Roles for Endoplasmic Reticulum–Associated Degradation and the Novel Endoplasmic Reticulum Stress Response Gene Derlin-3 in the Ischemic Heart
Rationale: Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response, which restores efficient ER protein folding, thus leading to protection from stress. In part, the unfolded protein response alleviates ER stress and cell death by increasing the degradation of terminally misfolded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor (ATF)6, which can induce genes that encode components of the ERAD machinery.
Objective: Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart.
Methods and Results: In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia–induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia–induced cardiomyocyte death.
Conclusions: This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.
- endoplasmic reticulum stress
- unfolded protein response
- endoplasmic reticulum-associated degradation
Synthesis and folding of secreted, membrane-bound, and organelle-targeted proteins takes place in the endoplasmic reticulum (ER).1,2 The ER environment is sensitive to stresses that impair folding of ER proteins.2 The aggregation of misfolded proteins can be toxic and has been implicated in nearly sixty diseases,3 including desmin-related cardiomyopathy.4 In addition, a recent study has shown that transgenic overexpression of preamyloid oligomers is toxic to cardiomyocytes.5
An accumulation of misfolded ER proteins triggers the ER stress response (ERSR), also known as the unfolded protein response (UPR),6 in many different cell and tissue types.7–9 Initial ERSR signaling is oriented toward resolving the stress and reducing the protein folding load on the ER, leading to survival. Such prosurvival responses include ER-associated protein degradation (ERAD), through which terminally misfolded proteins that accumulate in the ER are degraded. Terminally misfolded ER proteins are translocated out of the ER lumen to be degraded by ERAD.10 Although the importance of ERAD in the heart has been discussed,11,12 few studies have analyzed the functionality of ERAD in the myocardium.
Although many details of ERAD remain to be elucidated, it is believed that in part, the activating transcription factor (ATF)6 branch of the ERSR induces some of the ERAD genes. ATF6 is an ER-transmembrane protein that is cleaved on ER stress; the N-terminal fragment of ATF6 translocates to the nucleus and acts as a transcription factor to induce numerous protective ERSR genes. ATF6 can bind to cis regulatory elements in ERSR genes, including the ER stress response element (ERSE), ERSE-II, and, to a lesser extent, the unfolded protein response element (UPRE).13
To investigate the effects of ATF6 and ERAD in the heart we generated a line of transgenic (TG) mice featuring cardiac-restricted expression of a novel protein in which the mutant mouse estrogen receptor (MER) was fused to the C terminus of the active fragment of ATF6. This fusion protein, ATF6-MER, is constitutively expressed in ATF6-MER TG mouse hearts, but is active only on tamoxifen administration.14 Activation of ATF6 in TG mouse hearts enhances function and reduces necrosis and apoptosis during ex vivo I/R,14 suggesting that ATF6 contributes to reducing damage and enhancing recovery from I/R. It is possible that some of these effects might be exerted through ATF6-mediated enhancement of ERAD.
Whole-genome microarray analyses of ATF6-MER TG mouse hearts showed that 381 transcripts exhibited ATF6-dependent increases.15 One of the most induced genes was Derlin-3 (Derl3), a member of a family comprised of Derl1, -2, and -3. The Derlins were named “der” for “degradation in the ER.”16 Derl1 facilitates the retro-translocation of misfolded proteins from the ER lumen to the cytosolic face of the ER.17 Thus, it is possible that the other Derl family members, including Derl3, exert similar functions.
Although studies of Derl1, -2, and -3 have been done with noncardiac cell types,18 the mechanism of Derl3 induction in any cell type is unknown, and the Derlin family has not been studied in the heart. Accordingly, we examined the mechanism of induction and function of Derl3 in the heart.
The transgenic mice used in this study have been described previously.14 Approximately 100 neonatal rats and 24 adult male mice were used in this study. All procedures involving animals were in accordance with the San Diego State University Institutional Animal Care and Use Committee.
