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Previous Article | Next Article 
Molecular and Cellular Biology, September 2004, p. 8007-8017, Vol. 24, No. 18
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.18.8007-8017.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Molecular Embryology,1 Department of Pharmacology,2 Department of Anesthesiology, Chiba University Graduate School of Medicine, Chuo-ku, Chiba City, Chiba,4 Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo,3 Laboratory for Developmental Genetics, RIKEN Research Center for Allergy and Immunology, Tsurumi, Yokohama,5 Center for Animal Resources and Development, Kumamoto University, Kumamoto City, Kumamoto, Japan6
Received 19 February 2004/ Returned for modification 29 March 2004/ Accepted 25 June 2004
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ABSTRACT |
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INTRODUCTION |
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Misfolded proteins had been believed to remain in the ER, but recent genetic analyses in Saccharomyces cerevisiae have indicated that the unfolded protein response involves the whole secretory pathway (56) and that some misfolded proteins require transport between the ER and the Golgi complex for ER-associated degradation (17, 41, 53, 58). In addition, certain misfolded proteins in mammalian cells have also been reported to exit the ER and recycle between the ER and post-ER compartments, associating with ER chaperones. The KDEL receptor mediates this retrieval, suggesting that the secretion of misfolded proteins from the ER and their retrieval may contribute to ER quality control (12, 62).
The KDEL receptor has been identified as a retrieval receptor for luminal ER chaperones that have a carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) sequence (28, 29, 36). These ER chaperones, when secreted from the ER, are recognized by the KDEL receptor in post-ER compartments, and then both the chaperones and the receptor are sorted into coat protein complex (COP) I vesicles for retrograde transport to the ER. Besides being a retriever, the KDEL receptor has been recognized as a regulator of membrane trafficking in the early secretory pathway. The deletion of ER retention-defective complementation group 2 (ERD2), the yeast homologue of the KDEL receptor, causes the accumulation of membrane structure and disturbs transport through the Golgi complex (49). The activation of the KDEL receptor by the ligand accelerates the formation of COPI-coated vesicles (2, 32).
The KDEL receptor has been studied extensively in yeast and mammalian cells; however, its function and the outcomes of its dysfunction in animals and humans in vivo are totally unknown. Impairment of the KDEL receptor is expected to perturb ER quality control, which may cause diseases associated with ER stress. We took advantage of previous studies on the KDEL receptor and made stable cell lines expressing a transport-mutant human KDEL receptor (55). We found that the mutant KDEL receptor disturbed the circulation of misfolded proteins between the ER and the Golgi complex, resulting in accumulation of misfolded proteins in the ER. As a result, these cells became sensitive to ER stress. This finding prompted us to make transgenic mice expressing a mutant KDEL receptor possibly sensitive to ER stress. The mutant mice developed dilated cardiomyopathy and heart failure. We discuss the possible role of ER stress on the pathogenesis of dilated cardiomyopathy.
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MATERIALS AND METHODS |
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(62), a mouse monoclonal antibody against Golgi p58, a mouse monoclonal antibody against 
-tubulin (Sigma Chemical, St. Louis, Mo.), mouse monoclonal antibody SPA-827 against immunoglobulin heavy chain-binding protein (BiP) (KDEL sequence), mouse monoclonal antibody SPA-810 against HSP72 (Stressgen, Victoria, Canada), mouse monoclonal antibody AF8 against calnexin (kindly provided by M. Brenner, Boston, Mass.) (18), a rabbit antiserum against C/EBP homologous protein (CHOP), a goat polyclonal antiserum against troponin T, a rabbit antiserum against X-box binding protein 1 (XBP-1), and a rabbit antiserum against ubiquitin (Santa Cruz Biotechnology, Santa Cruz, Calif.). The following reagents were used: endoglycosidase H (New England Biolabs, Beverly, Mass.), dithiothreitol (Sigma Chemical), and tunicamycin (Nacali Tasque, Kyoto, Japan). Plasmids and transfection. The Myc-tagged wild-type human KDEL receptor 1 and a transport mutant, D193N (55), were gifts from H. R. B. Pelham (Cambridge, United Kingdom). CD8E19 and CDE193S were kindly provided by M. R. Jackson (San Diego, Calif.). Transfection was performed by the calcium phosphate method (62). To generate a stable cell line, Myc-tagged D193N cDNA was cotransfected with a construct containing a neomycin resistance gene into HeLa cells by the calcium phosphate method. Cells were selected in complete medium containing 0.5 mg of geneticin (Invitrogen, Carlsbad, Calif.) ml–1. Stable transfectants were screened by immunofluorescence microscopy. The mutant KDEL receptor stable cell line expressed as much Myc-tagged protein as the wild-type KDEL receptor stable cell line (63).
