The serine/threonine-protein kinase/endoribonuclease IRE1α protects the heart against pressure overload–induced heart failure

IRE1α induces adaptive and transient UPR in cardiac myocytes

To investigate the functional impact of IRE1α activity, we generated adenoviral vectors expressing WT IRE1α. To validate the functionality of this expression vector, we tested the adenoviral vector in INS-1 cells. As shown in Fig. 1, IRE1α expression in INS-1 cells led to significant cell death 2 days after transfection (Fig. 1A). Xbp1 splicing was induced in a dose-dependent manner by IRE1α expression, similar to tunicamycin (TM) treatment (Fig. 1B). In addition, IRE1α autophosphorylation, BiP expression, phospho-EIF2α (Fig. 1C), and JNK kinase activity (Fig. 1D) were also induced in a dose-dependent fashion by IRE1α expression in INS-1 cells. These data are consistent with previous literature where IRE1α expression promotes cell death, associated with elevated RNase and stress kinase signaling via oligomerization in INS-1 cells (42, 43). In contrast, neonatal rat ventricular myocytes (NRVMs) with IRE1α overexpression did not show significant difference in morphology or cell sizes (Fig. 2A), although autophosphorylation and downstream Xbp1 activation were detected as in INS-1 cells (Fig. 2, B and C). Other ER stress effectors, such as BiP expression, were also significantly induced by IRE1α overexpression, whereas the pro-apoptotic ER stress gene CHOP was not (p = 0.09) (Fig. 2D). As expected, expression of a kinase-dead IRE1α-KA mutant did not affect any of the ER stress molecules (Fig. 2E). These results revealed an unexpected myocyte-specific response to IRE1α expression, where only a subset of cytoprotective ER stress response was triggered in primary cardiomyocytes without activating the pro-apoptotic downstream pathway. Remarkably, prolonged IRE-1α expression (5 days) failed to sustain downstream Xbp1 activation or other UPR gene expression, although IRE1α autophosphorylation remained elevated (Fig. 2, F–H). These observations have revealed two specific features in IRE1α function in cardiomyocytes; one is a selective induction of IRE1α-mediated cytoprotective response versus stress signaling, and another involves the transient nature of the IRE1α-induced downstream Xbp1 activation, most likely due to a yet to be identified negative feedback inhibition.

Heart-specific and inducible expression of IRE1α in mice

To elucidate IRE1α function in intact heart, we generated an animal model where IRE1α expression was both inducible and heart-specific using a Cre-loxP–mediated gene switch strategy (Fig. 3A) (44). Cre-dependent expression of IRE1α was demonstrated in 293 cells, validating the efficacy of the construct (Fig. 3B). As shown in vitro, the Flox-GFP-IRE1α transgenic mice showed widespread expression of GFP but not the IRE1α transgene. After cross-breeding with αMHC-Mer-Cre-Mer mice (45–47), the transgenic gene IRE1α was induced only in ventricular tissue only after tamoxifen treatment (Fig. 3, C–E). Four weeks after transgene induction, no abnormal phenotype was observed in the IRE1α-expressing hearts compared with the littermate controls, including non-transgenic or single-transgenic mice, based on morphometric, histological, and functional analysis (Fig. 4). Furthermore, mRNA levels of sXbp1, BiP, and CHOP (Fig. 5A) were not affected by IRE1α expression, consistent with our in vitro observation after long-term IRE1α expression. The molecular profiles of ANF, β-MHC, and TNFα were all unchanged following IRE1α overexpression (Fig. 5B). Together, these observations suggest that IRE1α expression was not sufficient to sustain ER stress response and did not exert any detrimental effect in the adult mouse heart.

IRE1α preserves heart function after transverse aortic constriction (TAC)

To test the effect of IRE1α expression on pathological stress response in heart, IRE1α transgenic mice were subjected to pressure overload implemented by TAC (48, 49). Pressure overload led to significant loss of cardiac function over time in the control mice. In contrast, the IRE1α transgenic mice demonstrated preserved function even at 4 weeks post-TAC (Fig. 6A), associated with reduced chamber dilation at the systole (Fig. 6B) based on echocardiograph measurements. Furthermore, the induction of pathological marker genes, including ANF and βMHC, was significantly blunted in response to TAC in the IRE1α transgenic heart versus the controls (Fig. 6C). However, cardiac hypertrophy as measured from tissue weight and histology was induced by TAC to the same levels in the control versus the IRE1α transgenic hearts (Fig. 6, D and E). Interestingly, fibrotic induction in myocardium was also significantly blunted in response to TAC in the IRE1α transgenic heart versus the controls (Fig. 6, E and F).

To investigate the potential mechanism, we analyzed the expression of UPR genes and stress-signaling molecules in post-TAC hearts. Although there was a trend for lower BiP expression in the post-TAC hearts, the IRE1α heart showed a modestly but significantly higher level of BiP expression than controls after TAC (Fig. 7A). In contrast, the mRNA level of CHOP was not affected by IRE1α expression, whereas there was trend for lower expression of ATF4 in the post-TAC IRE1α transgenic heart (38) (Fig. 7A). Therefore, IRE1α expression in the heart leads to better preserved heart function following the TAC with only modest impact on ER stress downstream molecules. ER stress signaling by IRE1α and TRAF2 has been reported to activate inflammatory signals (50). As shown in Fig. 7B, IRE1α transgenic hearts showed significantly reduced TAC-induced mRNA expression for both TNFα and IL-6 cytokines after comparing with the controls. Furthermore, whereas NF-κB induction was not affected by IRE1α expression in the post-TAC hearts, the IkB kinase β induction was significantly blunted in the IRE1α transgenic hearts, suggesting that attenuated NF-κB signaling may in part contribute to reduced TNFα/IL-6 induction in the IRE1α TAC heart. In summary, IRE1α expression in the heart protects cardiac dysfunction associated with blunted induction of pro-inflammatory genes.

