Optical metabolic imaging of irradiated rat heart exposed to ischemia-reperfusion injury.

Whole thoracic irradiation (WTI) is known to cause deterioration in cardiac function. Whether irradiation predisposes the heart to further ischemia and reperfusion (IR) injury is not well known. The aim of this study is to examine the susceptibility of rat hearts to IR injury following a single fraction of 15 Gy WTI and to investigate the role of mitochondrial metabolism in the differential susceptibility to IR injury. After day 35 of irradiation, ex vivo hearts from irradiated and nonirradiated rats (controls) were exposed to 25-min global ischemia followed by 60-min IR, or hearts were perfused without IR for the same protocol duration [time controls (TC)]. Online fluorometry of metabolic indices [redox state: reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and NADH/FAD redox ratio] and functional variables [systolic left ventricular pressure (LVP), diastolic LVP (diaLVP), coronary flow (CF), and heart rate were recorded in the beating heart; developed LVP (dLVP) and rate pressure product (RPP)] were derived. At the end of each experimental protocol, hearts were immediately snap frozen in liquid N2 for later three-dimensional imaging of the mitochondrial redox state using optical cryoimaging. Irradiation caused a delay in recovery of dLVP and RPP after IR when compared to nonirradiated hearts but recovered to the same level at the end of reperfusion. CF in the irradiated hearts recovered better than the control hearts after IR injury. Both fluorometry and 3-D cryoimaging showed that in WTI and control hearts, the redox ratio increased during ischemia (reduced) and decreased on reperfusion (oxidized) when compared to their respective TCs; however, there was no significant difference in the redox state between WTI and controls. In conclusion, our results show that although irradiation of rat hearts compromised baseline cardiovascular function, it did not alter cardiac mitochondrial redox state and induce greater susceptibility of these hearts to IR injury.

Optical Cryoimaging Reveals a Heterogeneous Distribution of Mitochondrial Redox State in ex vivo Guinea Pig Hearts and Its Alteration During Ischemia and Reperfusion.

Oxidation of substrates to generate ATP in mitochondria is mediated by redox reactions of NADH and FADH2. Cardiac ischemia and reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation. We hypothesize that IR alters the metabolic heterogeneity of mitochondrial redox state of the heart that is only evident in the 3-D optical cryoimaging of the perfused heart before, during, and after IR. The study involved four groups of hearts: time control (TC: heart perfusion without IR), global ischemia (Isch), global ischemia followed by reperfusion (IR) and TC with PCP (a mitochondrial uncoupler) perfusion. Mitochondrial NADH and FAD autofluorescence signals were recorded spectrofluorometrically online in guinea pig ex vivo-perfused hearts in the Langendorff mode. At the end of each specified protocol, hearts were rapidly removed and snap frozen in liquid N2 for later 3-D optical cryoimaging of the mitochondrial NADH, FAD, and NADH/FAD redox ratio (RR). The TC hearts revealed a heterogeneous spatial distribution of NADH, FAD, and RR. Ischemia and IR altered the spatial distribution and caused an overall increase and decrease in the RR by 55% and 64%, respectively. Uncoupling with PCP resulted in the lowest level of the RR (73% oxidation) compared with TC. The 3-D optical cryoimaging of the heart provides novel insights into the heterogeneous distribution of mitochondrial NADH, FAD, RR, and metabolism from the base to the apex during ischemia and IR. This 3-D information of the mitochondrial redox state in the normal and ischemic heart was not apparent in the dynamic spectrofluorometric data.