Document Type
Journal Article
Publication Date
3-31-2017
Journal
Scientific Reports
Volume
7
Inclusive Pages
45744
DOI
10.1038/srep45744
Abstract
Reduction of NAD(+) by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
APA Citation
Moreno, A., Kuzmiak-Glancy, S., Jaimes, R., & Kay, M. (2017). Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts.. Scientific Reports, 7 (). http://dx.doi.org/10.1038/srep45744
Peer Reviewed
1
Open Access
1
Included in
Medical Pharmacology Commons, Medical Physiology Commons, Pharmacology Commons, Physiology Commons
Comments
Reproduced with permission of Macmillan Publishers Ltd. Scientific Reports