Stromal interaction molecule 1 is essential for normal cardiac homeostasis through modulation of ER and mitochondrial function

Abstract

The endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) has been implicated as a key mediator of store-dependent and store-independent Ca(2+) entry pathways and maintenance of ER structure. STIM1 is present in embryonic, neonatal, and adult cardiomyocytes and has been strongly implicated in hypertrophic signaling; however, the physiological role of STIM1 in the adult heart remains unknown. We, therefore, developed a novel cardiomyocyte-restricted STIM1 knockout ((cr)STIM1-KO) mouse. In cardiomyocytes isolated from (cr)STIM1-KO mice, STIM1 expression was reduced by ∼92% with no change in the expression of related store-operated Ca(2+) entry proteins, STIM2, and Orai1. Immunoblot analyses revealed that (cr)STIM1-KO hearts exhibited increased ER stress from 12 wk, as indicated by increased levels of the transcription factor C/EBP homologous protein (CHOP), one of the terminal markers of ER stress. Transmission electron microscopy revealed ER dilatation, mitochondrial disorganization, and increased numbers of smaller mitochondria in (cr)STIM1-KO hearts, which was associated with increased mitochondrial fission. Using serial echocardiography and histological analyses, we observed a progressive decline in cardiac function in (cr)STIM1-KO mice, starting at 20 wk of age, which was associated with marked left ventricular dilatation by 36 wk. In addition, we observed the presence of an inflammatory infiltrate and evidence of cardiac fibrosis from 20 wk in (cr)STIM1-KO mice, which progressively worsened by 36 wk. These data demonstrate for the first time that STIM1 plays an essential role in normal cardiac function in the adult heart, which may be important for the regulation of ER and mitochondrial function.

Keywords: ER stress; STIM1; cardiomyocytes; mitochondria; store-operated Ca2+ entry.

Fig. 1.

Stromal interaction molecule 1 (STIM1) protein expression in whole heart tissue (A) and isolated cardiomyocytes (B) from adult male control and STIM1 knockout (^crSTIM1-KO) mice. C: STIM2 and Orai1 expression in cardiomyocytes from adult male control and ^crSTIM1-KO mice. D: calsequestrin protein expression in 20-wk cardiomyocytes isolated from adult male control and ^crSTIM1-KO mice. Control, n = 4; ^crSTIM1-KO, n = 4. Representative immunoblots (A–D: left) and mean densitometric data (A–D: right) from 4 individual experiments normalized to GAPDH (A) and β-actin (B–D) **P < 0.01 and *P < 0.05 vs. aged-matched control; Student's t-test. ns, Not significant. E: heat maps from unbiased gene expression array data were performed using the Illumina mouse WG-6 2.0 expression bead chip kit on hearts from ^crSTIM1-KO (n = 6) and control (n = 6) mice of 12 wk of age. Full gene name corresponds to *IGHV1S35_M12376_Ig_heavy_variable_1S35_13 and ^#IGHV1S120_AF025443_Ig_heavy_variable_1S120_8.

Fig. 2.

Immunoblots (A) and densitometry analysis (B) of endoplasmic reticulum (ER) stress proteins in 12-, 20-, and 36-wk adult control (12, 20, and 36 wk, n = 6) and ^crSTIM1-KO heart (12 and 20 wk, n = 6; 36 wk, n =7) are shown. **P < 0.01 and ***P < 0.001 vs. age-matched control (ANOVA/Bonferroni post hoc test). IRE1α, inositol-requiring enzyme 1α; CHOP, C/EBP homologous protein; BiP (GRP78), binding immunoglobulin protein (78 kDa glucose-regulated protein); PDI, protein disulfide isomerase; WT, wild-type; KO, knockout. C: immunoblot (left) and densitometry (right) of ER stress proteins in 20-wk cardiomyocytes isolated from adult control (n = 4) and ^crSTIM1-KO (n = 4) hearts.

Fig. 3.

Transmission electron micrograph images taken from left ventricular (LV) tissue isolated from 20-wk-old control (A) and ^crSTIM1-KO (B) mice. Scale bar equals 500 nm, and red arrows highlight ER dilatation. C: mean measurements of mitochondrial length and number of mitochondria per 750 μm² grid taken at 1,100× magnification from 20-wk ^crSTIM1-KO and control mice. Measurements were taken using ImageJ from at least 10 different images per 3 different hearts. **P < 0.001 and ***P < 0.0001, Student's t-test. D: Western blot and densitometry analysis of mitochondrial fusion proteins Mfn1 and Mfn2 and the mitochondrial fission protein dynamin-related protein 1 (DRP-1) in 20-wk control and ^crSTIM1-KO heart. *P < 0.05 vs. control (Student's t-test). Control, n = 6; ^crSTIM1-KO, n = 6.

Fig. 4.

Serial echocardiography was performed from 12 to 36 wk of age for quantification of cardiac function and histology in control and ^crSTIM1-KO mice. A: representative M-mode echocardiography traces taken from 12 and 36 wk control and ^crSTIM1-KO. B: ejection fraction (in %) as a function of age. C and D: diastolic LV volume (C) and LV posterior wall thickness at diastole (LVPWd) at 20 and 36 wk of age. *P < 0.05 and **P < 0.001 vs. age-matched control (ANOVA/Bonferroni post hoc test). E: transverse whole heart images of hematoxylin and eosin (H&E) staining from 20- and 36-wk-old control and ^crSTIM1-KO, taken at 1.25×. Scale bar represents 2 mm. F: H&E, Masson trichrome, and picric acid Sirius red stained sections of hearts from 20- and 36-wk-old control and ^crSTIM1-KO. Images taken at 40×, and scale bar equals 50 μm. Control (20 wk, n = 6; and 36 wk, n = 7) ^crSTIM1-KO (20 wk, n = 14; and 36 wk, n = 8). G: Kaplan-Meier survival curve of ^crSTIM1-KO vs. age-matched control mice (χ², P = 0.0001, n = 20 per genotype).