Immunofluorescence microscopy and biochemistry. Immunofluorescence microscopy, immunoprecipitation, RNA isolation, and RNA blot analysis were conducted as previously described (62, 63).
Metabolic labeling and chase experiment. Cells were transiently transfected with CD8E19 or CD8E193S. Forty hours later, the cells were incubated in labeling medium (Dulbecco's modified Eagle's medium without methionine supplemented with 2% fetal bovine serum, 2 mM glutamine, 50 µg of streptomycin ml–1 and 50 U of penicillin G ml–1) for 20 min at 37°C, labeled with [35S]methionine at 250 µCi ml–1 for 10 min, washed, and then chased in complete medium for 0 to 60 min. The cells were collected, lysed, and subjected to immunoprecipitation with an anti-CD8 monoclonal antibody (OKT8). The immunoprecipitates were washed, boiled in sample buffer, and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. The gels were then analyzed with BAS 2500 and Image Gauge software (Fuji Photo Film Co. Ltd., Tokyo, Japan).
Sucrose gradient experiment.
The cells were washed twice with ice-cold phosphate-buffered saline and once with homogenization buffer (10 mM triethanolamine, 1 mM EDTA, 250 mM sucrose), removed from the dishes, and pelleted by centrifugation for 10 min at 800 x g. The pellet was resuspended in 1 ml of homogenization buffer containing 10 µg of aprotinin, 10 µg of leupeptin, and 30 µg of N-acetyl-L-leucinal-L-lecinal-L-norleucinal (ALLN; Sigma Chemical Co.) ml–1 and homogenized by passing it four times through a ball-bearing homogenizer (EMBL, Heidelberg, Germany). A postnuclear supernatant was obtained by centrifugation for 10 min at 800 x g at 4°C. The postnuclear supernatant was loaded on a continuous sucrose gradient (20 to 50%) prepared with a Gradient Master (Biocomp Instruments, Inc., Fredericton, Canada). The postnuclear supernatant on the gradient was centrifuged for 2 h at 40,000 rpm with a Beckman SW41Ti rotor. Twelve fractions were obtained from each sample. One twentieth of each fraction was separated by SDS-PAGE under reducing conditions and then transferred onto polyvinylidene fluoride membranes. The distribution of TCR, calnexin, and Golgi p58 was determined by Western blotting.
Generation of transgenic mice. All animal experimental procedures were in accordance with a protocol approved by the Institutional Animal Care Committee of Chiba University, Chiba, Japan. We made an inducible expression vector of the wild-type and mutant KDEL receptors with the Cre-loxP-based recombination system (see Fig. 2A). The Myc-tagged wild-type and mutant (D193N) KDEL receptor cDNAs were cloned into the transgenic construct after the chicken beta-actin (CAG) promoter-loxP-CAT-poly(A)-loxP sites. The construct was linearized and injected into the male pronucleus of fertilized single-cell embryos of C57BL/6 mice to produce transgenic mice. The transgene-positive mice were mated with mice generating Cre, driven by a CAG promoter. The offspring's tail DNA was screened by PCR with oligonucleotides 3 (5'-CTGCTAACCATGTTCATGCC-3') and 4 (5'-AGGAATCGGAAGAGATTCAT-3'), which were within the CAG promoter and the first exon of human KDEL receptor 1, respectively. Recombination occurred in all mice that had both the transgene and the Cre gene, and this was passed on to the offspring regardless of the Cre gene (see Fig. 2B and C).
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Ultrastructural analysis. The mice were sacrificed by cervical dislocation under pentobarbital anesthesia, and the hearts were removed. The left ventricle was dissected, sliced, and immersion fixed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate-buffered saline. Thin sections of the slice were osmicated, dehydrated, and embedded in epoxy resin. Ultrathin sections were prepared, stained with lead citrate and uranyl acetate, and observed under a Hitachi (Tokyo, Japan) H-600 transmission electron microscope.
