Die Rolle von Mitochondrien bei kalter Ischämie/Reperfusion und chronischer Abstoßung im Rahmen der Herztransplantation

Abstract

Introduction: Prolonged cold organ storage as a non-immunologic risk factor in the pathogenesis of chronic rejection was evaluated in the Lew to F344 rat cardiac allograft model. Material and Methods: Hearts were transplanted without cold storage (CR, n=20) or subjected to 10 h of cold ischemia at 0–1 °C and then transplanted (CRI, n=28). Both groups were compared with corresponding syngeneic control groups. After evaluation of graft function (cardiac score), hearts were retrieved at 2, 10, 40 and 60 days. Degree of vasculopathy was investigated by means of H & E histology. For investigation of underlying mechanisms intragraft gene expression was analysed employing cDNA microarray technology (“MWG Rat 10k Array”, MWG®containing 9715 cDNA sequences) and RT-PCR. A cluster of transcripts regulated in response to cold ischemia as well as chronic rejection were genes encoding for enzymes involved in mitochondrial activity. We therefore analysed mitochondrial function using high- resolution respirometry of permeabilized myocardial fibers. In addition, enzymatic defects were assessed in myocardial homogenates. Results: Cardiac score declined from score 4 at 2 days after reperfusion to score 1.6 ± 0.5 at 60 days. Impairment of organ function was faster in the CRI when compared to the CR group and was nearly absent in isografts. Histology revealed myointimal proliferation and fibrosis after 40 and 60 days in allogeneic groups. Both findings were significantly more pronounced in the CRI group. Microarray analysis allowed for detection of a set of 82 genes regulated at 2 and 10 days in response to cold ischemia in allogeneic groups. In contrast, only 20 genes were regulated in corresponding isografts. 60 Genes were up- or downregulated during chronic rejection and 22 genes were regulated only in chronically rejected grafts undergoing 10 h of cold ischemia prior to transplantation. Investigated genes mainly comprised transcripts involved in myocyte function, humoral immune response and enzymes involved in mitochondrial activity. Mitochondrial respiration declined with all substrates used, indicating the general loss of mitochondria rather than specific defects of respiratory chain complexes. The activity of the mitochondrial matrix enzyme citrate synthase was relatively stable in syngeneic grafts, but decreased substantially (up to 80% at 60 days) in both experimental groups and correlated well with cardiac score. However, enzymatic changes were more evident in CRI group. In contrast, the activity of mitochondrial respiratory chain enzyme complex I equally declined in all groups and did not match with cardiac score. Activity of cytosolic enzyme lactate dehydrogenase was similar in CRI and CR, decreasing to 50% of syngeneic controls at 60 days. Conclusion: Prolonged CI substantially contributes to the progression of chronic rejection and loss of organ function in this model. Patterns of gene expression as well as enzymatic injuries reflect severe mitochon-drial damage in response to CI and progression of chronic rejection.