CARD6: Warm Ischemic Time, Cold Static Storage and Impairment of Cardiac Endoplasmic Reticular Stress in Human Hearts Donated After Circulatory Death and Brain Death

Background: Endoplasmic reticulum (ER) plays an important role in maintaining normal myocardial function at the cellular level. Overexpression of ER stress-associated proteins during ischemia impact cardiac function. Initial result of heart transplantation using donation after circulatory death (DCD) heart with short warm ischemic time (WIT, less than 30 minutes) was encouraging. However, little is known about the impact of various WIT on donor heart and with subsequent cold storage as used in normothermic regional perfusion(NRP) on the DCD hearts. In this study we investigated expression of myocardial ER stress response proteins in the DCD hearts to evaluate the impact of warm ischemic time (WIT) and subsequent cold storage. We also studied expression of myocardial ER stress response proteins in the brain death donor (DBD) hearts as a control group. Methods: A total of 23 human hearts, DCD group, n=17, with 3 WIT sub-groups [WIT 20 (n=6), 40 (n=6), ≥60 (n=5) minutes] and DBD group, n=6, no warm ischemia (control: WIT:0) were studied. All the DCD hearts received del Nido cardioplegia and all DBD hearts received Celsior solution before they were placed at cold storage for 6 hours. Left ventricular biopsies were performed at hour 0 (T0), 2 (T2), 4 (T4) and 6 (T6). Myocardial ER stress response proteins ATF6 (Activating Transcription Factor 6) and GRP78 (78-kDa glucose-regulated protein) were measured through immunoblotting after normalizing to GAPDH loading control in all cardiac biopsies. Results: Representative western blots showing the protein expressions patterns among the DBD and three different groups of DCD hearts under increasing time points (T0:0hrs, T2:2hrs, T4:4hrs, T6:6hrs) of cold static storage conditions (Fig.1A). At baseline, the proteins expression found to be comparable between DBD, DCD:20 and 40 groups, while DCD:≥60 group remained significantly higher (Fig.1B,C). We noticed differential temporal increase of ER stress proteins after 2 hours of cold storage and became prominent at hour 6 in DCD groups when compared to DBD group; while the DCD:60 group always remained highly significant throughout the cold storage compared to all other groups (Fig.1D,E). Conclusions: Our study concluded that duration of warm ischemia and subsequent cold storage condition in DCD hearts were associated with elevated ER stress response in myocardium. The length of cold storage was also found to be linked with elevated ER stress in DCD hearts. The DCD hearts with more than 60 min warm ischemic has the highest ES stress response suggesting profound alteration in ER homeostasis which may promote cardiac damage. Such findings are important in guiding clinical decision making on selecting DCD donor hearts. On the other hand the DBD hearts with up to 6 hour cold storage exerted minimal ER stress, supporting current DBD heart transplant donor preservation practice.