Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study

The aim of this post-mortem ultrastructural and immunohistochemical study is to explore the characteristics of acute myocardial ischemia in the context of sudden death, using the combination of two different methods, both more insightful than ordinary histology. Transmission electron microscopy and immunohistochemistry, in addition to the traditional histology, were applied to study human heart specimens collected during forensic autopsies. The whole series was sub-grouped into cases (n=17) and controls (N=10). The control group consisted of unnatural death with a short agonal period (immediately lethal injuries). Heart samples of the two cohorts of subjects were prepared for electron microscopy. On the other hand, each specimen, formalin fixed and paraffin embedded, was stained with haematoxylin and eosin and immunoreacted with the following primary antibodies: anti-Fibronectin, anti- Connexin-43, anti-npCx43 (dephosphorylated form of Connexin43), anti-Zonula occludens-1. Immunopositivity for each marker in the myocardium was semi-quantitatively graded. Electron microscopy revealed a number of interesting differences, statistically significant, between acute myocardial ischemia and controls, regarding the morphology of nucleus, mitochondria and intercellular junctions. By immunohistochemistry, fibronectin was found to be increased in the extracellular matrix of the acute myocardial ischemia cases, with a statistically significant difference compared to the controls. Connexin 43 staining disclosed a slight increase (not statistically significant) in the cytoplasm of acute myocardial ischemia cases compared to the controls, whereas no significant differences were seen between cases and controls at intercellular junctions. npCx43 showed an evident difference of intensity and pattern (even though not statistically significant) in cases compared to controls and overall this difference was more evident in the cytoplasm. Zonula occludens 1, described as an important marker for functional modification of cardiac muscle fibers, resulted negative or very weak in the vast majority of both cases and controls. The present study attempts to simultaneously apply electron microscopy and immunohistochemistry, in order to figure out the morphological changes that might lead to pathological processes underlying the sudden, unexpected death due to acute myocardial ischemia, and consequently to find useful diagnostic markers of very early ischemic injury. Both methods showed significant differences between acute myocardial ischemia and controls, regarding, overall nuclei, mitochondria, and intercellular junctions.