Developmental differences in myocardial protection in response to 5'-nucleotidase inhibition.

Age-related differences in the activity of 5'-nucleotidase, an enzyme responsible for conversion of high-energy phosphates to their the diffusible precursors, may help to explain age-related differences in tolerance of global myocardial ischemia. Postischemic function and high-energy phosphate content were measured in the hearts of rabbits 7 to 10 days old (neonate), 30 to 40 days old (1 month), and 6 to 12 months old (adult). Hearts in each age group were subjected to 60 minutes of ischemia at 34 degrees C either with no cardioplegia, with unmodified St. Thomas' Hospital cardioplegic solution, or with St. Thomas' Hospital cardioplegic solution with pentoxifylline, a 5'-nucleotidase inhibitor. These groups were compared with one another and with control hearts that were continuously perfused for 1 hour. In adults, addition of pentoxifylline to St. Thomas' Hospital cardioplegic solution restored adenosine triphosphate and total nondiffusible nucleotide levels to control values and improved recovery of cardiac output and developed pressure compared with results with unmodified St. Thomas' Hospital cardioplegic solution. In contrast, biochemical and functional parameters in neonatal hearts were not affected by either unmodified St. Thomas' Hospital cardioplegic solution cardioplegia or St. Thomas' Hospital cardioplegic solution with pentoxifylline. Functional recovery in neonatal hearts subjected to unprotected ischemia was superior to that in the older age groups. In 1-month-old hearts, St. Thomas' Hospital cardioplegia improved recovery compared with recovery after unprotected ischemia, but no incremental improvement in function or high-energy stores was seen with addition of pentoxifylline. The lack of effect of pentoxifylline on neonatal hearts suggest that there is a relative deficiency of 5'-nucleotidase in this age group. This may contribute to the improved functional recovery observed in unprotected hearts. Furthermore, addition of pentoxifylline to adult hearts appears to confer the benefits of low 5'-nucleotidase activity occurring naturally in the neonate.

Ultrastructural demonstration of peroxidative activity and peroxidation in ischaemic and ischaemic-reperfused rabbit hearts.

OBJECTIVE: The aim was to characterise subcellular histochemical evidence of the involvement of peroxidation and peroxidases in myocardial reperfusion injury. The histochemical technique involved the use of 3,3'-diaminobenzidine (DAB), which reacts with peroxides and proteins with peroxidase activity to form an electron dense polymer. METHODS: Isolated rabbit hearts were perfused (Langendorff method) for 30 min with oxygenated physiological saline solution. Some were subjected to 30 min of normothermic global ischaemia, with or without 30 min reperfusion. Non-ischaemic control hearts were perfused continuously for 90 min. Hearts were fixed with glutaraldehyde and cut into 100-150 microns sections that were incubated for 1 h in buffered DAB (1 mg.ml-1) with or without added KCN or H2O2. They were processed further for transmission electron microscopy. Planimetry was done on micrographs taken from random fields (approximately 500 photos). RESULTS: The total amount of DAB polymer in non-ischaemic control heart sections incubated with DAB alone occupied 1.19(SEM 0.44) micron 2 x 1000 micron-2 total cell area. For ischaemic-nonreperfused hearts, the value was 2.32(0.90) micron 2 x 1000 micron-2 (p = 0.223 v control); DAB occupied 7.49(1.42) micron 2 x 1000 micron-2 in ischaemic-reperfused hearts (p = 0.001 v control). DAB positive staining of mitochondria and lipid droplets, but not of peroxisomes, was significantly increased in reperfused hearts compared with non-ischaemic controls. CONCLUSIONS: Reperfusion, but not ischaemia, was associated with increased DAB staining. This suggests a reperfusion induced increase in myocyte peroxidation. Increased staining may be due to the actions of haem proteins with peroxidase activity on peroxidized lipid.