Endoplasmic Reticulum Chaperone GRP78 Protects Heart From Ischemia/Reperfusion Injury Through Akt Activation

Abstract

Rationale: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s (spliced X-box binding protein 1), the most highly conserved branch of the unfolded protein response, is protective in response to cardiac I/R injury. GRP78 (78 kDa glucose-regulated protein), a master regulator of the UPR and an Xbp1s target, is upregulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined.

Objective: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches.

Methods and results: Simulated I/R injury to cultured neonatal rat ventricular myocytes induced apoptotic cell death and strong activation of the UPR and GRP78. Overexpression of GRP78 in neonatal rat ventricular myocytes significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific overexpression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GRP78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the antioxidant activity and cardioprotective effects of GRP78.

Conclusions: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.

Keywords: cell death; myocardium; reactive oxygen species; reperfusion injury; unfolded protein response.

Figure 1. I/R injury induces GRP78

A. sI/R elicited significant cell death. NRVM were treated for either 3 hours or 6 hours of ischemia before reperfusion. Lactate dehydrogenase (LDH) was determined in culture medium and in cell lysates. Relative cell death was calculated based on the ratio of released LDH into the medium. N = 6 per group. B. Cell death by sI/R was determined using flow cytometry to detect annexin V- and propidium iodide (PI)-positive cells. Significant increases in Annexin V-positive apoptotic cells were observed, which accounted for the preponderance of cell death. N = 3 for each group. C. sI/R led to strong induction of ER resident chaperones at mRNA levels. NRVM were subjected to ischemia for 6 hours, followed by overnight reperfusion. Real-time PCR was conducted to measure mRNA levels with 18s as an internal control. N = 3 per group. D. sI/R induced expression of the ER chaperones at protein levels. Immunoblotting showed that GRP78 and GRP94 were significantly increased at protein levels (left). GAPDH was used as a loading control. Quantification is shown at right. N = 3. NS; not significant; *, p < 0.05; ***, p < 0.001.

Figure 2. Over-expression of GRP78 protects cardiomyocytes from I/R-induced cell death

A. Adenoviral over-expression increased GRP78 protein levels. A representative immunoblot is depicted on the left, and quantitation with GAPDH as loading control is shown on the right. N = 3. B. Over-expression of GRP78 blunts I/R-induced cell death. After adenoviral infection, NRVM were subjected to ischemia for 6 hours followed by overnight reperfusion. LDH levels were measured to determine relative cell death. N = 7 per group. C. Over-expression of GRP78 conferred significant protection against I/R-induced cell death as determined by flow cytometry for Annexin V- and PI-positive cells. A representative histogram is shown. D. Quantification of data depicted in panel C revealed that over-expression of GRP78 protected NRVM from I/R injury. N = 3 per group. NS, not significant; *, p < 0.05; **, p < 0.01.

Figure 3. Over-expression of GRP78 protects heart from I/R injury

A. Strategy of cardiomyocyte-restricted over-expression of GRP78. A mouse model harboring a CAG-loxP-STOP-loxP-GRP78 transgene was engineered and bred to a cardiomyocyte-specific Cre mouse line. In the double transgenic mouse, Cre recombines and cleaves the STOP region, which leads to GRP78 expression exclusively in cardiomyocytes. B. GRP78 expression is increased in the transgenic hearts. Protein lysates were extracted in control or GRP78-expressing transgenic (TG) hearts and Western blotting was performed to detect GRP78 protein levels. GAPDH was used as a loading control. C. Over-expression of GRP78 in cardiomyocytes protected hearts from I/R damage. Control and TG mice 12–14 weeks of age were subjected to myocardial ischemia for 45 minutes, followed by overnight reperfusion. TTC staining was conducted to detect, localize, and quantify the injured tissue. White region depicts ischemic infarct zone; pink/red area is the border zone; blue region represents the remote zone. Scale bar: 1mm. D. The relative ratios of infarct region (IF) to area at risk (AAR, ischemic and border) were compared between control and TG hearts. The relative ratios of AAR to left ventricle did not differ between control and TG groups, suggesting the I/R surgery was performed similarly across genotypes. N = 5 per group. E. TG mice manifested a decrease in fibrosis after I/R. Hearts from control and TG mice were harvested 21 days after I/R. Trichrome C staining was conducted to compare fibrosis between the two groups. Scale bar: 100 μm. NS, not significant; *, p < 0.05.

Figure 4. Over-expression of GRP78 ameliorates ROS production by I/R

A. Over-expression of GRP78 in vitro and in vivo led to decreases in I/R-induced ROS levels. Protein lysates from NRVM or cardiac tissues were incubated with DNPH, which interacts with carbonyl groups in proteins. Immunoblotting was then conducted to detect carbonylation using anti-DNP antibodies. sI/R increased carbonyl modifications in NRVM, which was significantly reduced by GRP78 over-expression. Likewise, over-expression of GRP78 in heart suppressed I/R-induced protein carbonylation. B. Quantification of data depicted in panel A revealed significant decreases in protein carbonylation triggered by GRP78 expression in NRVM or hearts. N = 4 per group. C. Flow cytometry for dihydroethidium, a superoxide indicator, showed that over-expression of GRP78 led to strong reduction of I/R-elicited ROS levels. N = 3 for each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5. Over-expression of GRP78 stimulates Akt signaling

A. Akt phosphorylation was augmented by GRP78 over-expression. NRVM were infected with adenovirus expressing GFP or GRP78. After ischemia for 6 hours and reperfusion for 1 hour, NRVM were collected and protein lysates were prepared for immunoblotting. GAPDH was used as loading control. B. Quantification revealed that GRP78 over-expression elicited significant increases in Akt phosphorylation at both Thr 308 and Ser 473 residues. However, Erk phosphorylation was not altered. N = 3 per group. C. Over-expression of GRP78 in vivo in hearts stimulated Akt phosphorylation. Protein lysates from control or GRP78-expressing TG hearts were subjected to Western blotting. GAPDH was used as loading control. D. Relative quantification showed that GRP78 over-expression in vivo significantly increased Akt phosphorylation. N = 3 for each group. NS, not significant; *, p < 0.05.

Figure 6. Akt signaling is required for GRP78-mediated cardioprotection against I/R

A. Akt inhibitors suppressed Akt signaling. Two structurally distinct Akt inhibitors, LY294002 and Akt inhibitor VIII, were used to treat NRVM for 24 hours. Both inhibitors elicited dose-dependent inhibition of Akt signaling as indicated by decreases in Akt phosphorylation. B. Suppression of Akt signaling abolished GRP78-mediated protection against I/R damage. Control or GRP78 adenovirus was used to infect NRVM for 48 hours. Cells were then subjected to sI/R. Akt inhibitors were incubated during ischemia and reperfusion. Relative cell death was determined by LDH assay. N = 4 per group. C. Akt signaling was required for GRP78-mediated suppression of ROS production. After treatment with Akt inhibitors during sI/R, flow cytometry for dihydroethidium, a superoxide indicator, was conducted to determine relative ROS levels. N = 3 for each group. *, p < 0.05.