Western blotting of heart lysates. The hearts were removed from the mice and homogenized in a buffer containing 0.4% Nonidet P-40, 0.2% N-lauroylsarcosine, 30 mM Tris-HCl pH 8.0, 1 mM EDTA, 10 µg of aprotinin ml–1, 10 µg of leupeptin ml–1, and 30 µg of ALLN ml–1. The lysates were boiled in sample buffer and separated by SDS-PAGE under reducing conditions. For Western blotting, gels were transferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore Corp., Bedford, Mass.), blocked, incubated with primary antibody followed by peroxidase-conjugated donkey anti-goat, -mouse, or -rabbit immunoglobulin G antibody, and developed by chemiluminescence (ECL kit; Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Imaging was obtained by LAS1000 and Image Gauge software (Fuji Photo Film Co. Ltd.).
Myocyte isolation and culture. Primary cardiac myocyte cultures were prepared as previously described (64) with modifications. Each ventricle from 0-day-old mice was treated. The ventricles were first perfused with nominally Ca2+-free solution for 5 min; this was immediately followed by 40 min of recirculating perfusion with the same solution containing 0.9 mg of class II collagenase (Worthington Biochemicals, Lakewood, N.J.) ml–1. Purified yachts from each mouse were plated on gelatin-coated culture dishes in minimal essential medium (Sigma Chemical) with 5% iron-fortified bovine calf serum (JRH Biosciences, Lenexa, Kans.) and maintained for 48 h.
Electrophysiology. Mice aged 19 to 24 weeks were used for the functional experiments. Single cells were isolated by conventional enzymatic digestion, and whole-cell membrane currents were recorded by the patch-clamp method, as previously described (52). Recordings of the L-type Ca2+ current were performed at 36.0°C with a glass patch pipette filled with 120 mM CsOH, 110 mM L-aspartame, 20 mM CsCl, 1 mM MgCl2, 5 K2-ATP, 1.42 mM CaCl2, 10 mM EGTA, and 5 mM HEPES (pH 7.4). The external solution contained 143 mM NaCl, 5.4 mM CsCl, 0.33 mM NaH2PO4, 0.5 mM MgCl2, 1.8 mM CaCl2, 5.5 mM glucose, and 5 mM HEPES (pH 7.4). Membrane currents were elicited by 300-ms depolarizing pulses from a holding potential of –40 mV, delivered at 0.1 Hz. Membrane capacitance was measured and used as an index of cell surface area.
Mechanical function study. The heart was quickly removed from mice anesthetized with urethane (1.5 mg g–1, intraperitoneally), and the left ventricular pressure of the Langendorff-perfused hearts was continuously measured as previously described (52).
Reverse transcription-PCR. The nucleotide sequences of the primers used in this study were follows: mouse atrial natriuretic peptide, 5'-CCGTGACAAGCTTTGCCGAA-3' and 5'-GATCTGTGTTGGACACCGCA-3'; mouse brain natriuretic peptide, 5'-CAAGATGCAGGTGAGCACTG-3' and 5'-GGGGCCTCTAAGGGTAGCAT-3'; and mouse glyceraldehyde-3-phosphate dehydrogenase, 5'-ATGGGGTGAGGCCGGTGCTG-3' and 5'-CTTGATGTCATCATACTTGG-3'.
Aortic constriction of rats. Sprague-Dawley male rats weighing 290 to 300 g were anesthetized with 2% isoflurane. The right carotid artery was cannulated with a polyethylene catheter connected to a pressure transducer, and the mean carotid arterial pressures were measured. Under sterile conditions, the abdominal aorta was exposed and constricted at the suprarenal level with a 25-gauge needle to establish the diameter of ligature as previously described (34). Thereafter, the abdominal incision was closed. A similar mock procedure was performed except for the constriction.