Cultured Cardiac Myocytes
Primary neonatal rat ventricular myocyte cultures (NRVMCs) were prepared and maintained in culture, as previously described.19
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Promoter Sequence Analysis of ATF6-Regulated Genes in the Heart Identifies Derl3
We previously identified ATF6-regulated genes in ATF6-MER TG mouse hearts.15 To determine which of the genes from the microarray analyses of ATF6-MER TG mouse hearts might be direct targets of ATF6, the regulatory regions of each were analyzed for the ATF6 binding sites, ERSE, ERSE-II and UPRE.20–22 ERSE, ERSE-II and UPRE sequences were found 9.6-, 8.4- and 2.1-fold more frequently, respectively, than in the whole mouse genome (Figure 1). Among the 607 ATF6-regulated genes in the heart, 16 have canonical ERSEs, ERSE-IIs, and/or UPREs (Online Tables I through III), whereas 211 have elements, with 1 mismatch (Online Tables IV through VI); mismatches of 1 bp have been shown in other studies to be potential ATF6-binding elements.13,20 Thus, 227 genes are likely to be direct targets of ATF6.
One of only 2 ATF6-regulated genes with 2 canonical ERSEs is Derlin-3 (see Online Table I; Derl3, NM_024440). Because it may play a role in ERAD, we investigated the mechanism of induction and the function of Derl3 in the heart and cultured cardiac myocytes. To examine transcriptional regulation cultured cardiac myocytes were transfected with several luciferase reporter constructs (Figure 2A) with or without subsequent infection with either a control (AdVCon) or an adenovirus that encodes activated ATF6 (AdVATF6). Luciferase activation in cultures infected with AdVATF6 and transfected with reporter construct 1 was 200-fold of control (Figure 2B, bar 4). ATF6-mediated luciferase induction was reduced by 75% to 80% in constructs 2 and 3, which each contain 1 mutated ERSE (Figure 2B, bars 5 and 6). The ERSR activator tunicamycin (TM) conferred robust induction of construct 1 (Figure 2B, bar 7); however, constructs 2 and 3 exhibited decreased induction (Figure 2B, bars 8 and 9). Thus, maximal ATF6- or TM-mediated induction of the Derl3 promoter was dependent on both ERSE1 and ERSE2.
ATF6 Induces Derl3 in Mouse Hearts
The ability of ATF6 to induce the other Derlin family members was also examined. Whereas neither Derl1 nor Derl2 was induced by tamoxifen in the ATF6 TG mouse hearts (Figure 3A, bars 1 through 8), Derl3 was induced by 400-fold by tamoxifen, but only in the TG mouse hearts (Figure 3A, bars 9 through 12); thus, only Derl3 was ATF6-inducible in the heart, consistent with the lack of ERSEs in the Derl1 and Derl2 genes (Online Table I).
Derlin levels were relatively low in sections from vehicle-treated ATF6-MER TG hearts (Figure 3B, Derl3) and tamoxifen-treated nontransgenic (NTG) mouse hearts (not shown). In contrast, tamoxifen-treated ATF6-MER TG mouse hearts exhibited robust Derl3 expression that colocalized primarily with actin-positive cardiomyocytes (Figure 3C, overlay). Thus, ATF6 induces Derl3 expression in cardiac myocytes, in vivo.
Derl3 Is an ATF6-Inducible ER Stress Response Gene
The effects of ATF6 or ER stress on Derlin mRNA in cultured cardiac myocytes were examined. Derl3 was the only family member that exhibited ATF6-inducibility, in cultured cardiac myocytes (Figure 4A, bars 1 through 6). When cells were treated with TM, Derl1 and Derl2 mRNA levels were 3- and 8-fold of control, respectively (Figure 4B, bars 2 and 6); however, neither was affected by dominant-negative ATF6 (Figure 4B, bars 4 and 8). In contrast, on TM treatment, Derl3 mRNA was 200-fold of control (Figure 4B, bar 10), and this induction was attenuated by more than half by dominant-negative ATF6 (Figure 4B, bar 12).
Simulating ischemia (sI) activates ER stress in cardiac myocytes19; accordingly, the effect of sI on Derlin expression was examined. Whereas Derl1 and Derl2 have been shown to be inducible by TM in XBP1 knockout MEFs,18 in the present study in NRVMCs, sI had no effect on Derl1, and Derl2 was only slightly increased, but this increase did not reach significance (Figure 4C, bars 2 and 6). In contrast, sI significantly increased Derl3 to ≈2.5-fold of control (Figure 4C, bar 10); moreover, sI-mediated Derl3 induction was completely blocked by dominant-negative ATF6 (Figure 4C, bar 12), which was different than the partial blockage of Derl3 induction following TM treatment. This could be attributable to the higher dynamic range of induction experienced with TM or attributable to other potential mediators of Derl3 induction during TM but not sI. Thus, Derl1 and Derl2 were induced by the prototypical ER stressor, TM, independently of ATF6. Moreover, only Derl3 was induced by the physiological ER stressor, sI, in an ATF6-dependent manner.
