is known of the physiological role of stromal interaction molecule 1 (STIM1) in the heart. Here, we demonstrate, for the first time, that hearts lacking cardiomyocyte STIM1 exhibit dysregulation of both cardiac glucose and lipid metabolism. Consequently, these results suggest a potentially novel role for STIM1 in regulating cardiac metabolism.

INTRODUCTION

In cardiomyocytes, the most widely studied Ca²⁺ entry pathway is voltage-dependent Ca²⁺ entry across the sarcolemma followed by Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) (6); thus, in the heart, the widely accepted view is that the ER/SR is the predominant source of Ca²⁺ for both excitation-contraction coupling as well as Ca²⁺ signaling. However, in many nonexcitable cell types, store-operated Ca²⁺ entry (SOCE) is the primary mechanism of Ca²⁺ signaling. SOCE occurs in response to a decrease in ER/SR Ca²⁺, which triggers a subsequent influx of extracellular Ca²⁺. In 2005, stromal interaction molecule 1 (STIM1), an ER/SR Ca²⁺ sensor, along with plasma membrane Orai1 channels were identified as essential mediators of the SOCE pathway (48), with STIM1 coupling to Orai1 being required to facilitate Ca²⁺ entry (46). STIM1 expression was first detected in the heart in 2001 (64); however, its function in cardiomyocytes has been understudied. Apart from its potential role in cardiomyocyte hypertrophy (26, 35, 61), little is known about the physiological role of STIM1-mediated SOCE in adult cardiomyocytes. Despite this, we have recently shown that cardiomyocyte-restricted deletion of STIM1 (^crSTIM1-KO; i.e., >90% deletion in isolated cardiomyocytes) results in a dilated cardiomyopathy by 36 wk of age, demonstrating that STIM1 is essential for cardiomyocyte homeostasis (10). Of note, we found that STIM1 deletion was not accompanied by any compensatory changes in either STIM2 or Orai1 (10), supporting the fact that the observed changes are STIM1 specific.

Ca²⁺ signaling is widely recognized as playing a key role in the regulation of cardiac metabolism through its direct activation of Ca²⁺-dependent proteins, kinases, and phosphatases as well as via activation of several metabolic transcription factors and genes. Increases in intracellular Ca²⁺ have been shown to stimulate glycolysis and glucose oxidation (51). Cytoplasmic Ca²⁺ is also known to positively regulate the activities of several mitochondrial dehydrogenases, including pyruvate dehydrogenase (PDH), NAD-isocitrate dehydrogenase (IDH), and oxoglutarate dehydrogenase (OGDH) and the activity of the key regulators of PDH, including pyruvate dehydrogenase kinases (PDKs) (24) and pyruvate dehydrogenase phosphatases (PDPs) (3, 11). In addition, Ca²⁺ regulates a number of protein kinases that are known to play a key role in regulating cardiac energy metabolism, including 5′-AMP-activated protein kinase (AMPK) (31), protein kinase B (PKB/AKT) (20, 67), PKC isoforms (53), Ca²⁺/calmodulin-dependent protein kinase II (CAMKII) (16), phosphoinositide-dependent protein kinase 1 (PDPK1), and glycogen synthase kinase-3β (GSK-3β) (34), as well as protein phosphatases (PPs) such as PP2A, PP2B, PP2C, and PP1 in the heart (56, 68).

The relationship between fatty acid oxidation (FAO) and Ca²⁺ signaling is also well established, as increased Ca²⁺ increases FAO (40) and, interestingly, long-chain fatty acids can directly activate Ca²⁺ channels in cardiomyocytes (25). Ca²⁺ has also been shown to regulate the expression of genes encoding FAO enzymes via transcriptional regulators including the members of the peroxisome proliferator-activated receptor (PPAR) family (21, 27, 32, 66) and PPAR-γ coactivator-1α (PGC-1α) (30). In addition, the activation of both hormone-sensitive lipase (HSL) (62) and lipoprotein lipase (LPL) (63, 70) is Ca²⁺ dependent. Reduced HSL activity has been associated with increased Ca²⁺ levels, lipid formation, and storage (62). Furthermore, Ca²⁺ signaling has been shown to play an essential role in the membrane trafficking of receptors to the plasma membrane either directly or through modulation by AMPK; these include glucose transporters 1 and 4 (GLUT1/4) and fatty acid translocase (CD36/FAT) (2), both of which play key roles in glucose uptake and fatty acid metabolism, respectively.

Despite the known roles of Ca²⁺ in regulating cardiac energy metabolism, the specific Ca²⁺-handling pathways involved are not well characterized. Interestingly, recent studies using fibroblasts from patients with loss-of-function mutations in STIM1 have indicated that SOCE may play a role in regulating lipid metabolism and lipolysis (36), although whether this is the case in the heart remains unknown. Therefore, we used our established ^crSTIM1-KO mouse model to determine the effect of STIM1 deletion on cardiac metabolism. In the present study, we show, for the first time, that lack of cardiomyocyte STIM1 alters substrate utilization in the heart, resulting in reduced glucose oxidation and upregulation of lipogenic proteins associated with lipid accumulation.

MATERIALS AND METHODS

Materials.

Unless otherwise stated, all reagents and chemicals were obtained from Fisher Scientific.

Cardiomyocyte-restricted STIM1-KO mice.