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RESULTS |
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HeLa cells and cells stably expressing either wild-type or mutant Myc-tagged KDEL receptor (63) were transiently transfected with CD8E19 or CD8E193S and subjected to metabolic pulse-chase labeling. CD8E19 acquired N-glycosylation immediately (Fig. 1A, i) and O-glycosylation gradually (Fig. 1A, ii) (21). The fraction acquiring O-glycosylation may represent the circulating fraction of the CD8E19 that reaches the cis-Golgi. Interestingly, the O-glycosylation of CD8E19 in the cells stably expressing the mutant KDEL receptor was suppressed in comparison to that in HeLa cells and in the wild-type KDEL receptor stable cells (Fig. 1A, lower panel). On the other hand, CD8E193S was transported through the medial Golgi complex and acquired a complex N-linked glycosylation to a similar extent in all cell types (Fig. 1A, iii).
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We previously showed that the subunit of the T-lymphocyte antigen receptor complex (TCR) was circulating as a misfolded protein between the ER and the Golgi complex when it expressed itself in a cell without other subunits (62). To evaluate the effects of the mutant KDEL receptor on the circulation of misfolded proteins, we transfected TCR into stable cells and examined its subcellular localization by sucrose gradient experiments. Calnexin is an integral membrane protein and one of the ER chaperones located in the ER (18). Thus, we used it in the sucrose gradient experiment as a marker for the ER fraction. In the wild-type KDEL receptor stable cells, TCR was distributed in both the ER and post-ER compartments (Fig. 1B, upper panel). Even the TCR in the ER acquired a complex Golgi glycosylation that was resistant to endoglycosidase H digestion (Fig. 1B, lower panel, p43), suggesting that the TCR was circulating between the ER and the Golgi complex. On the other hand, most of the TCR in the mutant KDEL receptor stable cells was located in the ER as a core-glycosylated ER form and remained sensitive to digestion (Fig. 1B, lower panel, p38).
These findings show that the mutant KDEL receptor might disturb the circulation of misfolded proteins in the early secretory pathway. As a result, the mutant KDEL receptor stable cells became sensitive to ER stress when treated with an agent such as dithiothreitol that perturbed protein folding and caused the accumulation of misfolded proteins in the ER (Fig. 1C); this suggests that the secretion of misfolded proteins from the ER and their retrieval may contribute to ER quality control and that impairment of the KDEL receptor may sensitize cells to ER stress.
Generation of transgenic mice. In order to investigate the function of the KDEL receptor in vivo, we created transgenic mice expressing the human wild-type and transport mutant KDEL receptors with the Cre-loxP-based recombination system (48). We established two lines of the wild-type and two lines of the mutant KDEL receptor transgenic mice after crossing them with CAG promoter-Cre transgenic mice (Fig. 2A). The recombinant transgene was passed on to the offspring and expressed its products without the Cre gene. The Northern blot revealed that the expression level of the mutant KDEL receptor was as high as that of the endogenous one (Fig. 2B). Western blotting confirmed that the mutant KDEL receptor was expressed ubiquitously (Fig. 2C).
Transgenic mice expressing the mutant KDEL receptor developed dilated cardiomyopathy. The transgenic mice expressing the mutant KDEL receptor seemed to grow normally until early adulthood. In the course of the study, we found that the transport mutant KDEL receptor transgenic mice of both lines died sporadically after attaining the age of more than 14 weeks. They appeared dyspneic, lethargic, and motionless. These mice developed peripheral edema, ascites, and cardiomegaly and seemed to die due to heart failure (Fig. 3A).
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Ultrastructural analyses revealed the accumulation of protein aggregates in the expanded sarcoplasmic reticulum of the mutant cardiomyocytes. In order to gain insight into the mechanism responsible for dilated cardiomyopathy in the mutant KDEL receptor transgenic mice, we examined the ultrastructure of the mutant cardiomyocytes. The contractile apparatus appeared intact, i.e., the arrangement of myofibril structures and the banding of myofilaments were little different in the myocardia of control C57BL/6 mice (Fig. 4A) and mutant transgenic mice (Fig. 4B). The arrangement of mitochondria was also intact in the transgenic mice. On the other hand, the proliferation of sarcoplasmic reticulum was prominent around the transverse tubule area, and the transverse tubule structure was narrowed significantly in mutant transgenic mice with (Fig. 4C) and without (Fig. 4D) apparent cardiomegaly, in comparison to the transverse tubules and surrounding the sarcoplasmic reticulum in the control (Fig. 4A and E). Furthermore, an aggregation of degenerate membrane structures was found among the proliferated sarcoplasmic reticulums (Fig. 4F), and lamellated or further fused membrane structures were found in the transverse tubule area in the mutant mice (Fig. 4D). We observed electron-dense materials, possibly protein aggregates, in the expanded sarcoplasmic reticulum (Fig. 4G and H). These aggregates were associated with polyribosomes, suggesting that they were part of the ER network. On the other hand, the ultrastructure of the cardiomyocytes from the wild-type KDEL receptor transgenic mice appeared normal.
