The Effect Of Clusterin On Cold Ischemia/Reperfusion Injury In Cardiac Transplantation

PartⅠReduction of Cold Ischemia-Reperfusion Injury by Graft-Expressing Clusterin in Heart Transplantation【Objective】Cold ischemia-reperfusion injury (IRI) is one of primary factors for early graft dysfunction and is associated with rejection episodes in heart transplantation. Clusterin (CLU) is a cytoprotective protein with chaperone activity. This study was designed to examine the impact of donor-expressing clusterin on cold IRI.【Methods】Donor hearts from wild type C57BL/6J (H-2b; B6 WT) versus CLU knockout C57BL/6J (H-2b; CLU-KO) mice were stored at 4"C saline for 8 hours followed by heterotopical transplantation to B6 WT mice. The expression of CLU in the hearts of WT B6 mice or CLU-KO mice was determined by RT-PCR and immunohistochemical analysis. At the end of the preservation, the severity of cardiac injury was determined by release of creatine kinase (CK) and lactate dehydrase (LDH). The function recovery of heart grafts was determined by palpation at 15 minutes and 24 hours after transplantation. At 24 hours after transplantation, tissue injury was tested by HE stain, the expression of MPO was detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.【Results】WT B6 mice hearts constitutively expressed clusterin, and mature clusterin protein was localized mostly in the endothelium as well as on the cell surface of cardiac myocytes. As compared to CLU-KO hearts, WT hearts were more resistant to cold injury during cold preservation. Compared with the CLU-KO hearts, the CK and LDH levels released from WT hearts were significantly lower, (CK:784.25±90.71 U/L VS 610.96±60.02 U/L, p= 0.0226; LDH:0.744±0.129 VS 0.548±0.076, p= 0.0134). WT hearts had a better function recovery from a prolonged cold preservation following transplantation, indicated by the median score 2.3 in WT group compared to 1 in CLU-KO group at 15 minutes (p= 0.002), or 3.5 in WT group compared to 3 in clusterin deficient group at 24 hours (p= 0.0271). The better graft function of clusterin-expressing grafts was significantly correlated with less degree of both neutrophil infiltration and cardiac injury including myocytic apoptosis and necrosis.【Conclusion】Clusterin expression renders donor hearts resistance to cold IRI in transplantation, suggesting that up-regulation of clusterin expression in donor hearts may have potential for protecting heart grafts from cold IRI. PartⅡProtection of Cultured Myocytes from Cold-Induced Cell Death by Clusterin Expression【Objective】We have demonstrated that clusterin expression renders donor hearts resistance to cold IRI in transplantation. But the mechanism by which CLU prevents cell death at cold temperature is not completely understood. This study was designed to investigate the cytoprotective activity of clusterin at cold temperature in cultured myocytes.【Methods】CLU-KO myocyte line (MHC) established from primary myocytes isolated from the heart of a CLU-KO mouse, followed by immortalization with origin deficient SV40 DNA. The phenotype of cloned MHC was confirmed by RT-PCR. MHC was converted to clusterin-expressing myocytes (MHC-CLU) by stable expression of pHEX6300 vector containing human CLU isoform 1 cDNA, while MHC with stable expression of empty pHEX6300 vector were used as clusterin negative control cells (MHC-Con). Clusterin protein in protein extracts of cultured myocytes was examined by Western blot. Cell death/necrosis at 4℃in saline was measured using the levels of LDH release. The change of membrane fluidity in CLU-expressing MHC-CLU cells versus CLU null MHC-Con cells was investigated following exposure to cold temperature.【Results】The phenotype of cloned MHC was confirmed by its positive expression of both a-cardiac myosin heavy chain and connexin 43 transcripts, and negative expression of (3-cardiac myosin heavy chain and a-skeletal actin, using RT-PCR. Ectopic expression of clusterin protein in cellular extracts of MHC-CLU versus MHC-Con was determined by Western Blot. Only a slight increase in LDH release was seen in CLU-expressing MHC-CLU cell cultures, from 22.66±7.53% after 1 hr of incubation to 28.24±1.78% after 8 hrs of incubation, while in CLU null MHC-Con cell cultures, LDH release was largely induced following the exposure to 4℃, indicated by the increase of LDH release from 31.39±3.42% after 1 hr to 92.16±4.07% after 8 hrs of incubation (p<0.0001). Further study also shows that the membrane fluidity in MHC-CLU cells increased following cold exposure, evidenced by approximately 30% increase at the end of 6 hrs of incubation, while there was a trend of a slow decrease of membrane fluidity in MHC-Con cells.【Conclusion】Ectopic expression of CLU in CLU-KO myoytes stabilizes the cell membrane fluidity, resulting in protection of cells from cell membrane disruption, indicated by LDH release, at cold temperature, suggesting that CLU may function as a chaperone and can stabilize the cell membrane by its chaperone activity at cold temperature.PartⅢReduction of Cold Ischemia/Reperfusion Injury of Donor Organs by Supplement of Recombinant Clusterin Protein in Cold Preservation Solution【Objective】Donor organ injury during cold preservation prior to transplantation negatively impacts graft survival. Clusterin (CLU) is a chaperonic protein, and its expression confers donor hearts resistance to cold ischemic injury. This study was to evaluate if supplement of recombinant CLU protein (rCLU) protects donor organs from injury during cold storage with University of Wisconsin (UW) solution.【Methods】Western blot analysis of recombinant cluterin (rCLU) protein, purified from HEK 293 cell cultures. Human endothelial cell cultures were used as an in vitro model. Heart transplantation in mice was used as an in vivo model. Cell death was measured using lactate dehydrogenase (LDH) release or using a fluorescence microscope after stained with fluorescein diacetate-ethidium bromide solution. The intensity of rCLU on cell surface was measured using a flow cytometer. The cell membrane fluidity of endothelial cells was measured at different time points. Donor injury was determined by biochemical evaluation, its functional recovery and HE stain, the expression of MPO was detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.【Results】Western blot analysis showed that rCLU protein, isolated from HEK 193 cell cultures, was a heterodimer. Supplement of rCLU to UW solution remarkably protected cultured human endothelial cells from cold-induced cell necrosis, evidenced by a decrease in LDH release or in the number of ethidium bromide-stained necrotic cells. The protective activity of rCLU was associated with enhanced membrane fluidity at cold temperature. During cold storage of heart organs in UW solution, supplement of rCLU significantly reduced LDH release from heart tissue. In preclinical model of transplantation, heart grafts after cold preservation with rCLU-containing UW solution had a better functional recovery and less severity of perivascular inflammation, neutrophil infiltration and cardio injury including apoptosis and necrosis that correlated with lower levels of serum creatine kinase and LDH in recipients.【Conclusion】Our data suggest that supplement of CLU protein in a cold preservation solution may have potential in the improvement of cold preservation of donor organs in transplantation.