Cardiac-specific Overexpression Of E3 Ligase Nrdp1 Increases Ischemia And Reperfusion-induced Cardiac Injury

Background and aims:Cardiomyocyte death is a major event of myocardial infarction and heart failure. Previous studies have demonstrated that ubiquitin E3 ligase-mediated protein degradation plays a critical role in cell apoptosis and inflammation. However, the role of Nrdp1 in regulating cardiac injury remains unclear. In this study, we sought to determine the role of a newly identified E3 ligase, neuregulin receptor degradation protein-1 (Nrdp1), in cardiac ischemia/reperfusion (I/R) injury.Methods:(1) Mice were subjected to left anterior descending coronary artery occlussion to induce myocardial infarction. (2) Neonatal rat cardiomyocytes were isolated and infected with Ad-GFP, Ad-Nrdp1 or Ad-Dn-Nrdp1 and exposed to 2-hour ischemia followed by reperfusion for 24 hours. TUNEL assay and Western blot and RT-PCR analysis were used to examine the effect of Nrdp1 adenoviral transfection on cardiomyocyte apoptosis and inflammation in cells. (3) Transgenic mice expressing Flag-tagged Nrdp1 in the heart were generated and identified by PCR. Cardiac function was measured by Echocardiography. After I/R injury, TTC staining was performed to measure myocardial infarct size and survival rates were compared. TUNEL assay and Western blot were used to examine cardiomyocyte apoptosis. H&E-staining and immunohistochemical staining were used examine cardiomyocyte injury and inflammatory cell infiltration in mouse hearts. (4) To elucidate the molecular mechanism of cardiac-specific expression of Nrdp1 enhancing I/R injury, Western blot analysis was used to examine the effect of Nrdp1 expression on ErbB3,BRUCE,TBK1,MyD88,AKT,STAT3 and MAPKs in vivo and in vitro after I/R.Results:We found that I/R markedly upregulated Nrdp1 expression in the heart tissue; Neonatal cardiomyocytes were infected with adenoviral constructs expressing wild-type, dominant-negative Nrdp1 genes. Infection of neonatal cardiomyocytes with adenovirus wild-type Nrdp1 (Ad-Nrdp1 WT) markedly enhanced I/R-induced cardiomyocyte apoptosis and inflammation as compared with the adenovirus green fluorescent protein (Ad-GFP) control, whereas these effects were attenuated by overexpression of a dominant-negative Nrdp1 (Ad-DnNrdp1 C34S/H36Q). Furthermore, cardiac-specific Nrdp1 expression in vivo significantly increased infarct size, the number of TUNEL-positive nuclei and infiltration of inflammatory macrophages, as well as mouse mortality, as compared with wild-type mice after I/R injury. Finally, overexpression of Nrdp1 WT significantly downregulated Nrdp1 substrate, ErbB3, decreased the activation of its downstream targets AKT, STAT3 and ERK and increased the activation of p38 and JNK.Conclusion:Together these results provide evidence for an important role for Nrdp1 in regulating I/R-induced cardiac injury. Nrdp1 may constitute a new therapeutic target in ameliorating the I/R-induced cardiac injury.