ATF6 Is Activated and Derl3 Is Induced in an In Vivo Model of Myocardial Infarction
We previously showed that ER stress is activated in the mouse heart by myocardial infarction (MI).19 During ER stress, full-length, 90-kDa ATF6 is converted to 50-kDa ATF6, which is an active transcription factor.6,23 Accordingly, the effects of ischemia on ATF6 and Derl3 after MI were examined in vivo. An MI time course indicated that the 50-kDa form of ATF6 increased significantly after 16 hour of MI, remained elevated after 1 day and 3 days and decreased at 4 days of MI, consistent with activation of ATF6 (Figure 5A and 5B).24 The 50-kDa band migrated slightly further than a 3xFlag-tagged form of cleaved, N-terminal ATF6, consistent with its identity as cleaved ATF6 (Online Figure I, A).
The mRNA levels of all Derl family members increased in 4 days MI mouse hearts, compared to sham, with Derl1 and Derl3 reaching significance and Derl3 exhibiting the most robust upregulation of ≈6-fold (Figure 5C, bars 2, 4, and 6). An MI time course showed that although Derl3 mRNA showed a trend of being increased after 1 day MI, it was significantly increased after 4 days and 7 days of MI (Online Figure I, B; Online Data Supplement). The increase in p50 ATF6 by 16 hours of MI and continued elevation after 1 and 3 days of MI were consistent with the possibility that ATF6 could induce Derl3 mRNA as early as 1 day after MI. However, because the elevation of p50 ATF6 after 1 and 3 days of MI, and the elevated Derl3 mRNA after 1 day of MI did not reach statistical significance, it is formally possible that ATF6 activation/inactivation may precede Derl3 induction, suggesting that ATF6 may induce Derl3 expression indirectly. For example, ATF6 is known to induce the ER stress response transcription factor, XBP1, which could induce Derl3. These limitations leave open the question of whether ATF6 directly induces Derl3 mRNA in the heart, which we intend to investigate in future studies; nonetheless, ATF6 is a potent inducer of Derl3 gene expression in the myocardium.
Derl3 was low in sham mouse hearts (Figure 5D, image 1) but elevated in surviving myocytes in the infarct zone (Figure 5E, images 1 and 3, arrow 1), as well as other tropomyosin-negative cells, which were most likely nonmyocytes (Figure 5E, images 1 and 3, arrow 2). Derl3 staining was perinuclear in the myocytes, consistent with expression in the ER.
Derl3 Protects Cardiac Myocytes From Cell Death
ERAD reduces ER stress by degrading misfolded ER proteins; accordingly, the effect of Derl3 on ER stress was examined. Following sI or simulated ischemia/reperfusion (sI/R), control cells exhibited a significant increase in the prototypical ER stress response proteins glucose-regulated protein (GRP)94 and GRP78 (Figure 6A, lanes 1 through 3 versus 7 through 9 and 11 through 13; Figure 6B and 6C, bars 1, 3, and 5). In contrast, cells infected with AdV-Derl3 exhibited reduced GRP94 and GRP78 (Figure 6A, lanes 4 through 6, 10 through 12 and 14 through 16; Figure 6B and 6C, bars 2, 4, and 6). GRP94 and GRP78 mRNA levels were also assessed following sI and sI/R (Online Figure II, A and B). Because neither GRP94 nor GRP78 mRNA levels were elevated following 20 hours of sI (Online Figure II, A and B, bars 3 through 4), we hypothesized that these mRNAs were increased at shorter times of sI. As indicated in Online Figure II (C and D), GRP94 and GRP78 mRNAs increased at shorter times of sI, reaching a maximum at 12 hours, and AdV-Derl3 reduced the induction seen at these sI time points, consistent with the reduced levels of these proteins following sI seen in Figure 6A through 6C. In addition, sI/R significantly increased GRP78 and GRP94 mRNA in AdVCon infected cells by ≈15 and 17 fold, respectively (Online Figure II, A and B, bar 5). AdV-Derl3–infected cells exhibited a significant reduction in sI/R-mediated GRP78 and GRP94 mRNA (Online Figure II, A and B, bar 5 versus 6). Together, these results suggest that overexpression of Derl3 attenuated sI- and sI/R-activated ER stress.
