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.

Concepts and Core Principles of Fragment-Based Drug Design

“…For NADH-dependent enzymes, an enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) assay can also be used [124]. It relies on the photobleaching of a fluorophore as a product of an enzymatic reaction; in this case, NADH.…”

Light and Autofluorescence, Multitasking Features in Living Organisms

“…Changes in NADH autofluorescence are also reflecting the response of the respiratory chain to exposure to metabolic substrates or poisons, or to changes in oxygen availability, with implications also for the set-up of strategies in the monitoring of organ functionality during the transplantation phases [47,295,[306][307][308][309][310]]. An improvement in fluorescence studies of cytoplasmic metabolic functions is also promised by technological advances in imaging microscopy based on fluorescence lifetime detection, allowing to exploit the differences in lifetime between NADH and NADPH in living cells to study separately the metabolic engagement of the two derivatives, mainly as to energy production and antioxidant defense [173,291].…”

Cardioprotective Effects of 1-(3,6-Dibromo-carbazol-9-yl)-3-Phenylamino-Propan-2-Ol in Diabetic Hearts via Nicotinamide Phosphoribosyltransferase Activation

“…Reperfusion injury is often characterized with a return of blood supply resulting in cellular cardiac death, and is often characterized with infarct zones. Reperfusion injury has been associated with a significant increase in NADH levels (Moreno et al, 2017). We demonstrated that the pre-and post-exposure to P7C3 in an ex vivo Langendorff system provided significant protection in db/db hearts.…”