All experimental protocols used in this study were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee and adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). All animals received standard chow and water on an ad libitum basis (with the exception of the insulin experiments, where animals were fasted for 6 h before experimentation), and lighting was maintained on a 12:12-h light-dark cycle. ^crSTIM1-KO mice were generated and their respective genotypes were determined as previously described (10). Littermate STIM1 floxed mice without Cre recombinase were used as controls. Male control and ^crSTIM1-KO mice (20 wk old) were used in the following experiments (with the exception of the 12-wk data shown in Fig. 8), a time point that we have shown to occur before any decline in cardiac function and development of dilated cardiomyopathy (10). All experiments (with the exception of the isolated working perfused heart experiments; data in Fig. 1) were performed in freshly isolated hearts from control and ^crSTIM1-KO mice. Consistent with our previous study (10), there were no significant differences between genotypes in the weight of the mice. Given the known impact of time of day on the regulation of metabolism in the heart (9, 58), all experimental data end points were collected at the same time of day to minimize any impact of circadian regulation (i.e., 10 AM).

Fig. 8.

Effect of cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO) on glucose, lipid, and mitochondrial metabolism at 12 wk. A, top: immunoblots illustrating glucose transporter 4 (GLUT4; left) and acyl-CoA thioesterase 1 (ACOT1; right) expression in freshly isolated hearts from 12-wk control and ^crSTIM1-KO mice. A, bottom: densitometric analysis of the immunoblots shown at the top. n = 6 mice/genotype. B, top: immunoblots illustrating phosphorylated (p-)AMP-activated protein kinase (AMPK)/total (t-)AMPK expression in freshly isolated hearts from 12-wk control and ^crSTIM1-KO mice. B, bottom: densitometric analysis of the immunoblots shown at the top. n = 6 mice/genotype. C, top left: representative transmission electron micrographs taken from left ventricular tissue isolated from 12-wk control (left) and ^crSTIM1-KO (right) mice. C, bottom left: quantification of average mitochondrial length, average number of mitochondria, and number of lipids per 4,400× grid square (average number of measurements per biological sample). Analysis was performed using ImageJ. n = 4 mice/genotype. C, top right: immunoblots illustrating mitofusin 2 (Mfn2) and dynamin-related protein 1 (DRP-1) expression in freshly isolated hearts from 12-wk control and ^crSTIM1-KO mice. C, bottom right: densitometric analysis of the immunoblots shown at the top. n = 6 mice/genotype. *P < 0.05 vs. age-matched control mice (Student’s t-test).

Fig. 1.

Glucose and fatty acid metabolism in ex vivo isolated perfused hearts with cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO hearts). Ex vivo isolated working heart perfusion experiments performed on hearts from control and ^crSTIM1-KO mice. A−D: mean measurements of glucose oxidation (A), net lactate release (B), total glycolysis (glucose oxidation and net lactate release combined; C), and oleate oxidation (D). n = 3–4 mice/genotype. *P < 0.05 versus age-matched control mice (Student’s t-test).

Metabolic analysis of mouse blood plasma.

Blood (500–600 µl) obtained from the vena cavae of unfasted control and ^crSTIM1-KO mice was collected using EDTA-coated blood collection tubes. Blood samples were kept on ice for no longer than 30 min after collection until centrifugation to extract plasma. Collected blood plasma was immediately snap frozen and kept at −80°C until subsequent analysis. Blood plasma was examined for levels of the following circulating humoral factors: glucose, triglyceride, glycerol, free fatty acid, and insulin. Glucose was measured using a Sirrus Stanbio automated analyzer; triglyceride and glycerol were measured using commercially available colorimetric assay kits from Cayman Chemicals, free fatty acid was measured using a colorimetric kit from Cell Biolabs, and insulin was measured using a sensitive rat insulin kit from Millipore.

Isolated working heart perfusion.

Myocardial substrate utilization and contractile function were measured ex vivo using isolated working perfused mouse hearts, as previously described (9, 13, 58, 59). All hearts were perfused in the working mode (nonrecirculating manner) for 30 min with a preload of 12.5 mmHg and an afterload of 50 mmHg. Standard Krebs-Henseleit buffer was supplemented with 8 mM glucose, 1.2 mM oleate conjugated to 3% BSA (fraction V, fatty acid free, dialyzed), 10 μU/ml insulin (basal/fasting concentration), 0.05 mM l-carnitine, and 0.13 mM glycerol. Metabolic flux was assessed through the use of the following distinct radiolabeled tracers: 1) [U-¹⁴C]glucose (0.12 mCi/l) and 2) [9,10-³H]oleate (0.067 mCi/l). Measures of cardiac metabolism (e.g., glucose and oleate utilization) and function (i.e., heart rate, cardiac power, and rate-pressure product) were determined as previously described (9, 13, 58, 59). Data are presented as steady-state values (i.e., values during the last 10 min of the perfusion protocol).

Transmission electron microscopy.

As previously described (10), left ventricular tissue from control and ^crSTIM1-KO hearts was isolated, cut into longitudinal sections, and placed in 0.1 mol/l cacodylate buffer containing 2% glutaraldehyde-paraformaldehyde and then heat fixed for 30 min to cross link proteins and aldehydes. Lipid fixation was performed using 2% osmium tetroxide in 0.1 mol/l cacodylate buffer and 1% aqueous uranyl acetate, and tissue was embedded in Epon. Transmission electron microscopy was performed at the University of Alabama at Birmingham High Resolution Imaging Facility, and longitudinal sections of tissue were assessed at the level of the ER/SR membrane, mitochondria, and contractile filaments. Mitochondrial length measurements were taken as an index of mitochondrial size, as previously described (10).

