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.
Highlights
NADH fluorescence is typically monitored from cardiac tissue by illuminating tissue with low intensity ultraviolet (UV) light and acquiring the resulting fluorescence between 450–475 nm[5,8,11,13,14,18]
Studies were conducted (n = 8) to measure the percentage of the total NADH pool ([NAD+] +[NADH]) that was photobleached during a typical NADH ED-Fluorescence recovery after photobleaching (FRAP) measurement
These values are consistent with previous studies in isolated hearts where levels of fNADH indicated the redox poise to be within the range of 50% reduction when glucose was the only exogeneous substrate[10]
Summary
NADH fluorescence is typically monitored from cardiac tissue by illuminating tissue with low intensity ultraviolet (UV) light and acquiring the resulting fluorescence between 450–475 nm[5,8,11,13,14,18]. Using cellular preparations and isolated mitochondria suspensions, Combs and Balaban measured the rate of fNADH recovery after NADH photolysis to introduce NADH ED-FRAP as an assessment of dehydrogenase activity[2,3]. We tested the efficacy of NADH ED-FRAP for measuring changes in NADH production rate in hearts perfused at low temperatures, in the presence and absence of mechanical contraction, and before and after ischemia/reperfusion injury. These new results provide insight into how NADH production rate is adjusted to couple with the energy demand of specific physiologic conditions
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