Poster H9 - Impact of Heart Rate on Cardiac Energy Metabolism and Oxygen Utilization During Ischemia in a Perfused Rat Heart Model

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Lilly Carter

Undergraduate Research Assistant
The George Washington University, United States
Sarah Shectman (she/her/hers)

Undergraduate Research Assistant
The George Washington University
Mahopac, New York, United States

Co-Author(s)

BA

Bridget Alber

PhD Student
George Washington University, United States

Last Author(s)

MK

Matthew Kay, DSc

Professor
George Washington University, United States

Introduction: Cardiovascular disease (CVD) is the leading cause of death globally (about ⅓ of the population), with ischemic heart disease (IHD) being the common cause of these deaths. When untreated, ischemia causes myocyte death and myocardial infarction, a primary cause of heart failure and sudden cardiac arrest. NADH is reduced in the Krebs cycle to harvest electrons from fuels and is oxidized in the electron transport chain (ETC) to establish mitochondrial membrane potential, the electromotive force driving ATP synthesis. When the myocardium is deprived of oxygen, NADH is not oxidized by the ETC and the intracellular pool of NAD+ is quickly reduced to NADH by anaerobic respiration. NADH fluoresces (450-470nm) when excited by UV light (350-370nm), enabling the rate and amount of NAD+ reduction to be measured during ischemia as increases in NADH fluorescence recorded using a photometric device, such as a CMOS camera. This approach is typically used as a mitochondrial-specific assay of the balance of oxygen supply and myocardial demand. Our goal was to investigate how increased heart rate modulates the relationship between tissue oxygenation and mitochondrial production of NADH during ischemia. Excised perfused rat hearts were subjected to global ischemia testing the hypothesis that during higher pacing rates, tissue oxygen is consumed faster due to increased workload and NADH reaches peak fluorescence faster. This study aims to fill current research gaps by providing a detailed analysis of how cardiac energy metabolism and oxygen utilization respond to varying workloads during ischemia, potentially leading to new therapies for IHD.

Materials and

Methods: Female rats were anesthetized by inhalation of 5% isoflurane until lack of pedal response. Hearts were then rapidly excised, cannulated via the aorta, and perfused at constant pressure (70 mmHg) and temperature (37℃). Hearts were allowed to stabilize for 10 minutes. A platinum pacing electrode was placed on the right atria and connected to a constant current source to deliver 3.6mA of current with 5ms pulse duration at 5 pacing rates. An Oxylite probe was inserted into the left ventricle to measure tissue oxygenation while endogenous NADH fluorescence was excited with two Mightex 365nm UV lights and measured with a CCD camera (475 nm, iXon DV860, Andor Technology). Hearts were subjected to a pacing protocol consisting of 300, 350, 400, 450, and 500 bpm during global ischemia, induced by pausing the aortic flow. Flow was restored after 30 seconds and hearts were allowed to recover. Data was collected using Andor SOLIS and ADInstruments Labchart. Data was analyzed using excel and GraphPad Prism.

Results, Conclusions, and Discussions: During nonischemic conditions, NADH fluorescence increased with each increase in pacing rate. Interestingly, there was no observed difference in tissue oxygenation level. While inducing global ischemia, under higher pacing rates (500 BPM), tissue oxygenation decreased more rapidly (11 seconds vs 13 seconds), reached zero %O2 faster (20 seconds vs 21 seconds), and took longer to fully recover (21 seconds vs 17 seconds) compared to slower pacing rates (300 BPM). Under the same ischemic conditions, higher pacing rates (500 BPM) caused the NADH fluorescence to increase more rapidly, but plateaued at the same fluorescence level compared to slower pacing rates (300 BPM). Regardless of the pacing rate, all hearts that underwent global ischemia exhibited the same general patterns. Initially, NADH fluorescence increased over a 15-second period, then started to plateau. Once perfusate flow resumed, NADH fluorescence showed a steep decline over 5 seconds. All hearts demonstrated an exponential decay in tissue oxygenation during ischemia followed by a plateau, while the increase in tissue oxygenation after restoring flow linearly increased. A novel observation is that tissue oxygenation is fully depleted before NADH fluorescence plateaus. These results suggest that differences in pacing rates change the kinetics of tissue oxygenation, and the rate of NAD+ reduction, as measured by the rate of increased NADH fluorescence, suggesting that cardiac energy metabolism and oxygen utilization respond to the level of workload during ischemia, which could inform the development of new therapeutics for IHD.

Acknowledgements (Optional):