Animal models and surgical procedures

The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication number 85-23, revised 1985). All procedures were performed in accordance with UCLA animal welfare guidelines and approved by the UCLA institutional animal care and use committee. IRE1α was cloned into a vector for generation of transgenic animals with Cre-regulated expression of the transgene of interest (61, 62). Transgenic animals were generated in C57/Bl6 background through collaboration with the UCLA Molecular Genetics Technology Center. Founder animals were identified by PCR with transgene-specific primers.

Animals with heart-specific, inducible IRE1α overexpression were generated by crossing transgenic founder animals with previously established αMHC-Mer-Cre-Mer (MCM) transgenic mice (47) (45). IRE1α transgene overexpression was induced by intraperitoneal injection of tamoxifen citrate salt (Sigma), 20 mg/kg body weight/day for 5 days (45). WT and floxed single-transgenic littermate animals treated with tamoxifen or double-transgenic flox-GFP/CRE animals treated with vehicle were also used as controls. Both male and female mice age 12–16 weeks were included in this study.

TAC was performed as described previously with modifications (49). Mice were anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) by intraperitoneal injection. Respiration was provided by mechanical ventilation with 95% O₂ (tidal volume 0.5 ml, 130 breaths/min). Left parasternal thoracotomy was performed to access the transverse aorta, which was tied with a 5-0 nylon suture on a 27-gauge needle. The needle was removed, leaving in place a 65–70% constriction of the aortic lumen. Constriction of the aorta was confirmed by measuring differential blood flow through the right and left carotid arteries 1 week after surgery.

Animals were continuously anesthetized with 1.5% isoflurane and 95% oxygen. VisualSonics Vevo 770 and Vevo 2100 imaging systems and a 30-mHz scan head (Toronto, Canada) were used to collect short and long axis B-mode and M-mode views. Reported values refer to short-axis measurements and calculations.

Histology

Hearts were perfused and fixed in 10% formalin before embedding in paraffin. All short-axis sections were prepared from mid-ventricle. Sections of 4-μm heart were deparaffinized and rehydrated before staining by hematoxylin and eosin (H&E) or Masson trichrome and Verhoeff–Van Gieson stain. Stained tissue sections were recorded as digital images by the Aperio XT whole-slide scanning system, and snapshot images were taken using the ImageScope software (Aperio Technologies, Vista, CA).

Cell culture

293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Lipofectamine reagent (Life Technologies, Inc.) was used according to the manufacturer's protocol to achieve overexpression of the flox-GFP-IRE1α construct. INS-1 cells were cultured in RPMI1640 according to published methods (57). NRVMs were harvested from 1–3-day-old Sprague–Dawley rat pups as described previously (63) and cultured in serum-free Dulbecco's modified Eagle's medium supplemented with 1% penicillin/streptomycin and 1× Insulin-Transferrin-Selenium (ITS). NRVMs were infected with adenovirus (multiplicity of infection of 10) and incubated for 2 days before additional treatment with 5 g/ml TM for 4 h. Experiments with prolonged IRE1α expression were incubated for 5 days before RNA or protein analysis.

Western blotting

Cells were harvested for protein analysis with standard lysis buffer containing 1% Triton X-100, 1 mm β-glycerophosphate, 2.5 mm Na₄P₂O₇, 20 mm NaF, 1 mm Na₃VO₄, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche Applied Science). Proteins were boiled for 5 min in LDS loading buffer containing 0.1% β-mercaptoethanol and separated on a 4–12% BisTris SDS-PAGE (Life Technologies). Specific proteins were detected with antibodies directed against p-IRE1α (Novus Bio), IRE1α, actin (Santa Cruz Biotechnology), BiP/Grp78 (Stressgen), p-p38, p38, p-JNK, JNK, p-IEF2α, eIF2α, GFP, and CHOP (Cell Signaling Technologies), as shown in Table 1.

RNA and RT-PCR analysis

Total RNA was isolated from heart or cells with TRIzol (Life Technologies). For animal studies, cDNA was prepared using iScript Reverse Transcription Supermix and amplified with SsoFast EvaGreen Supermix on a CVX96 thermal cycler (all from Bio-Rad). For cell studies, cDNA was prepared using Superscript II (Invitrogen) and amplified with SYBR Green supermix on a MyIQ system (Bio-Rad). Melt curves were generated for each primer set during each experiment, and analysis was performed using the ΔΔCT method (Table 2).

Xbp1 activation

IRE1α RNase activity toward Xbp1 mRNA was monitored by semiquantitative PCR. Both unspliced and spliced Xbp1 mRNA were amplified with primers targeting the region surrounding the IRE1α-dependent splicing site (forward, 5′-GTTCCAGAGGTGGAGGCCA-3′; reverse, 5′-CATGACAGGGTCCAACTTGTCC-3′). Products were amplified with the cycling protocol of 95 °C for 30 s, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 25 s, followed by 72 °C for 10 min. PCR products were separated on 4% agarose gel.

Statistical analysis

Data are presented as mean ± S.D. Means of two groups were compared by two-tailed Student's t test. Means of more than two groups were compared by analysis of variance and Turkey post hoc test. Differences between groups were considered statistically significant when p < 0.05.