P537Characterisation of electrical activity in the infarct region of rat hearts using widefield epifluorescence and 2-photon excitation techniques

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Abstract

Interruption of the coronary blood supply due to occlusion of coronary vessels leads to ischaemia in the tissue supplied by these vessels. If left untreated, the myocardium becomes necrotic and is replaced by a connective tissue scar. This scar is maintained by a newly developed vasculature that supports the intrinsic fibroblasts and few surviving myocytes. The infarct scar acts as a non-contractile region of the myocardium, but the electrical consequences for the heart are poorly understood. In this study, electrical activity in the infarct scar was investigated using widefield epifluorescence and 2-photon (2P) excitation techniques.

Male Wistar rats were anaesthetised and the left anterior descending coronary artery was ligated to induce myocardial infarction. 4 weeks post-procedure, hearts were isolated and Langendorff-perfused with oxygenated Tyrode's solution containing 10mM BDM and 10µM blebbistatin to inhibit contraction. Hearts were paced at 5Hz and loaded with the voltage sensitive dye di-4-ANEPPS. Widefield recordings were made using a CMOS camera and LED excitation at 470nm. 2P-excitation was at 920nm, with emission collected at 2 PMTs to enable ratiometric fluorescence measurements. 2P line scan recordings were started ~50µm below the epicardial surface and repeated at 50µm intervals through the LV wall. Recordings were made in areas of non-infarct, border zone and infarct tissue.

2P imaging of non-infarct tissue revealed normal myocardial structure and normal action potential recordings at all levels from 50-500µm below the surface within the LV free wall. 2P imaging in the border zone revealed connective tissue structures close to the surface. No electrical activity was observed. However, optical APs were recorded deeper into the tissue, associated with intermittent myocardial structures. In the majority of hearts (n=4), electrical activity was recorded within the central infarct region using widefield recordings. In some hearts (n=2), subsequent optical sectioning through the LV with 2P recordings identified discrete layers of electrical activity, but these signals were not associated with obvious myocyte structure.

This preliminary data suggests that structures other than myocytes (e.g. electrically coupled fibroblasts) may be able to support electrical signals from distant surviving myocardium. Alternatively, it may be the result of discrete areas of surviving myocardium within the infarct scar that cannot be easily identified as such from structure. Further work is being carried out to investigate the cellular origin of the electrical activity within the infarct.

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