In vivo insulin administration.

Control and ^crSTIM1-KO mice were fasted for 6 h (from 4 to 10 AM), after which mice received an in vivo injection (via the vena cava) of either saline or insulin (0.167 U/kg, Humulin R U-100, Eli Lilly) as previously described (37). Hearts were excised 5 min after insulin treatment, rinsed in ice-cold PBS, snap frozen, and stored for subsequent analysis.

Immunoblot analysis.

Whole heart tissue was homogenized/sonicated in lysis buffer, as previously described (10). Protein (25 µg) was separated on 7.5%, 10%, or 12% SDS-PAGE gels and subsequently transferred to polyvinylidenedifluoride (PVDF) membranes. PVDF membranes were blocked for 1 h at room temperature with 5% nonfat milk/Tris-buffered saline with Tween 20 (TBST) or 5% BSA/TBST and incubated overnight at 4°C with primary antibodies specific for the following phospho-specific proteins: phospho-AKT (Ser⁴⁷³, no. 9271, Cell Signaling), phospho-AKT (Thr³⁰⁸, no. 9275, Cell Signaling), phospho-AS160 (Thr⁶⁴², no. 8881, Cell Signaling), phospho-acetyl-CoA-carboxylase (ACC; Ser⁷⁹, no. 3661, Cell Signaling), phospho-GSK-3β (Ser⁹, no. 9336, Cell Signaling), phospho-AMPKα (Thr¹⁷², no. 2535, Cell Signaling), and phospho-PDH (Ser²⁹³, AP1062, Millipore). In addition, membranes were incubated with primary antibodies specific for GLUT4 (no. 07-1404, Millipore), GLUT1 (no. 07-1401, Millipore), PDK4 (ab38242, Abcam), PDK2 (sc100534, Santa Cruz Biotechnology), PDH (no. 2784, Cell Signaling), CD36/FAT (no. 5525, Cascade Biosciences), AMPKα (no. 2532, Cell Signaling), liver kinase B1 (LKB1; sc32245, Santa Cruz Biotechnology), GSK-3α/β (sc7291, Santa Cruz Biotechnology), hexokinase II (HK2; AB3279, Millipore), phosphofructokinase 1 (PFK1; sc67028, Santa Cruz Biotechnology), PGC-1α (sc13067, Santa Cruz Biotechnology), PPAR-β/δ (sc7197, Santa Cruz Biotechnology), PPAR-α (ab8934, Abcam), AKT (no. 9272, Cell Signaling), AS160 (no. 07-741, Millipore), ACC (no. 3662, Cell Signaling), fatty acid transporter 1 (FATP1; ab167099, Abcam), fatty acid synthase (FAS; no. 3180, Cell Signaling), fatty acid-binding protein 4 (FABP4; ab66682, Abcam), adipose triglyceride lipase (ATGL; no. 2439, Cell Signaling), HSL (no. 4107, Cell Signaling), acyl-CoA thioesterase 1 (ACOT1; ab133948, Abcam), acyl-coenzyme A:diacylglycerol acyltransferase (DGAT2; sc293211, Santa Cruz Biotechnology), mitofusin 2 (Mfn2; 1:500, Abcam), and dynamin-related protein 1 (DRP-1; 1:1,000, Thermo Fisher Scientific), after which membranes were washed in TBST. Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 2 h at 4°C. Blots were exposed to ECL chemiluminescence detection, and images were obtained using autoradiograph film. Expression was normalized to the loading controls of GAPDH (1:5,000, ab8245, Abcam), β-actin (1:1,000, ab8227, Abcam), or calsequestrin (1:5,000, ab3516, Abcam), where necessary.

Quantification of cardiac triglyceride content.

Cardiac triglyceride content was measured from homogenate extracts from control and ^crSTIM1-KO hearts using the Triglyceride Colorimetric Assay kit (Cayman Chemicals) as per the manufacturer’s protocol.

Quantification of cardiac glycogen content.

Cardiac glycogen content was measured spectrophotometrically in homogenate extracts from control and ^crSTIM1-KO hearts, as previously described (12). The hearts used for these experiments were collected rapidly after euthanasia to ensure minimal postmortem glycogen release.

Statistical analyses.

Statistical significance was calculated using ANOVA followed by a Tukey post hoc test or Student’s t-test using GraphPad Prism 4 and 7, as appropriate, where P values of < 0.05 were deemed statistically significant. All data are presented as means ± SE. ROUT analysis was used to determine and remove any significant outliers. The present experiments were performed using a minimum of 3 mice/experiment, typically between 3 and 8 mice/manipulation. Densitometric analysis of Western blot data and quantification of lipid droplet images were performed using NIH ImageJ software.

RESULTS

Cardiomyocyte-restricted STIM1-KO hearts exhibit impaired glucose metabolism.