Prolonged ER stress activates apoptosis6,23,25,26; accordingly, we assessed whether Derl3 overexpression decreased the ER stress-inducible, proapoptotic protein, C/EBP homologous protein (CHOP) and caspase-3 activation. AdV-Con cells exhibited ≈25- and 5-fold increase in CHOP following sI and sI/R, respectively (Figure 6D, lanes 1 and 2 versus 5 and 6 and 9 and 10; Figure 6E, bars 1, 3, and 5). In contrast, AdV-Derl3 cells exhibited decreased CHOP induction following sI and sI/R (Figure 6D, lanes 3 and 4, 7 and 8, and 11 and 12; Figure 6E, bars 2, 4, and 6). sI did not activate caspase-3 in AdV-Con– or AdV-Derl3–infected cells, which is consistent with the lack of ATP required to activate caspase during sI (Figure 6F, bars 1 and 2 versus 3 and 4). However, sI/R resulted in a ≈2-fold increase in caspase-3 in AdV-Con–infected cells, which was attenuated in AdV-Derl3–infected cells (Figure 6F, bars 5 and 6). In addition, AdV-Con–infected cells exhibited a ≈2-fold and 3.5-fold increases in cell death following sI and sI/R, respectively (Figure 6G, bars 1 versus 3 and 5). In contrast, AdV-Derl3–infected cells exhibited significantly less cell death in response to sI/R (Figure 6G, bar 6). Thus, overexpression of Derl3 attenuated ER stress and apoptosis in cardiac myocytes subjected to these stressors.
Derl3 Enhances ERAD and Reduces ER Stress
The effect of Derl3 on ERAD was examined using a mutant form of the α-1 anti-trypsin protein (A1ATmut), which is constitutively misfolded in the ER.27 HeLa cells were cotransfected with plasmids expressing A1ATmut and Derl3-encoding plasmid. Derl3 overexpression decreased A1ATmut (Figure 7A and 7B), indicating that Derl3 augmented clearance of misfolded proteins in the ER during ERAD.
The effect of A1ATmut overexpression on ER stress was determined by measuring GRP78 promoter activation. Cells were transfected with plasmids expressing a GRP78-luciferase promoter and A1ATwt, which folds properly or A1ATmut. Whereas A1ATwt had no effect, promoter activity was ≈2-fold of control in cells transfected with A1ATmut (Figure 7C, bars 2 and 3). Moreover, cotransfecting Derl3 decreased A1ATmut-mediated GRP78 promoter activation (Figure 7C, bars 3 versus 5). Thus, Derl3 enhanced the removal of A1ATmut and, in so doing, decreased ER stress.
Dominant Negative Derl3 and microRNA Directed to Derl3 Enhance sI/R-Mediated Cardiac Myocyte Death
An expression construct encoding an inactive, dominant-negative (DN) form of Derl3 was designed based on previous studies using Derl1 and 2 DN constructs.17 HeLa cells were transfected with A1ATmut, control, Derl3, or Derl3-DN plasmids. Derl3 conferred an approximate 70% reduction in the level of A1ATmut, whereas Derl3-DN did not significantly change the levels of A1ATmut (Figure 8A and 8B). Thus, unlike wild-type Derl3, Derl3-DN does not increase the clearance of A1ATmut.
Cultured cardiac myocytes were transfected with Derl3-DN, subjected to sI or sI/R, and then analyzed for cell death by flow cytometry. Compared to control, cells transfected with Derl3-DN exhibited slight increases in cell death under control conditions, although this did not reach significance (Figure 8C, bars 1 versus 2). Cells transfected with Derl3-DN exhibited significant increases in cell death on sI and sI/R (Figure 8C, bars 3 versus 4 and 5 versus 6).
