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Purpose: We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage., Methods: Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products., Results: During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used., Conclusions: Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

Low density lipoprotein cholesteryl [14C]oleate (LDL-[14C]CO) was used as a tool to label lysosomes with cholesteryl [14C]oleate (CO) and to follow subsequently the metabolic processing of oleic acid released by acid lipase. Liberated [14C]oleate was incorporated into glycerolipids, mainly into phosphatidylcholine. Incubations in the presence of various concentrations of exogenously added oleic acid and double label experiments showed that oleic acid derived from lysosomal degradation of CO and exogenously added oleic acid distributed in a similar fashion among triacylglycerol and various phospholipids. To further study the metabolism of LDL-derived oleic acid, experiments were performed in which fibroblasts were prelabeled with LDL-[14C]CO. The subsequent processing of lysosome-derived oleic acid was followed with time without LDL-[14C]CO in the medium. From these experiments it became clear that apart from the esterification into glycerolipids a substantial part of lysosome-derived oleic acid was released into the medium. The efflux of oleic acid into the medium preceded the incorporation into glycerolipids, was dependent on the composition of the extracellular medium, and was energy-independent. Our data are compatible with a mechanism in which lysosome-derived fatty acids are transported to the plasma membrane prior to transport to endoplasmic reticulum for esterification. Intra- and extra-cellular factors influence the distribution of lysosome-derived oleic acid among cells and medium.