Mitochondria transmit apoptosis signalling in cardiomyocyte-like cells and isolated hearts exposed to experimental ischemia-reperfusion injury

Abstract

Ischemia-reperfusion (I/R) is a condition leading to serious complications due to death of cardiac myocytes. We used the cardiomyocyte-like cell line H9c2 to study the mechanism underlying cell damage. Exposure of the cells to simulated I/R lead to their apoptosis. Over-expression of Bcl-2 and Bcl-x_L protected the cells from apoptosis while over-expression of Bax sensitized them to programmed cell death induction. Mitochondria-targeted coenzyme Q (mitoQ) and superoxide dismutase both inhibited accumulation of reactive oxygen species (ROS) and apoptosis induction. Notably, mtDNA-deficient cells responded to I/R by decreased ROS generation and apoptosis. Using both in situ and in vivo approaches, it was found that apoptosis occurred during reperfusion following ischemia, and recovery was enhanced when hearts from mice were supplemented with mitoQ. In conclusion, I/R results in apoptosis in cultured cardiac myocytes and heart tissue largely via generation of mitochondria-derived superoxide, with ensuing apoptosis during the reperfusion phase.     

Apparent superoxide dismutase-like activity of immunoglobulin

Summary

Using various superoxide generating systems and nitroblue tetrazolium or cytochrome c as superoxide detector molecules it is possible to assess the superoxide dismutase activity of proteins. Intact antibodies raised to different antigens, the Fab’ fragment of anti-TNF [M632] and well-characterized recombinant Fv fragments of the murine antibody NQ11.7.22 appear to possess superoxide dismutase (SOD)-like activity.

Kinetic characteristics of the SOD-like activity of NQ11.7.22-Fv fragments suggest an enzymatic property and these fragments behave in an analogous manner to human erythrocyte Cu-Zn SOD. Furthermore, the SOD-like activity of the NQ11.7.22-Fv fragment is affected by certain single-point mutations in the amino acid composition and has a pH optimum of 6.2–6.6 which is unlike Cu-Zn SOD (pH 7.8–8.2). A change in tyrosine at the 32 position in the heavy chain and histidine at position 27 of the light chain of the NQ11.7.22-Fv fragment results in a profound reduction in SOD-like activity. Tyrosine at the 32 position in the heavy chain is known to play a significant role in antigen binding suggesting that the SOD-like activity occurs at the antigen-binding site itself. Single-point mutations at the periphery of the antigen combining site on the NQ11.7.22-Fv fragment had little or no effect on SOD-like activity.

Further studies show that immunoglobin (lgG-1), a commercially available murine monoclonal antibody, can also enhance the generation of hydrogen peroxide, the product of superoxide dismutation, when present in superoxide producing systems. The generation of hydrogen peroxide was increased by low pH (pH 6.25) with lgG-1 but reduced with Cu-Zn SOD.