Hypothermia Attenuates Brain Injury via HECTD1-Mediated VDAC3 Degradation in a Rat Model of CPR

Introduction: Neurological injury significantly contributes to poor outcomes after return of spontaneous circulation (ROSC) following cardiac arrest (CA). Prior in vitro studies showed that hypothermia promotes VDAC3 ubiquitination, reducing microglial apoptosis under ischemia/reperfusion (I/R) conditions. This study explored the in vivo role of the E3 ubiquitin ligase HECTD1 in mediating VDAC3 degradation and neuroprotection during therapeutic hypothermia.


Methods: Adult male Sprague–Dawley rats underwent 8 minutes of asphyxial CA followed by standardized cardiopulmonary resuscitation (CPR). Rats were randomized into normothermia (T37, core temperature 37 ± 0.5°C) and hypothermia (T33, 33 ± 0.5°C) groups, initiated 5 minutes post-ROSC and maintained for 6 hours. Each temperature group was subdivided into PBS control, si-NC (non-targeting siRNA), and si-Hectd1 subgroups. The si-Hectd1 group received intracerebroventricular injection of adeno-associated virus encoding Hectd1-targeting siRNA. Survival was monitored for 72 hours. Neurological function were assessed at 72 hours after ROSC, including neurological deficit score (NDS), serum S100B and NSE levels, and histopathological evaluation (H&E, electron microscopy). Apoptosis-related protein expression (VDAC3, cleaved caspase-3, BAX, BCL-2) was measured by western blot. HECTD1–VDAC3 interaction and colocalization were analyzed via co-immunoprecipitation and immunofluorescence.


Results: Hypothermia significantly improved 72-hour survival and neurological outcomes compared to normothermia. The T33 group demonstrated reduced expression of VDAC3, cleaved caspase-3, and BAX, and increased HECTD1 and BCL-2 levels (p < 0.05). Hypothermia enhanced the interaction between HECTD1 and VDAC3 and promoted VDAC3 ubiquitination, which were abrogated by Hectd1 knockdown. The T37+si-Hectd1 subgroup exhibited the worse survival and neurological outcomes. No significant differences were observed between PBS and si-NC subgroups at either temperature.


Conclusions: Therapeutic hypothermia mitigates brain injury following CA/CPR by promoting HECTD1-dependent VDAC3 degradation and suppressing apoptosis.These findings suggest a novel molecular mechanism underlying hypothermic neuroprotection and identify HECTD1 as a potential therapeutic target.