High energy phosphate depletion in leaflet matrix cells during processing of cryopreserved cardiac valves

Preimplantation preparation of cardiac valves includes three major steps: (1) harvesting with accompanying ischemia (warm time from cessation of donor heart beat), (2) antibiotic disinfection, and (3) controlled-rate cryopreservation. To define the interdependent injury effects of these manipulations on leaflet matrix cells and specifically the potential for prolonged harvest-related ischemia to predispose greater injury by the subsequent steps, 96 semilunar valves were harvested from pigs in a manner analogous to human heart valve retrievals and randomly allocated to study groups as follows: 48 control valves were exposed to increasing harvested-related ischemic times, (2, 6, 12, 24 hr) and immersed in liquid nitrogen to arrest metabolic activity (i.e., prior to cryopreservation) and conclude the ischemia; another 48 were similarly harvested, subjected to identical ischemic times, then disinfected in 4 degrees C RPMI medium with standard antibiotics for 24 hr and dimethylsulfoxide cryopreserved at -1 degrees C/min to -170 degrees C (i.e., formal cryopreservation protocol). At thawing, each valve was extracted in 12% trichloroacetic acid and assayed by high performance liquid chromatography for components of the adenine nucleotide pool including ATP, lower energy nucleotides (total adenine nucleotides, [TAN] = [ATP] + [ADP] + [AMP]), adenosine, and the diffusible purines. Results are reported as nanomoles metabolite/milligram of leaflet cell protein (Lowry) and reflect a maintenance of total high energy phosphates in the control groups (5.41 +/- 0.29 nmole TAN at 2 hr; 8.34 +/- 0.67 nmole TAN at 24 hr), which fell significantly in all cryopreserved groups (1.27 +/- 0.33 nmole TAN at 2 hr; 0.34 +/- 0.22 nmole TAN at 24 hr).(ABSTRACT TRUNCATED AT 250 WORDS)