To examine roles for Derl3 on cell survival, recombinant adenovirus encoding Derl3-targeted microRNA (miRNA) (AdVmiDerl3) was generated. Compared to control, AdVmiDerl3 decreased basal Derl3 mRNA levels by ≈70% (Figure 8D). To determine whether sI increased Derl3 protein, and whether AdVmiDerl3 could attenuate this increase, NRVMCs were treated with AdVmiDerl3, subjected to sI, and lysates were examined for Derl3 protein. Derl3 was increased with sI (Figure 8E, bars 1 through 3 versus 4 through 6; Figure 8F, bars 1 versus 2). This increase was significantly attenuated by AdVmiDerl3 (Figure 8E, lanes 4 through 6 versus 10 through 12; Figure 8F, bars 2 versus 4). AdVmiDerl3 slightly increased cell death under basal conditions, although this increase did not reach significance (Figure 8G, bars 1 versus 2); however, compared to control, AdVmiDerl3 significantly increased cell death during sI (Figure 8G, bars 3 versus 4) and sI/R (Figure 8G, bars 5 versus 6).
Thus, Derl3-DN or Derl3 knockdown attenuated clearance of misfolded ER proteins, and augmented sI and sI/R-mediated cell death, suggesting that the ability to clear misfolded proteins from the ER is especially critical during ischemic stress.
ER stress is activated in the heart during ischemia and that ATF6 protects the heart from ischemic damage, in vivo. ATF6 induces numerous known and novel ER stress response genes, however, only recently have potential ATF6 gene targets been identified in the heart15; however, it is unclear which genes contribute to myocardial protection. In the present study, a detailed promoter analysis identified 16 ATF6-regulated genes which contained canonical ATF6-binding elements (Online Tables I through III). Only 2 of these genes contain 2 canonical ER stress response elements: GRP94, the well-known ER stress responsive chaperone; and Derl3, which encodes a protein likely to be involved in ERAD. Neither Derl3 nor the functional consequences of ERAD have been studied in the cardiac context; therefore, the present study focused on this gene and roles for ERAD in cultured cells and in the mouse heart, in vivo.
The Derlins are all ER transmembrane proteins that associate with other proteins that participate in the degradation of misfolded proteins in the ER. Derl1 and Derl2 associate with the p97 AAA ATPase and VIMP,17 Derl2, and Derl3 associate with proteins known to be involved in ERAD, EDEM, and p97,18 and the Derl3 yeast homolog Der3p/Hrd1p interacts with Hrd3p and the retro-translocon pore complex protein Sec61p.28 Overexpression of Derl2 and Derl3 accelerates degradation of known misfolded substrates in HEK293 cells, whereas knockdown blocks degradation.18 In addition, the IRE1/XBP1 pathway was implicated to play a role in induction of Derl1 and Derl2; however, the specific mechanism of Derl3 induction was not determined.18
In the present study, it was shown that Derl3 was strongly induced by ATF6 in vivo, a finding that was replicated in cultured cardiac myocytes. Derl3 was also induced in the infarct border zone in an in vivo mouse model of myocardial infarction and by simulated ischemia in cultured cardiac myocytes.
Because Derl3 enhances ER-associated degradation of misfolded proteins in other cell types, we investigated the possibility that Derl3 may serve a potentially beneficial role during physiologically relevant stresses in cardiac myocytes, such as ischemia and/or reperfusion. We found that overexpressing Derl3 attenuated long-term ER stress response signaling and cell death in response to sI and sI/R, suggesting that enhancing elements of the ERAD machinery in the heart can protect from ischemic injury. Although the present study indicates that Derl3 is induced in the heart, in vivo, by physiological stress, such as myocardial infarction, additional studies must be carried out using overexpression or knockdown of Derl3 or other ERAD components, in vivo, to determine whether enhancing or inhibiting ERAD in the heart can result in attenuation or exacerbation of ischemic damage in cardiac tissue.
Sources of Funding
This work was supported by NIH grants HL-075573 and HL-085577. P.J.B. and N.G. are fellows of the Rees-Stealy Research Foundation and the San Diego State University Heart Institute. P.J.B. is a scholar of the San Diego Chapter of the Achievement Rewards for College Scientists (ARCS) Foundation and a recipient of an American Heart Association Western States Affiliate Predoctoral Fellowship (Award 0815210F). M.N.S.P. is a Minority Access to Research Careers (MARC) scholar and a recipient of a MARC fellowship (NIH/NIGMS SDSU MARC 5T34 GM08303).
Original received June 29, 2009; revision received November 12, 2009; accepted November 16, 2009.
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