title: Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts.
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.
authors.affiliation: Department of Biomedical Engineering, The George Washington University, Washington, DC 20052, USA.
authors.forename: Angel
authors.initials: A
authors.lastname: Moreno
authors.affiliation: Department of Biomedical Engineering, The George Washington University, Washington, DC 20052, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kuzmiak-Glancy
authors.affiliation: Department of Biomedical Engineering, The George Washington University, Washington, DC 20052, USA.
authors.forename: Rafael
authors.initials: R
authors.lastname: Jaimes
authors.affiliation: Department of Biomedical Engineering, The George Washington University, Washington, DC 20052, USA.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Kay
mesh_terms: D000818:Animals; D005260:Female; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006321:Heart / Q000528:radiation effects; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000032:analysis* / Q000737:chemistry; D010088:Oxidoreductases / Q000032:analysis* / Q000737:chemistry; D017207:Rats, Sprague-Dawley
chemical_list: D009243:NAD; D010088:Oxidoreductases