NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations

Abstract

Wengrowski AM, Kuzmiak-Glancy S, Jaimes R 3rd, Kay MW. NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations. Am J Physiol Heart Circ Physiol 306: H529 -H537, 2014. First published December 13, 2013; doi:10.1152/ajpheart.00696.2013.-Langendorff-perfused hearts and working hearts are established isolated heart preparation techniques that are advantageous for studying cardiac physiology and function, especially when fluorescence imaging is a key component. However, oxygen and energy requirements vary widely between isolated heart preparations. When energy supply and demand are not in harmony, such as when oxygen is not adequately available, the imbalance is reflected in NADH fluctuations. As such, NADH imaging can provide insight into the metabolic state of tissue. Hearts from New Zealand white rabbits were prepared as mechanically silenced Langendorffperfused hearts, Langendorff-perfused hearts, or biventricular working hearts and subjected to sudden changes in workload, instantaneous global ischemia, and gradual hypoxia while heart rate, aortic pressure, and epicardial NADH fluorescence were monitored. Fast pacing resulted in a dip in NADH upon initiation and a spike in NADH when pacing was terminated in biventricular working hearts only, with the magnitude of the changes greatest at the fastest pacing rate. Working hearts were also most susceptible to changes in oxygen supply; NADH was at half-maximum value when perfusate oxygen was at 67.8 Ϯ 13.7%. Langendorff-perfused and mechanically arrested hearts were the least affected by low oxygen supply, with halfmaximum NADH occurring at 42.5 Ϯ 5.0% and 23.7 Ϯ 4.6% perfusate oxygen, respectively. Although the biventricular working heart preparation can provide a useful representation of mechanical in vivo heart function, it is not without limitations. Understanding the limitations of isolated heart preparations is crucial when studying cardiac function in the context of energy supply and demand.Langendorff; biventricular working; blebbistatin; nadh imaging; workload THE HEART IS RESPONSIBLE FOR pumping blood with vital supplies to the entire body and therefore requires a constant high rate of energy supply. Additionally, the heart has a large dynamic range with the capacity to increase its workload 10-fold and maintain this increase for extended periods of time. This metabolic requirement necessitates acute regulation of energy supply to match demand, as well as a constant adequate oxygen supply. Any limitation in energy supply is rapidly detrimental to cardiac function.The demand for ATP and oxygen varies, depending on the amount of work being performed (12a, 17). The amount of available ATP to the heart at any given time is enough for only a few beats and must constantly be resynthesized to keep up with utilization (16). In the heart, 80 -90% of energy production occurs via oxidative phosphorylation (25). Fats and carbohydrates are broken down and electrons are harvested, reducing NAD ϩ to NADH, (and, to a lesser exte...

β-Adrenergic stimulation and rapid pacing mutually promote heterogeneous electrical failure and ventricular fibrillation in the globally ischemic heart

“…In contrast, another uncoupler blebbistatin in three experiments postponed the adverse electrophysiological outcomes of ischemia (not shown) consistent with results from our recent study (56). Lack of mechanical load in the Langendorff-perfused hearts could also distort the ischemic changes (62) …”

Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance

“…Blebbistatin was previously shown to affect electrophysiology (4) but not to interfere with the mechanism of VF (13) in normoxemic hearts. However, Wengrowski et al (32) have recently demonstrated that blebbistatin dramatically postpones NADH accumulation during acute ischemia, indicating a significant interference of this drug in the natural course of ischemic events (32). Clearly, our study extends their observation, demonstrating that blebbistatin also postpones ⌬⌿ m collapse and asystole.…”

“…Blebbistatin, a recently discovered selective inhibitor of myosin II ATPase, has become a "standard of care" electromechanical uncoupler for use in experimental studies involving optical mapping or confocal imaging, including those addressing effects of I/R on electrophysiological parameters and ⌬⌿ m dynamics (6, 23, 24). It was recently shown that blebbistatin postpones changes in cellular redox state associated with ischemia (32). So it was important to test whether blebbistatin also modulates the time of ischemic electrical failure and ⌬⌿ m loss.…”

A technical review of optical mapping of intracellular calcium within myocardial tissue

“…Blebbistatin is a popular uncoupling agent for optical mapping, especially when imaging Ca i 2ϩ because it specifically inhibits the myosin ATPase without altering sarcolemmal ion currents or SR Ca 2ϩ kinetics (28). However, myocyte ATP utilization is severely reduced by electromechanical uncoupling, potentially altering the time course of physiological changes, especially mitochondrial function, during experimental perturbations (47,105,114). Motion tracking algorithms (41,80,115) and ratiometry (42,44,93) are sometimes effective in removing motion artifact from optical action potentials, but there has been less work in the application of motion tracking algorithms to reduce artifact in optically mapped Ca i 2ϩ transients.…”