Purpose: We investigated the feasibility of measuring the reduction state of mitochondrial cytochromes in an intact, isolated heart using Raman spectroscopy. Methods: Isolated hearts of male Sprague-Dawley rats were perfused with oxygenated Tyrode solution (37°C). Raman spectra were recorded from a 2 mm spot on the left ventricle epicardial surface, using InVia Raman microscope with a 532 nm laser and 60 mm focal distance lens. Heart contractions were arrested by a Ca2+-free perfusion. Results: Raman spectra corresponded closely to those described in the literature on isolated cardiomyocytes (1). The mitochondrial peaks reflected essentially the reduced forms of b- and c-type cytochromes. Major peaks were mainly contributed by cytochromes c and c1 (750 cm-1) and b (1127 cm-1). Several less intense peaks were also assigned. Main myoglobin (Mb) peaks were present at 1587 and 1556 cm-1, showing maximal intensities with fully oxygenated and deoxygenated Mb, respectively. Perfusion with sodium dithionite (SDT) caused an increase of the intensity of cytochromal peaks. Mb deoxygenation by SDT was evident in downward frequency shifts of Mb peaks (1377 to 1358 cm-1, 1587 to 1556 cm-1 and 1640 to 1606 cm-1). As expected from a decreased reduction of electron transport chain upon uncoupling, the presence of FCCP in the perfusion caused a decrease in the intensity of cytochromal peaks with no change of Mb peaks. Global stop-flow ischemia of 35 min elicited a progressive increase in the intensity of the 750 cm-1 peak and an appearance of a 604 cm-1 peak, both associated with reduced cytochromes c-c1. Interestingly, peaks representing reduced b-cytochromes (1127 and 1337 cm-1) showed no significant increase, giving a decrease in reduced cytochromes b/c ratio. Measurement of the Mb peaks at 1358 and 1377 cm-1 allowed a hypoxia estimate to approximately 3 mm Hg pO2 (≈P50 for Mb). All changes were reversed upon reperfusion. Conclusions: Raman spectroscopy of the subepicardial surface of an isolated, crystalloid-perfused heart may offer new insights to mitochondrial function under various oxygenation conditions. For instance, the observed reversible decrease in the reduced cytochromes b/c ratio during ischemia suggested a lack of complex III damage, expected to be associated with an increase in this ratio (2). Effects on mitochondria of ischemia-reperfusion, with or without cardioprotective maneuvers (conditioning, pharmacological interventions etc.), may be studied in an intact organ by this method.
European Heart Journal, 2013, Vol 34, Issue Suppl 1, p. 607-608