Cold ischemia contributes to the development of chronic rejection and mitochondrial injury after cardiac transplantation.

Chronic rejection (CR) remains an unsolved hurdle for long-term heart transplant survival. The effect of cold ischemia (CI) on progression of CR and the mechanisms resulting in functional deficit were investigated by studying gene expression, mitochondrial function, and enzymatic activity. Allogeneic (Lew→F344) and syngeneic (Lew→Lew) heart transplantations were performed with or without 10 h of CI. After evaluation of myocardial contraction, hearts were excised at 2, 10, 40, and 60 days for investigation of vasculopathy, gene expression, enzymatic activities, and mitochondrial respiration. Gene expression studies identified a gene cluster coding for subunits of the mitochondrial electron transport chain regulated in response to CI and CR. Myocardial performance, mitochondrial function, and mitochondrial marker enzyme activities declined in all allografts with time after transplantation. These declines were more rapid and severe in CI allografts (CR-CI) and correlated well with progression of vasculopathy and fibrosis. Mitochondria related gene expression and mitochondrial function are substantially compromised with the progression of CR and show that CI impacts on progression, gene profile, and mitochondrial function of CR. Monitoring mitochondrial function and enzyme activity might allow for earlier detection of CR and cardiac allograft dysfunction.

Evaluation of mitochondrial respiratory function in small biopsies of liver.

Mitochondrial respiratory function was studied in permeabilized pig liver biopsies. The cell membrane was permeabilized mechanically in tissue samples of 2-7 mg, for application of a standardized substrate/inhibitor titration protocol in high-resolution respirometry. Specific respirometric tests demonstrated complete plasma membrane permeabilization and accessibility of substrates to intact mitochondria. High respiratory adenylate control ratios and cytochrome c conservation in the tissue preparation were comparable or even better than in isolated mitochondria. Citrate synthase and cytochrome c oxidase activities remained at 85% of controls after up to 98 h storage of liver tissue at 0 degrees C in histidine-tryptophan-ketoglutarate solution. Multiple mitochondrial defects, however, were indicated after 48 h cold storage by the decline in respiratory capacity, which was lowered to a larger extent with complex I substrates compared to respiration with substrates for complex II or IV, measured in the absence of cytochrome c. After prolonged ischemia, the adenylate control ratio was significantly reduced, and cytochrome c depletion was detected by the stimulatory effect of cytochrome c. High-resolution respirometry allows the assessment of mitochondrial function in a few milligrams of permeabilized liver tissue, without isolation of mitochondria. This provides a basis for the analysis of mitochondrial function in human liver biopsies.