Therapeutic Hypothermia Ameliorates Apoptosis and Cerebral Injury by Upregulating HECTD1-mediated Ubiquitination and VDAC3 Degradation in a Rat CPR Model.

Background and Objective: Post-cardiac arrest (CA) brain injury is a leading cause of death and disability in patients with return of spontaneous circulation (ROSC). Our previous study showed that hypothermia-induced VDAC3 ubiquitination attenuated apoptosis of microglia cells following simulated ischemia/reperfusion (I/R). This study aimed to further investigate the role of E3 ubiquitin ligase, HECTD1, in the ubiquitination and degradation of VDAC3, which is involved in hypothermic neuroprotection. Methods: Sprague-Dawley rats were randomized into two groups, namely the normothermic (T37) group and hypothermic (T33) group, which were further divided into three subgroups: PBS, si-NC, and si-Hectd1. All the rats were subjected to 8 min of asphyxia-induced CA and cardiopulmonary resuscitation (CPR). The core temperature in the T33 and T37 groups were maintained at 33 ± 0.5 °C and 37 ± 0.5 °C, respectively, after 5 min following ROSC and maintained for 6 h. The si-Hectd1 subgroups were administered adeno-associated viral vector with siRNA against rat Hectd1 (si-Hectd1). The PBS and si-NC subgroups were administered the same volume of PBS and nonspecific siRNA, respectively, as controls. Survival time was measured for the resuscitated rats. At 72 h after ROSC, the neurological injuries to rats were evaluated using neurological deficit scores, Serum S100B and neuron-specific enolase levels, hematoxylin and eosin staining, and transmission electron microscopy analysis. Apoptosis-related proteins were measured using western blot assay. The interaction and colocalization of HECTD1 and VDAC3 were evaluated by immunoprecipitation and double immunofluorescence assays, respectively. Results: Survival time and neurological outcomes, as well as ultrastructural damage, were significantly improved in the T33 group, with the T37 + si-Hectd1 subgroup showing the worst outcome at ROSC 72 h. Compared to the T37 group, the T33 group showed reduced expression of VDAC3, cleaved caspase-3, and BAX but increased expression of HECTD1 and BCL-2 at ROSC 72 h. Additionally, compared to the T37 group, the T33 group showed increased VDAC3 and Hectd1 interaction, resulting in increased VDAC3 ubiquitination at ROSC 72 h; Hectd1 knockdown reversed these effects. The PBS and si-NC subgroups showed no significant differences under both targeted temperatures. Conclusions: Hypothermia treatment contributed to greater antiapoptotic neuroprotection in the rat CA/CPR model, which may be attributed to the promotion of HECTD1-mediated ubiquitination and degradation of VDAC3.