Introduction Iron overload-cardiomyopathy (IOCM) is an increasing clinical problem worldwide. 70% of patients who receive compulsory blood transfusion die of IOCM, with increased susceptibility to arrhythmias and sudden death. We have previously found that iron exposure impairs cardiomyocyte Ca homeostasis. However, the cellular mechanisms responsible are unknown. Iron is known to participate in the Fenton reaction to produce reactive oxygen species (ROS), which mediate oxidative damage. We therefore tested the hypothesis that increasing the anti-oxidant capacity of cardiomyocytes, with the ROS scavenger Tempol, could be cardioprotective in the presence of iron.

Methods Single rat ventricular cardiomyocytes were loaded with fluo-3 to monitor intracellular Ca changes upon stimulation while bathed in control Tyrode solution and after adding ferrous iron (iron II). Sarcoplamic reticulum (SR) Ca load and sarcolemmal Ca extrusion rates were estimated during exposure to caffeine, which empties SR Ca stores. The ROS scavenger tempol was used to dissect ROS-mediated pathways from the direct effects of iron II on Ca handling. Data are provided as mean±SEM. Significance was tested using paired student t test and defined as p<0.05.

Results Iron II exposure significantly increases systolic Ca transient amplitude (mean increase 82.8±21.8%, n=9) and causes spontaneous arrhythmogenic Ca release events (SACRE). These changes corresponded with increased SR Ca content (mean increase 21.0±5.7%, n=8), which is known to impact on systolic Ca release and spontaneous activity in cardiomyocytes. Sarcolemmal Ca extrusion rate was also significantly reduced upon iron II exposure (measured as the rate of fall of the caffeine response; mean decrease 48.7±5.5%, n=8), consistent with an overall gain of Ca by the cardiomyocyte. The onset of these Ca disruptions was significantly delayed in the presence of the ROS scavenger tempol (p<0.001). Without tempol, SACRE onset occurred after 6.9±0.6 min (n=22) following iron II exposure. The same manoeuvre in in tempol delayed the onset of SACRE to 17.8±1.8 min (n=7). Furthermore, increasing ROS scavenging reversed the increase in systolic Ca transient amplitude, as well as SACRE upon washout of iron II. In contrast, in cardiomyocytes not exposed to tempol, the effects of iron II were irreversible.

Conclusions Our data show that iron II disrupts cardiomyocyte Ca handling. This is mediated via inhibition of sarcolemmal Ca extrusion, leading to SR Ca overload and SACRE. These are the initiators of most fatal non-reentrant arrhythmias and cardiac sudden death in experimental models. The observed effects are partly due to iron II-mediated oxidative damage. This was confirmed by the presence of a ROS scavenger delaying the onset of the effects of iron II, and crucially rendering the effects reversible upon iron-washout. These effects of tempol suggest a novel therapeutic target for the treatment of IOCM patients.