To determine the impact of STIM1 deletion on cardiac metabolism, hearts from control and ^crSTIM1-KO mice were isolated and perfused in working mode in the presence of radiolabelled glucose and oleate. We found that hearts from ^crSTIM1-KO mice exhibited a significant reduction in glucose oxidation, net lactate release, and total glycolysis compared with littermate control mice (Fig. 1, A−C). Myocardial glycogen levels did not exhibit differences between genotypes (0.30 ± 0.05 vs. 0.27 ± 0.04 mg/ml for control vs. ^crSTIM1-KO mice). There was also no significant genotype effect for oleate oxidation (Fig. 1D). There were no differences in rate-pressure products (5,492.72 ± 627.28 vs. 4,772.80 ± 465.18 beats·min⁻¹·mmHg for control vs. ^crSTIM1-KO mice) or cardiac power (1.06 ± 0.17 vs. 1.02 ± 0.11 mW for control vs. ^crSTIM1-KO mice) between control and ^crSTIM1-KO hearts, suggesting that alterations in metabolism were not secondary to functional perturbations. In addition, there were no significant differences in either oxygen consumption (45.79 ± 5.29 vs. 50.34 ± 5.61 μmol·min⁻¹·g dry wt⁻¹ for control vs. ^crSTIM1-KO mice) or heart rate (315.0 ± 16.26 vs. 297.1 ± 22.13 beats/min for control vs. ^crSTIM1-KO mice). These heart rates are lower than those measured in vivo in mice; however, they are consistent with other studies of the ex vivo isolated working mouse heart (9, 58).

To identify potential factors contributing to decreased glucose utilization in ^crSTIM1-KO hearts, we examined protein levels of several regulators of glucose uptake, glycolysis, and pyruvate oxidation. Analysis of glucose transporters revealed a marked reduction in whole cell GLUT4 protein expression in ^crSTIM1-KO hearts; however, there were no differences in GLUT1 levels between groups (Fig. 2A). There was no significant difference in the expression of HK2 between control and ^crSTIM1-KO hearts; however, PFK1 expression was significantly increased (Fig. 2B). In addition, we observed a significant increase in the ratio of phospho-PDH (Ser²⁹³) to total PDH (Fig. 2C), concomitant with an increase in PDK4 expression levels in the ^crSTIM1-KO group (Fig. 2D). The increase in phospho-PDH/total PDH was due, in part, to the ~50% decrease in total PDH levels (P < 0.05) in ^crSTIM1-KO hearts. There was also a modest decrease in PDK2 expression in ^crSTIM1-KO versus control hearts (Fig. 2D).

Fig. 2.

Expression of key proteins involved in regulating cardiac glucose metabolism. A, top: immunoblots illustrating glucose transporter (GLUT)1 (left) and GLUT4 (right) expression in freshly isolated hearts from control and cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO). A, bottom: densitometric analysis of the immunoblots shown at the top. B, top: immunoblots illustrating hexokinase 2 (HK2) and phosphofructokinase 1 (PFK1) expression in freshly isolated hearts from control and ^crSTIM1-KO mice. B, bottom: densitometric analysis of the immunoblots shown at the top. Protein expression was normalized to GAPDH expression. C, top: immunoblots illustrating phosphorylated (p-)pyruvate dehydrogenase (PDH)/total (t-)PDH and t-PDH/GAPDH expression in freshly isolated hearts from control and ^crSTIM1-KO mice. C, bottom: densitometric analysis of the immunoblots shown at the top. Protein expression was normalized to GAPDH expression. D, top: immunoblots illustrating pyruvate dehydrogenase kinase (PDK)4 and PDK2 expression in freshly isolated hearts from control and ^crSTIM1-KO mice. D, bottom: densitometric analysis of the immunoblots shown at the top. Protein expression was normalized to GAPDH expression. n = 5/6 control mice and n = 8 ^crSTIM1-KO mice (with the exception of the GLUT1 blot, where n = 5 control mice and n = 5 ^crSTIM1-KO mice). *P < 0.05 vs. age-matched control mice (Student’s t-test).

AMPK is an important regulator of cardiac metabolism, and phosphorylation of AMPK at Thr¹⁷² was markedly reduced in ^crSTIM1-KO hearts (Fig. 3A). AMPK phosphorylation can be regulated via LKB1, which showed a trend to being lower in ^crSTIM1-KO hearts (Fig. 3A). In the random fed state, there were no significant differences in the phosphorylation of AKT at Ser⁴⁷³ or GSK-3β at Ser⁹ between control and ^crSTIM1-KO hearts (Fig. 3B).

Fig. 3.

Effect of cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO) on key kinases that regulate cardiac metabolism. A, top: Immunoblots illustrating phosphorylated (p-)AMP-activated protein kinase (AMPK)/total (t-)AMPK and liver kinase B1 (LKB1) expression in freshly isolated hearts from control and ^crSTIM1-KO mice. A, bottom: densitometric analysis of the immunoblots at the top. n = 5–8 mice/genotype. B, top: immunoblots illustrating p-AKT/t-AKT and p-glycogen synthase kinase-3β (GSK-3β)/t-GSK3β expression in freshly isolated hearts from control and ^crSTIM1-KO mice. B, bottom: densitometric analysis of immunoblots shown at the top. Protein expression was normalized to GAPDH expression. n = 5 control mice and n = 5 ^crSTIM1-KO mice. *P < 0.05 vs. age-matched control mice (Student’s t-test).

Impact of STIM1 deletion on cardiac lipid metabolism.

