Hypothermic Injury: the Mitochondrial Calcium, ATP and ROS Love-Hate Triangle out of Balance

Abstract

Background/Aims: Catecholamines prevent hypothermic cell death which accounts for severe tissue damage and impaired allograft function after prolonged organ preservation. Here, we identified cellular processes which govern hypothermia-mediated cell death in endothelial cells and how they are influenced by dopamine. Methods: Lactate dehydrogenase assay, intracellular ATP, reactive oxygen species and reduced thio-group measurement, intracellular calcium measurement and mitochondrial calcium staining were performed in the study. Results: Intracellular ATP was almost completely depleted within 12 hrs of hypothermic preservation in untreated human umbilical vein endothelial cells (HUVEC), while dopamine pre-treatment significantly delayed ATP depletion. 4 hrs after hypothermia a redox imbalance was observed in untreated cells, which increased with the duration of hypothermia. The redox imbalance was primarily caused by depletion of SH reduction equivalents and was significantly inhibited by dopamine. In addition, hypothermia-induced Ca²⁺ influx and mitochondrial Ca²⁺ accumulation were both prevented by dopamine. The protective effect of dopamine was abrogated by ionomycin and sodium azide and partly by oligomycin and CCCP. Conclusions: Our data demonstrated that loss of intracellular ATP, generation of a redox imbalance and accumulation of intracellular Ca²⁺ underlie cold preservation injury. Dopamine improves the redox balance, prevents intracellular Ca²⁺ accumulation and delays ATP depletion.

Dopamine and Lipophilic Derivates Protect Cardiomyocytes against Cold Preservation Injury

“…Another study on ischemia/ reperfusion injury in neonate rat cardiomyocytes supports that cardiac D 1 receptors are not involved in cytoprotection, because selective activation of D 1 receptors with the agonist SKF-38393 (1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol) induces apoptotic cell death (Li et al, 2008). As NOD has been shown not to act on dopamine receptors (Lösel et al, 2010), our data clearly support that DA's cytoprotective action does not require dopamine receptor engagement (Brinkkoetter et al, 2006(Brinkkoetter et al, , 2008Benck et al, 2011). Direct evidence to underpin this assumption is provided by the data demonstrating that the protective effects of DA and NOD are not abrogated by the D 1 /D 2 receptor antagonist fluphenazine.…”

Effects of Donor Pre-Treatment With Dopamine on Survival After Heart Transplantation

“…Strategies that reduce intracellular calcium with novel hyperpolarizing preservation solutions have been shown to ameliorate ischemic damage during cold preservation in experimental heart transplantation (27). We were able to demonstrate that dopamine decelerates the deleterious amplification loop of intracellular calcium accumulation, and subsequent ATP consumption by scavenging of reactive oxygen species (9,30). These findings have been recently expanded and confirmed by in vitro studies with cardiomyocytes in culture (31).…”

“…We recently presented a randomized, controlled clinical trial that showed that pre-treatment of brain-dead donors with low-dose dopamine of 4 g/kg/min reduces the dialysis requirement after kidney transplantation (8). The underlying mechanism is related to antioxidant properties of the dopamine molecule (9). In the particular setting of organ preservation, dopamine increases the tolerance of the kidney graft to withstand ischemic damage during cold storage.…”

Use of a Third-Generation Perfluorocarbon for Preservation of Rat DCD Liver Grafts

“…Even in this reduced metabolic state, prolonged ischemic time leads to depletion of energy stores and subsequent loss of cellular integrity [15][16][17]. Providing additional oxygen as substrate for metabolism during both warm and cold ischemia, third generation PFCs are able to extend the period of time where aerobic metabolism is maintained and, therefore, delay the onset of tissue damage associated with prolonged preservation [18,19].…”