Controlled regional hypoperfusion in Langendorff heart preparations

Abstract

We describe a new approach that combines several techniques to allow abnormal electrical and calcium activity to be visualized within hypoperfused myocardial tissue. A flexible microcannula was inserted into the left anterior descending artery of Langendorff perfused rat hearts, an air-tight seal between the coronary artery and the cannula was created, and an HPLC pump was used to deliver a specified flowrate through the microcannula. High resolution optical mapping of NADH/ calcium, NADH/voltage or calcium/voltage was then conducted using a dual camera system. The ECG was acquired using surface electrodes. This perfusion technique is superior to occluding a vessel by either a tie or a clamp because it allows precise control of the composition and amount of flow to a defined ischemic bed. Another advantage is that flow can be stopped and resumed remotely, without touching the heart. This allows ectopic beats, or other arrhythmogenic activity, such as alternans, to be recorded immediately after changes in flow are imposed. Altogether, the described method provides a powerful new tool to assess how coronary flow rate affects the degree of local ischemia by the ability to record abnormal patterns of electrical activity and associated intracellular calcium transients with high spatiotemporal resolution from epicardial areas as small as 100 × 100 μm.

Use of endogenous NADH fluorescence for real-time in situ visualization of epicardial radiofrequency ablation lesions and gaps

“…Oxygen serves as a final electron acceptor in the mitochondrial electron chain, and its decline leads to NADH accumulation (5). Thus, ischemia causes an increase in fNADH in a time-dependent manner (24,32). For example, if coronary perfusion is disrupted before the ablation, patches of ischemic tissue with elevated fNADH levels may be observed adjacent to the darker circular fNADH lesions.…”

“…Hearts were stained with the potentiometric dye RH237 (10 M final concentration; Molecular Probes). To reduce motion artifact, blebbistatin was added to the perfusate at a final concentration of 10 M. A dual optical mapping system comprised of two cameras (Andor IXON DV860s) fitted with a dual port adapter (Andor CSU Adapter Dual Cam) and a dichroic mirror (610 nm) was used to image the epicardial fluorescence of RH237 [250 -500 frames/s (fps)] and NADH (2 fps) from the same field of view (32). To record optical action potentials, the epicardium was illuminated using two light-emitting diodes (LumiLEDs, 530/35 nm).…”

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

“…This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide) [9][10][11] , a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration 12 and provides a measure of mitochondrial redox state 13 .…”

“…NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration 12 and provides a measure of mitochondrial redox state 13 . fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions 14,15 and to monitor the progression of hypoxic conditions over time 10 .…”

High resolution contrast ultrasound and NADH fluorescence imaging of myocardial perfusion in excised rat hearts

“…We used an animal model of controlled coronary artery flow [10] to deliver UCA to specific coronary beds while imaging transmural flow and epicardial NADH fluorescence ( Figure 1). Hearts from male Sprague-Dawley rats (400-600g, n=4) were excised and Langendorff-perfused with oxygenated Tyrode's solution.…”