There were no differences in fatty acid oxidation between groups (Fig. 1D); however, myocardial triglyceride content was significantly increased in ^crSTIM1-KO hearts (Fig. 4A). There was also a significant increase in lipid droplet accumulation in ^crSTIM1-KO hearts (Fig. 4B). Accumulation of lipids can occur due to an imbalance between fatty acid uptake and oxidation and/or altered triglyceride turnover. We therefore examined expression of a number of proteins involved in fatty acid metabolism. Immunoblot analysis revealed no alterations in CD36/FAT or FATP1 levels in ^crSTIM1-KO hearts (Fig. 5A), both of which contribute to fatty acid transport across the plasma membrane. In contrast, however, we observed increases in FAS and FABP4 (Fig. 5B) and a greater than fourfold increase in ACOT1 in ^crSTIM1-KO hearts (Fig. 5C). Both HSL and ATGL levels were also significantly increased in response to loss of STIM1 (Fig. 5D). ACC is another key regulator of fatty acid metabolism. We found that the ratio of phospho-ACC (Ser⁷⁹) to total ACC was increased in ^crSTIM1-KO hearts, which was associated with a small but significant decrease in total ACC (Fig. 5E). Carnitine palmitoyltransferase I (CPT1), a key regulator of fatty acid oxidation, was significantly increased in the hearts of ^crSTIM1-KO mice (Fig. 5F).

Fig. 4.

Effect of cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO) on lipid droplets and triglyceride levels. A: triglyceride content of whole heart lysates from control and ^crSTIM1-KO mice. n = 5 mice/genotype. B, top: representative transmission electron micrographs taken from left ventricular tissue isolated from control (left) and ^crSTIM1-KO mice (right). Arrows depict the lipid droplets. Scale bar = 500 nm. B, bottom: quantification of the average number of lipid droplets located in a 4,400× grid square (average number of measurements per biological sample). n = 3–4 mice/genotype. Analysis was performed using ImageJ. * P < 0.05 vs. age-matched control mice (Student’s t-test).

Fig. 5.

Effect of cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO) on proteins that regulate lipid metabolism. A, top: immunoblots illustrating fatty acid translocase (CD36/FAT) and fatty acid transporter 1 (FATP1) whole cell expression levels in freshly isolated control and ^crSTIM1-KO hearts. A, bottom: densitometric analysis of the immunoblots shown at the top. B, top: Immunoblots illustrating fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4) whole cell expression levels in freshly isolated control and ^crSTIM1-KO hearts. B, bottom: densitometric analysis of the immunoblots shown at the top. C, top: immunoblots illustrating acyl-CoA thioesterase 1 (ACOT1) whole cell expression levels in freshly isolated control and ^crSTIM1-KO hearts. C, bottom: densitometric analysis of the immunoblots shown at the top. D, top: immunoblots illustrating hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) whole cell expression levels in freshly isolated control and ^crSTIM1-KO hearts. D, bottom: densitometric analysis of the immunoblots shown at the top. E, top: immunoblots illustrating phosphorylated (p-)acetyl-CoA-carboxylase (ACC)/total (t-)ACC and t-ACC/calsequestrin (CSQ) in freshly isolated hearts from control and ^crSTIM1-KO mice. E, bottom: densitometric analysis of the immunoblots shown at the top. F, top: immunoblots illustrating whole cell carnitine palmitoyltransferase I (CPT1) expression in freshly isolated hearts from control and ^crSTIM1-KO mice. F, bottom: densitometric analysis of the immunoblots shown at the top. Protein expression was normalized to GAPDH or CSQ expression, where necessary. For FATP1, FAS, FABP4, and ACOT1 blots, n = 6 control mice and n = 7/8 ^crSTIM1-KO mice; for CD36/FAT, ACC, and CPT1 blots, n = 5/6 control mice and n = 5/6 ^crSTIM1-KO mice. *P < 0.05 vs. age-matched control mice (Student’s t-test).

Since the transcription of lipid metabolism genes is regulated by the Ca²⁺-sensitive PPAR family of receptors, we also examined whole cell expression of PPAR-α, PPAR-β/δ, and PGC-1α in control and ^crSTIM1-KO hearts. There were no significant differences in the expression of these proteins between the hearts of both genotypes (Fig. 6).

Fig. 6.

Effects of cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO) on the expression of peroxisome proliferator-activated receptor (PPAR) and PPAR-related proteins. Top: immunoblots illustrating PPAR-γ coactivator-1α (PGC-1α), PPAR-α, PPAR-β/δ and PPAR-γ in freshly isolated hearts from control and ^crSTIM1-KO mice. Bottom: densitometric analysis of the immunoblots shown at the top. Protein expression was normalized to GADPH expression. Data were analyzed by a Student’s t-test. For PGC-1α, PPAR-α, and PPAR-γ blots, n = 6 control mice and n = 8 ^crSTIM1-KO; for the PPAR-β/δ blot, n = 6 control mice and n = 6 ^crSTIM1-KO mice.

Table 1 shows that plasma glucose, free fatty acids, and triglycerides as well as glycerol and insulin were not different between ^crSTIM1-KO and littermate control mice. Thus, the observed changes in cardiac metabolism cannot be attributed to differences in available substrates between genotypes.

Table 1.

Analysis of circulating humoral factors in control and ^crSTIM1-KO plasma samples

	Control	crSTIM1-KO	P Value
Glucose, mg/dl	278 ± 10	279 ± 7	NS
Free fatty acids, meq/l	374.0 ± 25.2	396.2 ± 33.4	NS
Insulin, ng/ml	0.795 ± 0.249	0.748 ± 0.138	NS
Triglyceride, mg/dl	87.63 ± 12.52	121.85 ± 15.24	NS
Glycerol, mg/l	20.04 ± 1.64	21.69 ± 1.78	NS

Values are means ± SE.

