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.