ComplementC3 regulated cell death in myocardial ischemia/reperfusion injury

Abstract

Ischemic heart disease is a life-threatening condition and requires immediate treatment by unblocking the occluded blood vessel. However, the restoration of blood flow to the ischemic area causes additional damage, named ischemia/reperfusion injury (I/R injury).Currently, there is no clinically approved therapy available to reduce I/R injury. Basic research suggests that myocardial cell death, i.e., necrosis and apoptosis, are key events in I/R injury. Our group previously found that complement and ROS were involved in different stages of necrosis development during the early phase of I/R injury in a murine myocardial model. In particular, SOD1 overexpression significantly reduced necrosis at 1 hour of reperfusion, while catalase overexpression reduced necrosis at 3 hours’ reperfusion. The inhibition of I/R injury by SOD1 and catalase was transient, and I/R injury appeared to be restored at 24 hours’ reperfusion. Meanwhile, complement C3 deposition became significant at 3 hours’ reperfusion and reached the peak at 24 hours of reperfusion. Therefore, we hypothesized that SOD1 and catalase are important in regulation of ROS mediated I/R injury during the first 3 hours of reperfusion, while complement may contribute to I/R injury thereafter. This thesis investigated how complement C3 regulates cell death during I/R injury after 3 hours of reperfusion. In Aim 1 of this study using the I/R mouse model, we found that after 1 hour ischemia/ 24 hours reperfusion, the level of necrosis in C3-/- mice was significantly lower than that in WT mice, while the level of apoptosis in C3-/- mice was significantly higher than that in WT mice. Furthermore, we found that 4 weeks after the initial 1 hour of ischemia, C3-/- mice had significantly less cardiac fibrosis and better cardiac function than WT mice. Our comparative proteomics analyses showed that Cyt c, a key factor in the intrinsic apoptotic pathway, was preferentially present in the C3-binding complexes in WT mice after 3 hours of reperfusion. These results indicate that C3 may promote necrosis and inhibit apoptosis in myocardial I/R injury. In Aim 2 of our study, we further explored the mechanism of C3-mediated apoptosis using an in vitro system of AC16 human ventricular cardiomyocyte cell line. We found that although AC16 cells do not express C3, they can uptake exogenous C3 when exposed to it at 37°C. The uptake of C3 was not significant at low temperature(4°C), suggesting a receptor-mediated uptake of C3. However, AC16 cells do not express the known complement receptors 1, 2, 3 and 4. Thus, C3 uptake in AC16cells is likely through unknown receptor(s). Incubation of exogenous C3 could significantly reduce H2O2-induced-apoptosis in AC16cells. In a cell free apoptosis system, we found C3 could significantly reduce the intrinsic pathway apoptosis, possibly through interaction with factor(s)downstream of Cyt c. In a cell free pull-down assay, pro-C3 was able to bind with the apoptotic factor pro-caspase 3.

In summary, our results showed that 1)At early stage of I/R injury, complement C3 can promote necrosis and apoptosis. At the late stage of I/R injury, C3 promotes cardiac fibrosis and causes worse cardiac function. 2) Human cardiomyocytes (AC16 cells), which did not expressC3, readily uptake the exogenous C3 from extracellular milieu. The uptake of C3into AC16 cells is likely through a receptor-mediated endocytosis, although the identity of the receptor is still unknown. 3) The inhibition of oxidative-related apoptosis by exogenous C3 in AC16 cells is very likely through the binding with apoptotic factor(s) in the intrinsic apoptosis pathway.