^crSTIM1-KO, cardiomyocyte-restricted deletion of stromal interaction molecule 1; NS, not significant.

Impact of STIM1 deletion on cardiac insulin signaling.

Given that insulin signaling is known to directly impact glucose metabolism, the observed changes in ^crSTIM1-KO hearts could be the consequence of cardiac insulin resistance; therefore, control and ^crSTIM1-KO mice were treated with insulin or saline, and hearts were isolated as described above in methods. As anticipated, insulin significantly increased Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ in control and ^crSTIM1-KO hearts; however, the insulin-mediated increase in Ser⁴⁷³ was significantly blunted in the ^crSTIM1-KO group (Fig. 7A). The apparent increase in AS160 phosphorylation at Thr⁶⁴² with insulin in control and ^crSTIM1-KO hearts was not statistically significant using ANOVA (Fig. 7B); however, a Students t-test comparing saline-treated control and saline-treated ^crSTIM-1-KO groups indicated a significant increase in AS160 phosphorylation in the ^crSTIM-1-KO group.

Fig. 7.

Effects of cardiomyocyte-restricted deletion of stromal interaction molecule 1 (^crSTIM1-KO) on insulin-induced activation of Akt and AS160. A, top: immunoblots illustrating Akt phosphorylation in hearts isolated from control and ^crSTIM1-KO mice in response to either 5-min saline or insulin treatment (0.167 U/kg). A, bottom: densitometric analysis of the immunoblots shown at the top. Protein expression was normalized to calsequestrin (CSQ) expression. B, top: immunoblots illustrating AS160 phosphorylation in hearts isolated from control and ^crSTIM1-KO mice in response to either 5-min saline or insulin treatment (0.167 U/kg). B, bottom: densitometric analysis of the immunoblots shown at the top. n = 3–6 hearts/experimental group. $P < 0.05, saline-treated vs. insulin-treated control mice; *P < 0.05 insulin-treated control mice vs. insulin-treated ^crSTIM1-KO mice; #P < 0.05, saline-treated vs. insulin-treated ^crSTIM1-KO mice; &P < 0.05, saline-treated control mice vs. saline-treated ^crSTIM1-KO mice (ANOVA/Tukey post hoc test).

Mitochondrial alterations precede changes in the expression of GLUT4 and ACOT1.

To examine whether the changes in metabolism in ^crSTIM1-KO hearts are a consequence of mitochondrial dysfunction, we examined the expression of metabolic proteins that exhibited the largest changes at 20 wk in addition to mitochondrial parameters and lipid accumulation in 12-wk control and ^crSTIM1-KO hearts. We found that there were no significant differences in the expression of GLUT4 and ACOT1 between the hearts of both genotypes (Fig. 8A). However, we found that phosphorylation of AMPK at Thr¹⁷² was significantly reduced in 12-wk ^crSTIM1-KO versus control hearts (Fig. 8B). Interestingly, we observed significant changes in mitochondrial size and number at 12 wk in ^crSTIM1-KO hearts, which was also associated with lipid accumulation (Fig. 8C) and similar to previously reported findings at 20 wk (10). At 20 wk, we also previously observed significant differences in DRP-1 and Mfn2 expression in ^crSTIM1-KO hearts (10); however, in 12-wk ^crSTIM1-KO hearts, only DRP-1 was significantly increased without changes in Mfn2 expression (Fig. 8C).

DISCUSSION

It is well established that Ca²⁺ plays an important role in regulating cardiac metabolism; however, the specific Ca²⁺ signaling pathways that contribute have been poorly defined. Although STIM1-mediated SOCE is a highly conserved process (48), its role in the heart remains controversial. Consequently, we postulated that STIM1 may contribute to the regulation of cardiac metabolism. We found that lack of cardiomyocyte STIM1 resulted in decreased cardiac glucose metabolism and lipid accumulation. We also observed changes in the expression and phosphorylation of proteins involved in regulating carbohydrate and lipid metabolism and demonstrated that ^crSTIM1-KO hearts exhibit signs of insulin resistance.

Although the decrease in glucose metabolism in ^crSTIM1-KO hearts could lead to an energy deficit, it is unlikely that this is the case under these perfusion conditions since contractile function was similar between control and ^crSTIM1-KO hearts. The decrease in glucose metabolism is likely compensated by an increase in endogenous substrate utilization, most likely triglycerides; however, this was not evaluated in these studies. Changes in the relative contributions of glucose and fatty acids to oxidative metabolism can, in principle, lead to alterations in oxygen consumption; however, in these experiments, hearts were perfused with 1.2 mM oleate, and, consequently, fatty acids provided the primary substrate for oxidative energy metabolism. Thus, the reduction in glucose oxidation in ^crSTIM1-KO hearts would have minimal effect on overall oxygen consumption, accounting for the similarities in oxygen consumption between the groups. Myocardial substrate utilization is highly dependent on the specific carbon substrates available, the presence of hormones such as insulin, and workload. Therefore, future studies will be needed to assess the consequences of STIM1 deletion on the utilization of other substrates such as lactate, ketone bodies, and amino acids, in addition to evaluating the metabolic responses to physiological stimuli such as increased workload.

