NADH Fluorescence Recovery after Photobleaching (NADH-FRAP) for In-Situ Assessment of Cardiac TCA Cycle Enzyme Activity

Many elements can modify the proper performance of metabolic processes including during ATP production. The ability of mitochondria to produce energy is a function of both the capacity of electron harvesting in the TCA cycle and the ability to convert this into ATP. Current techniques such as standard molecular assays and fluorescence imaging do not isolate these components from each other, limiting not only the proper understanding of regional enzymatic activities but ultimately the progress of efficient therapies. The development of a technology based on the fluorescence recovery after photobleaching (FRAP) in a whole heart is proposed as a novel technique for measuring the replenishment of a fluorophore product of an enzymatic reaction after it has been photobleached with high intensity light under conditions that closely resemble in-vivo state. The assessment of the absolute rate of energy production through the visualization of the reduce form of nicotinamide adenine dinucleotide (NADH) is possible using NADH-FRAP. Hearts from adult rats were quickly excised and perfused. A low-power (1,5mW) and a high-power (500mW) UV light were used to monitor NADH-fluorescence and to photobleach the epicardium respectively; the signal was filtered (475±25nm) and captured using a CCD camera. Optimal parameters for imparting the photobleaching energy were determined and no tissue necrosis or unwanted phototoxicity was observed at the end of the studies, subjecting the non-destructive nature of NADH-FRAP. Likewise, three experiments were performed to study NADH-FRAP under different situations of energy demand. First, given the fact that temperature-dependency has been associated with enzymes dynamics, three temperatures ranging from hypothermia (22 and 30oC) to normothermia (37oC) were tested. Faster NADH production was observed during normothermia compared to hypothermia (8.05±1.02, 5.11±0.55 and 3.43±0.34 A.U./100msec, respectively). Second, considering that ATP is mostly used for contractions, the hearts were subjected to a contracting vs non-contracting situation. With a higher energy demand for contractions, NADH is produced faster than in an arrested heart (11.91±0.95 vs 8.91±0.53 A.U./100msec, respectively). Finally, the impaired ability of the TCA cycle to produce NADH after ischemia/reperfusion (IR) was measured using NADH-FRAP. NADH production is diminished after an episode of IR (8.17±1.82 vs 4.41±1.19 A.U./100msec, respectively). Accurate evaluation of TCA cycle activity in the heart is crucial for studying pathologies and therapies to enhance cardiac performance. NADH-FRAP could be used to study the pathology of a wide variety of metabolic diseases ranging from acute and chronic ischemic injury and infarction to heart failure and diabetes