Fumarate hydratase downregulation in myocardial tissue: a potential biomarker for death from mechanical asphyxia

The authentication of death from mechanical asphyxia (DMA) remains a significant challenge in forensic pathology because of the lack of specific biomarkers. The present study identified fumarate hydratase (FH), a key tricarboxylic acid (TCA) cycle enzyme, as a potential biomarker associated with DMA and investigated its regulatory mechanism in the context of acute severe hypoxia. Western blot analysis of 87 human myocardial samples revealed that FH protein expression was significantly downregulated in DMA cases than in controls (hemorrhagic shock, brain injury, and other causes). These findings were independently validated by immunohistochemistry (IHC) in a separate cohort of 118 human samples. The diagnostic performance of myocardial FH, evaluated by receiver operating characteristic curve analysis, yielded an area under the curve of 0.8561, with 84.38% sensitivity and 70.93% specificity. In vitro experiments using AC16 cardiomyocytes revealed that hypoxia led to decreased FH expression at both the mRNA and protein levels, and this effect was reversed by the N-acetylcysteine antioxidant. FH downregulation resulted in significant accumulation of its substrate, fumarate, in human DMA myocardium, hypoxic cells, and culture medium. This subsequent accumulation of fumarate triggered the activation of the cytoprotective KEAP1-NRF2 pathway, as evidenced by NRF2 nuclear translocation and the upregulation of downstream antioxidant genes. Collectively, the present results support myocardial FH downregulation as a promising adjunctive biomarker associated with DMA and reveal a novel hypoxia-FH-fumarate-NRF2 axis, providing crucial insights into cellular responses to fatal hypoxic injury. KEY POINTS: • FH expression is significantly reduced in myocardial tissue from mechanical asphyxia cases. • FH immunohistochemistry shows potential utility for the postmortem identification of mechanical asphyxia. • Hypoxia-induced FH suppression leads to fumarate accumulation in cardiomyocytes. • Fumarate accumulation activates the KEAP1-NRF2 antioxidant pathway.