The decrease in glucose metabolism in ^crSTIM1-KO hearts was associated with an ~75% reduction in GLUT4 expression with no change in GLUT1 expression (Fig. 2A). GLUT4 has a higher affinity for glucose than GLUT1 and is responsible for the majority of glucose uptake in the heart (52); therefore, it is likely that the lower GLUT4 protein levels in ^crSTIM1-KO hearts contributes, at least in part, to the reduction in glycolysis. Since GLUT4 and GLUT1 are regulated by different transcription factors (18, 49), the selective decrease in GLUT4 expression suggests that GLUT4 but not GLUT1 transcription is STIM1 dependent.

As PFK1 is the first rate limiting step in glycolysis, the twofold increase in PFK1 could be an adaptive response to decreased glucose availability. The activity of PFK1 is regulated allosterically by a number of factors; however, its transcriptional regulation is less well understood. The heart also expresses PFK2, a bifunctional enzyme that controls glycolytic flux through regulation of fructose-2,6-bisphosphate levels and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, both of which could contribute to the regulation of glycolysis (8). The effects of STIM1 deletion on these regulatory proteins were not determined. We also found a decrease in PDH phosphorylation concomitant with an increase in PDK4 expression, which would also contribute to lower glucose oxidation. Since PDK4 expression is known to be regulated in part by lipid availability, it is possible that the increase in PDK4 is secondary to the increase in lipid accumulation (Fig. 4b) rather than a direct response to the loss of STIM1.

Similar to reports in other systems (7, 31, 38, 54), lack of STIM1 resulted in a marked decrease in AMPK phosphorylation and also a trend toward educed LKB1 expression. While LKB1 is the primary upstream kinase for AMPK, AMPK can also be phosphorylated via Ca²⁺/calmodulin-dependent kinase kinase-β, which is activated by an increase in cytosolic Ca²⁺. It should also be noted that AMPK phosphorylation is also regulated by PP2A, which is also activated by Ca²⁺; thus, an increase in PP2A activity could also reduce AMPK phosphorylation (41). However, owing to the trend in decreased LKB1 levels, PP2A-mediated regulation seems unlikely to be a factor here. Consequently, the reduction in AMPK phosphorylation may be a direct result of an absence of STIM1 and decreased AMPK activation, which could contribute to the reduction in glucose metabolism. In contrast to other reports (5, 33, 50), loss of STIM1 in the heart had no significant changes in basal AKT or GSK-3β phosphorylation.

The accumulation of triglycerides and lipid droplets (Fig. 4) in ^crSTIM1-KO hearts is consistent with reports showing that STIM1 regulates lipogenesis and lipid storage in Drosophila (4) and that SOCE controls transcriptional regulation of lipid metabolism through modulation of lipolysis and FAO in fibroblasts (36). The fatty acid transporters CD36/FAT and FATP1 were not significantly different between groups, suggesting that increased fatty acid transport was not a factor contributing to increased lipid accumulation (Fig. 5a). However, since translocation of CD36/FAT is both insulin (60) and Ca²⁺ dependent (2) and since CD36 has been implicated in SOCE (29), it is possible this could be altered in ^crSTIM1-KO hearts and warrants further investigation. The lipases HSL and ATGL were both increased in ^crSTIM1-KO groups (Fig. 5D), which is in contrast with the report by Maus et al. (36), who found that both were lower in SOCE-deficient cells. Increased ATGL expression has also been observed in hearts from diabetic mice, where triglycerides are also elevated and cardiac-specific ATGL overexpression was found to ameliorate the effects of diabetes (44, 45, 55). It was concluded that in diabetes, the increase in cardiac ATGL expression was an adaptive yet insufficient response to the increase in triglycerides. It is likely that this is also the reason for increased HSL and ATGL in ^crSTIM1-KO hearts.

The expression of a number of other proteins involved in lipid metabolism in the heart were also increased in response to STIM1 deletion; these included FAS, FABP4, ACOT1, phospho-ACC, and CPT1 (Fig. 5). While the heart is not a lipogenic organ, it does contain FAS, and here we also showed a twofold increase in FAS levels in ^crSTIM1-KO hearts (Fig. 5B). Cardiac levels of FAS are increased in heart failure and are also associated with increased lipid accumulation (1) and may be linked to Ca²⁺ signaling in the heart via activation of CaMKII (47). The role of FABP4 in cardiomyocytes remains unclear, but it has been reported to play a role in transporting fatty acids inside the cell, and it has also been suggested that FABP4 might scavenge reactive lipids (23). ACOT1, a cytosolic enzyme responsible for the hydrolysis of fatty acyl-CoA thioester leading to CoA and free fatty acid (57), has been shown to increase in the heart under conditions of increased availability of fatty acids and elevated triglycerides (14, 42, 69) and in response to increased dietary fat (14, 65). The increases in FAS, FABP4, and ACOT1 expression are all consistent with dysregulation of lipid metabolism in ^crSTIM1-KO hearts.

Consistent with an overall upregulation of fatty acid metabolism pathways, we also found that CPT1 expression was increased in ^crSTIM1-KO hearts. This may suggest that alterations in the malonyl-CoA decarboxylase axis may contribute to metabolic changes in ^crSTIM1-KO hearts; however, since FAO was unchanged between genotypes, it is unlikely that it has a major contribution. The increased ACC phosphorylation in ^crSTIM1-KO hearts (Fig. 5E), which is indicative of increased mitochondrial β-oxidation, is paradoxical as there was no increase in exogenous FAO in ^crSTIM1-KO hearts. The increase in ACC phosphorylation is also inconsistent with the decreased AMPK phosphorylation in ^crSTIM1-KO hearts. However, it has been reported that, in the heart, ACC may be regulated by other kinases, including PKA (15). Lipid accumulation in ^crSTIM1-KO hearts could also be due to defects in lipophagy, thereby contributing to lipooxidative stress and lipotoxicity, which could be a contributing factor in the cardiomyopathy observed in these mice (10). However, the role of Ca²⁺ in regulating autophagy/lipophagy is complex, and further studies are needed to determine whether STIM1 contributes to the regulation of these processes in cardiomyocytes. It is somewhat surprising that there were no differences between groups in PGC-1α and PPAR expression given their essential role as transcriptional regulators of lipid metabolism.