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Mutant KDEL receptor transgenic mice revealed defective L-type Ca2+ current in the ventricular myocytes. We measured the L-type Ca2+ current (ICa) in the ventricular cells with patch-clamp techniques to evaluate functional changes in the mutant hearts, since L-type Ca2+ channels reside on the transverse tubules. The density of basal ICa in the mutant transgenic cells was significantly lower than in the control cells (C57BL/6). The ß-adrenoceptor agonist isoproterenol increased ICa in both control and transgenic cells. The percent increase in ICa after isoproterenol treatment in the transgenic cells was not significantly different from that in the control cells; the increase in ICa at –10 mV in the control cells (n = 8) and the transgenic cells (n = 12) was 168 ± 25% and 130 ± 13%, respectively, after 100 nM isoproterenol. However, the densities of ICa after the application of 10 or 100 nM isoproterenol in the transgenic cells were significantly lower than those in the control cells (Fig. 5A and B).
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These results suggest that the transduction system of ß-adrenoceptors remains intact in mutant transgenic ventricular cells, at least for the changes in ICa and chronotropic response. Although the mutant transgenic mice used in these experiments did not show overt signs of heart failure yet, their cardiac function may have been latently impaired due to a decreased density of functional L-type Ca2+ channels in the sarcolemma of the heart cells. In fact, reverse transcription-PCR analyses showed enhanced expressions of atrial and brain natriuretic peptides in the mutant hearts (Fig. 5E), which indicated that the mutant transgenic mice suffered from chronic heart failure.
Hearts in the mutant KDEL receptor transgenic mice might have suffered from ER stress. We took neonatal cardiomyocytes as a primary cell culture. When treated with tunicamycin, which disturbs protein glycosylation in the ER, neonatal cardiomyocytes from the control mice showed diffuse expression of BiP, apparently in the ER distribution. To the contrary, we observed cell shrinkage with uneven distribution of BiP in the cardiomyocytes of the mutant KDEL receptor transgenic mice, suggesting that those cardiomyocytes were sensitive to ER stress (Fig. 6A). Accumulation of misfolded proteins in the ER leads to protein degradation in a ubiquitin-proteasome system. Western blotting revealed marked accumulation of ubiquitinated protein aggregates in the hearts of the mutant KDEL receptor transgenic mice but not the wild-type KDEL receptor transgenic mice, suggesting that misfolded proteins might have saturated the ubiquitin-proteasome system during a circumstance in which the mutant KDEL receptor might have impaired the capacity for ER quality control (Fig. 6B). We found slightly increased expression of BiP and significant accumulations of CHOP in the adult mutant hearts (Fig. 6C). Since CHOP is induced by ER stress and has been acknowledged to cause apoptosis during the unfolded protein response (67), this might account for the enhanced apoptosis in the mutant hearts (Fig. 2E). These results suggest that the mutant hearts might have suffered from ER stress, which might have contributed to the pathogenesis of the cardiomyopathy found in the mutant mice.
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DISCUSSION |
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ER stress brings on human disorders such as neurodegenerative diseases (25); however, it is uncertain whether it also causes cardiac diseases. Cardiomyocytes are exposed to mechanical stress throughout their lives. While mechanical stress may be sensed by cardiomyocytes specifically through integrins that transduce signals from the extracellular matrix to the cytoskeletal proteins and cytosolic signal transduction molecules (8, 47), it may also cause deformation of the sarcolemma, which may lead to conformational changes in the proteins (30, 47). This effect seems to be nonspecific. Since newly synthesized proteins have not folded correctly and are unstable, their folding process might also be affected. In fact, mechanical stress induces in hearts the transcription of heat shock protein genes such as HSP70 (4). Heat shock proteins are molecular chaperones that assist in the folding of newly synthesized proteins. A perturbation of protein folding causes the accumulation of misfolded proteins in the cytosol, which dissociates HSP70 from the heat shock transcriptional factor and induces stress response (4). Similarly, the accumulation of misfolded proteins in the ER dissociates BiP, an ER-resident homologue of HSP70, from ER kinases such as IRE1, PERK, and activating transcription factor 6; this dissociation initiates the unfolded protein response, the stress response in the ER (6, 50). We observed an accumulation of HSP70 as well as BiP upon pressure overload in normal rat hearts. Thus, mechanical stress may disturb the folding of newly synthesized proteins, which in turn induces the stress response in the ER as well as in the cytosol.
