A human Troponin I diagnostic kit cross-reacts with cultured cardiomyocytes and heart tissues derived from other mammalian species.

Tissue injury leads to the disruption of cellular membrane integrity and loss of cytoplasmic components into the extracellular space. Myocardial damage results in elevated levels of cytosolic and structural proteins belonging to injured cells such as the wider expressed aspartate aminotransferase (AST) or the cardiac specific Troponin I (cTnI). In this study, we tested a human cTnI diagnostic kit developed by Siemens for cross-reactions with cultured cardiomyocytes (CMs) and heart tissues derived from several animal species. cTnI structure is highly conserved across species and the assays validated for human cTnI can represent a valuable tool to estimate cardiac suffering in animals. We collected post-mortem cardiac tissue and we tested the cross reactivity of anti-cTnI antibodies in animal heart homogenates. As expected, homogenates collected from mammals showed a robust cross-reactivity with human cTnI immunoassay. In order to reproduce in vitro a cardiac ischemia/reperfusion injury (IRJ) model using murine cells, we improved a CM isolation protocol starting from neonatal mouse hearts that results in primary culture of cardiac fibroblasts and CMs. Using human diagnostic laboratory kits we were able to detect murine AST by an enzymatic activity assay as well as murine cTnI by a sandwich immunoassay based on luminescent oxygen channeling (LOCI™) technology. Indeed, we confirmed the specificity of cTnI dosage for CMs, at variance with AST that recognize also fibroblasts, and we were able to correlate cTnI amount with CM number. Next, CMs were challenged in extreme cultural conditions such as hypoxia and reoxygenation and both these events will cause CM damage. Similarly to the evaluation of cardiac markers in circulating blood, released cTnI was directly estimated in culture supernatants. In conclusion we confirm the specificity and the sensitivity of cTnI quantification in identifying not only human but also mammalian CMs and we suggest the use of a high sensitivity cross-reactive diagnostic kit for an accurate evaluation. We are convinced that this finding will surely help in assessing hypothetical myocardial damage in mammals and in cellular cardiovascular-related models.