Impaired insulin signaling could also be a factor in decreased glucose metabolism in ^crSTIM1-KO hearts and could also occur in response to increased lipid accumulation. In support of this, we found that while insulin-stimulated phosphorylation of Thr³⁰⁸ on Akt was similar between control and ^crSTIM1-KO hearts, the increase in phosphorylation of Ser⁴⁷³, which is required for full activation of Akt and subsequent GLUT4 translocation, was significantly blunted in ^crSTIM1-KO hearts. This finding is consistent with a recent report (5) demonstrating that cardiomyocyte STIM1 knockdown significantly decreased the mammalian target of rapamycin complex 2-Akt signaling axis. However, in that study, there was no examination of insulin signaling. The selective decrease in Ser⁴⁷³ phosphorylation suggests a defect in phosphatidylinositol 3-kinase (PI3K)-mTORC2 signaling but not the PI3K-PDK1 pathway. Surprisingly, we found no significant changes in insulin-mediated activation of AS160 in ^crSTIM1-KO hearts, as determined by ANOVA (Fig. 7B). Interestingly, analysis of these groups using Student’s t-test indicated that the increases in basal AS160 phosphorylation between control and ^crSTIM1-KO was significant, although the implications of this are unclear. Measurements of insulin-mediated glucose uptake would provide further insights. Nevertheless, these observations raise the possibility that STIM1 contributes to insulin signaling in cardiomyocytes; however, further studies are clearly needed to better understand this relationship.

In our earlier study (10), we found that in 20-wk ^crSTIM1-KO hearts, there were alterations in mitochondrial morphology, suggestive of an increase in mitochondrial fission. This raises the question as to whether the metabolic changes seen could occur secondary to the mitochondrial changes; therefore, we looked at a number of parameters in 12-wk crSTIM1-KO hearts. There were no significant changes in GLUT4 or ACOT1 (Fig. 8A), both of which exhibited large differences between genotypes at 20 wk of age. Interestingly, however, AMPK phosphorylation (Fig. 8B) was already significantly lower in the ^crSTIM1-KO group. In addition, changes in mitochondrial morphology at 12 wk were similar to those seen at 20 wk, and there was also a significant increase in lipid droplets at 12 wk (Fig. 8C). We used mitochondrial length as an index of mitochondrial size in the present study and our previous study (10); however, quantification of mitochondrial perimeter may yield additional insights. Although additional studies are clearly required, these data suggest that alterations in mitochondrial function and resulting lipid accumulation could precede and lead to the impaired glucose metabolism and insulin signaling observed at 20 wk of age. It also seems likely that the progressive mitochondrial phenotype coupled with changes in metabolism contribute to the cardiomyopathy associated with these mice later in life.

Currently, the only accepted role of STIM1 in the heart is in the regulation of cardiac hypertrophy (5, 35, 39); however, here, we have shown that loss of STIM1 in the heart is associated with a reduction in glucose metabolism, impaired insulin signaling, and dysregulation of lipid metabolism. STIM1 mutations and variants observed in humans are associated with severe immunodeficiency (43); consequently, the role of STIM1 in the cardiovascular system is typically overlooked. Interestingly, the metabolic remodelling seen here is remarkably similar to that seen in the heart in response to diabetes, which leads to decreased STIM1 protein levels (17, 28). The specific mechanisms linking STIM1 to metabolic regulation in the heart clearly warrant further investigation. Nevertheless, our findings are consistent with recent reports of STIM1-dependent alterations in lipid metabolism in other cells (4, 19, 22, 36) and suggest, for the first time, that STIM1 could be a previously unrecognized regulator of cardiac metabolism.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-110366 (to J. C. Chatham), HL-106199, HL-074259, and HL-123574 (to M. E. Young), HL-122975 (to J. C. Chatham and M. E. Young) and HL-133011 (to A. R. Wende), a William W Featheringill Postdoctoral Fellowship (University of Alabama at Birmingham Comprehensive Cardiovascular Center; to H. E. Collins), American Heart Association Southeast Affiliate Postdoctoral Fellowship 15POST25260004 (to H. E. Collins), and American Diabetes Association Postdoctoral Fellowship 1-16-PDF-024 (to H. E. Collins). This work was also supported by a University of Alabama at Birmingham AMC21 reload multi-investigator grant (to J. C. Chatham, M. E. Young, and A. R. Wende).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

H.E.C., B.M.P., and L.Z. performed experiments; H.E.C. and S.H.L. analyzed data; H.E.C., S.H.L., A.R.W., M.E.Y., and J.C.C. interpreted results of experiments; H.E.C. prepared figures; H.E.C. drafted manuscript; H.E.C., S.H.L., A.R.W., M.E.Y., and J.C.C. edited and revised manuscript; H.E.C., B.M.P., L.Z., S.H.L., A.R.W., M.E.Y., and J.C.C. approved final version of manuscript.