We observed accumulation of CHOP and ubiquitinated proteins in the hearts of the mutant KDEL receptor transgenic mice. Hyperubiquitination of proteins has been observed in human dilated cardiomyopathy (59). Accumulations of both cytosolic and membranous misfolded proteins impair the ubiquitin-proteasome protein degradation system, which sensitizes cells to ER stress (3). Aggregates of cytosolic polyglutamine proteins and membranous Pael receptors have been thought to cause Huntington's disease and Parkinson's disease, respectively, via ER stress (20, 39). Similarly, misfolded proteins might saturate the ubiquitin-proteasome system and cause ER stress, especially in mutant KDEL receptor transgenic mice, in which the ER quality control system might be impaired. In fact, the presence of Cre seemed to enhance the phenotype of the mutant mice, although all the transgenic mice had the recombinant transgene and expressed its products even without the Cre gene. As foreign protein, Cre might also impair the ubiquitin-proteasome system. In support of this assumption, a large overexpression of cytosolic green fluorescence protein (19) or ß2-adrenoreceptor (31) in transgenic mice has been reported to cause dilated cardiomyopathy. As we showed that the increased pressure load to the hearts caused upregulation of BiP, CHOP, and XBP-1 as well as HSP70, a marker for general cellular stress, the phenotype of the mutant transgenic mice might be caused by the combined effects of general cellular stress and ER stress.
While the KDEL receptor has been considered a retrieval receptor for the KDEL proteins, we observed that the transport mutant KDEL receptor suppressed the recycling of CD8E19 with a dilysine retrieval motif. The dilysine motif is recognized directly by the COPI complex (27). Thus, our results support the idea that the KDEL receptor is not merely a retrieval receptor for the KDEL proteins but also a regulator for COPI transport (2, 32). Since bidirectional transport between the Golgi complex and the ER may be coupled to each other, we suppose that the mutant KDEL receptor suppresses not only COPI retrograde transport from the Golgi to the ER but also anterograde transport from the ER to the Golgi. This suppressive effect of the mutant KDEL receptor on anterograde transport from the ER to the Golgi seems to be selective, because it reduces the recycling of CD8E19 but not the secretion of CD8E193S, which does not have the dilysine retrieval motif.
Similar selective effects on transport have been reported in a study with S. cerevisiae showing that a COPI mutation allows certain proteins to be transported unhindered to the secretory pathway, while other secretions from the ER are disturbed (11). We propose that misfolded proteins to be degraded or refolded are recycling between the Golgi and the ER to some extent and that the mutant KDEL receptor may disturb this recycling, resulting in the accumulation of misfolded proteins in the ER and causing ER stress. Interestingly, a large overexpression of ß2-adrenoreceptor in transgenic mice (31) accompanied the upregulation of Rab GTPases (61), which regulate transport in the secretory pathway. Rab1 specifically plays a role in the recruitment of COPI proteins to the Golgi complex (1). Transgenic mice expressing Rab1a developed cardiac hypertrophy and failure (61), suggesting the critical role of transport between the ER and the Golgi complex in the pathogenesis of cardiomyopathy (37).
Although the precise molecular mechanism that leads to dilated cardiomyopathy in the mutant mice is still open, we speculate that there is a kind of vicious cycle. Pressure load may induce the accumulation of misfolded proteins in the sarcoplasmic reticulum, especially in the mutant cardiomyocytes, where the capacity for ER quality control is limited. Expanded sarcoplasmic reticulums and obstructed transverse tubules may disturb Ca2+ homeostasis, which may impair cardiac contractility and in turn increase the pressure overload. Persistent pressure overload causes further accumulation of misfolded proteins in the sarcoplasmic reticulum, which elicits apoptosis of the cardiomyocytes through an intense unfolded protein response and ultimately results in dilated cardiomyopathy and heart failure (Fig. 8).
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. Although this phenotype might represent the common phenotype of ER stress on mice, it might also reflect the distribution of PERK, which is expressed at high levels in the pancreas (51). Since activation of PERK leads to the induction of CHOP through ATF4 (13), the apoptosis of ß cells in PERK–/– mice may not be dependent on CHOP. We found that CHOP accumulated in the mutant transgenic hearts. If CHOP plays an important role in apoptosis in cardiomyopathy, it may be possible that the hearts of PERK–/– mice are less sensitive to ER stress because CHOP induction is impaired in PERK–/– mice.
Another animal model, IRE1ß–/– mice, revealed an increased sensitivity to gastrointestinal insults, resulting in the development of colitis, which might reflect the restricted expression of IRE1ß in the epithelial cells of the gastrointestinal tract (5). IRE1 is ubiquitously expressed, and IRE1–/– mice died as embryos (57). Information about ATF6–/– mice is not available at this time. XBP-1–/– (also called TREB5–/–) mice died as embryos between days 10.5 and 14.5 and displayed cellular necrosis of cardiac myocytes (33) as well as hypoplastic fetal livers and anemia (44). BiP is an ER chaperone that plays an important role in the unfolded protein response. Although information about BiP–/– mice is not available, they might also die as embryos because yeast cells deleted of Kar2 (a yeast homologue of BiP) are not viable (40). Calreticulin is another ER chaperone that plays a major role in ER quality control, and calreticulin–/– mice died as embryos between days 12.5 and 18.5 due to the failure of cardiac development (35).
Collectively, based on the above studies, we are not sure whether there are any established features of ER stress in adult mice in vivo at this time. Each animal seems to have a specific phenotype derived from the specific function and expression pattern of the molecule responsible. At least XBP-1–/–(TREB5–/–) mice and calreticulin–/– mice have a cardiac phenotype during their embyogenesis.
The mutant KDEL receptor transgene is driven by the CAG promoter and is believed to be ubiquitously expressed. The expression level is moderate, as high as the endogenous level. Although all tissues of the mutant KDEL receptor transgenic mice might be sensitive to ER stress, one of the most important factors that affect whether ER stress may cause a disease is the regenerative ability of the tissue concerned. Neurodegenerative diseases and diabetes mellitus are usually regarded as typical ER stress-associated diseases or conformational diseases (14, 25, 42) because a neuron and a ß cell live as long as the individual's life span and are therefore susceptible to the accumulation of misfolded proteins. In this regard, cardiomyocytes have the same life span and are exposed to harsh conditions, such as mechanical and oxidative stress. This study suggests that a cardiomyocyte may also be a target for ER stress. Whether a failing human heart shares this novel molecular mechanism caused by ER stress will be an interesting issue to explore.
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ACKNOWLEDGMENTS |
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This work was supported by Grants-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan to T.A.
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FOOTNOTES |
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, P < 0.05. (C) Histological findings from the hearts of the wild-type KDEL receptor transgenic mouse with the Cre gene (12 weeks old), the mutant KDEL receptor transgenic mouse with the Cre gene (line B, 12 weeks old), and the C57BL/6 mouse (12 weeks old) stained with hematoxylin and eosin. The top panels show low-power photomicrographs of longitudinal sections. The bottom panels show higher-power photomicrographs of axial sections. Arrows indicate vacuolization. Bar, 100 µm. (D) The diameters of individual myofilaments in the longitudinal section were examined for each group in panel C. A statistical analysis among the three groups was performed with analysis of variance and the Scheffe test. Mean values ± standard deviation are shown. *, P < 0.0001, n = 100. (E) Cardiomyocytes from wild-type transgenic mice (11 weeks old), mutant KDEL receptor transgenic mice (line F), and littermate C57BL/6 mice (12 weeks old) were evaluated for DNA fragmentation by the TUNEL assay. Bar, 100 µm. TUNEL-positive cells were scored, and values are means ± standard deviation for five experiments. A statistical analysis was performed with analysis of variance and the Scheffe test among the three groups. *, P < 0.0001, n = 100. All mice were Cre gene negative.
