This new protocol permits the investigator to monitor multiple metabolic parameters including cytosolic and mitochondrial oxygenation as well as cytosolic redox status. While simultaneously measuring the traditional measurements of myocardial function. The main advantage of this technique is a simple nondestructive dynamic monitoring of important elements of mitochondrial metabolism in the perfused heart that avoids the artifacts associated with reflection spectroscopy.
Those new to this technique may struggle with inserting the catheter without damaging the valve and avoid room light contamination. The catheter must be inserted and adjusted carefully. The collection fiber must also be positioned properly to maximize light transmitted across the left ventricular free wall which can be difficult.
To begin cut a small piece of the left atrial appendage and insert a fiber optic catheter into the left ventricle via the mitral valve. Then rotate it to achieve an illuminated left ventricle free wall. Connect the other end of the pick up fiber to a rapid scanning spectrometer.
Position the pick up fiber optic directly opposite the region of maximum illumination of the left ventricle at about one centimeter from the heart. Room light sources should be zero. The rotation of the catheter and positioning of the pick up optical fiber must be adjusted to get adequate light without saturation of the detector.
Turn off the lights in the experimental area to obtain complete darkness. Start the custom program incorporating spectrometer drivers to perform data acquisition and real-time analysis of the transmitted light. Navigate through all prompts selecting options for perfused heart spectroscopy acquisition mode.
On the next page indicate whether auxiliary data collection is occurring. Finally enter acquisition parameters including location of both chromophore reference spectra and data to be saved. Now enter a bandwidth of 490 to 630 nanometers.
Enter a sampling rate of two hertz. Collect a dark current or zero light spectrum to correct for background signal levels by turning the light source off. Click to select the desired chromophore references to be used in the fitting routine.
In the acquire data page adjust the position of both the catheter and the pick up fiber to maximize the transmitted light displayed on the software with specific attention to the signal amplitude in the 500 nanometer region where the oxygenated myoglobin absorbances should be observed. Make sure the transmitted light is not saturating the detector in the 600 nanometer region. Ensure no external light sources contribute to the collected spectrum by turning off catheter illumination and confirming no light is now detected.
Initiate the data collection by clicking on the Save Spectra button. Click on Set as Control to view the difference absorbance spectrum from future spectra relative to the current control spectrum. At this point perform any physiological perturbation as desired.
For example, to determine the effects of cyanidine on cardiac performance and chromophore absorption, stop the recirculation of the perfusate to avoid recycling cyanide. Use a syringe pump to inject cyanide at different rates into the perfusate just before the aortic canula to achieve the desired concentrations of cyanide in the perfusate flowing into the heart. Simultaneously monitor cardiac function and optical properties.
Stop the cyanide syringe pump when the effects of coronary flow and heart rate, along with the optical transmission through the heart wall are at steady state. Five minutes after stopping the cyanide infusion switch the bubbling gas from 100%oxygen to 100%nitrogen to remove oxygen from the system. After about 10 minutes stop the flow to simulate a total ischemic and hypoxic condition.
For spectral data analysis run the program in the perfused heart analysis mode. Select the appropriate analysis program. Enter the data file path and reference spectra file.
Select catheter light source which loads the pre-saved spectrum of the catheter light source. Select Read Bin Data. Then, select Set Minimum and Maximum Wavelength.
Enter the bandwidth for the data analysis as 490 to 630 nanometers. Select Return to Main Menu and then select Read References. Confirm the reference spectra to use in the analysis.
Select Return to Main Menu and then select Time Points in the main menu. Now select a time zero time point as the control and set the range to 100 points. Also select a time one time point as the experimental period at a range of 100 points.
Observe the raw difference spectrum in the Averaged Absorbance Spectrum tab. Select Calculate Fit Coefficients and then click on the Fit Coefficients tab to observe the time course of the retrospective fit. Return to the main menu and select Calculate Difference Absorption.
Select time zero and difference between time zero and time one at all positions. Observe the fitted spectrum in the different spectrum window and the fitting elements in the reference weight window. Repeat this procedure to compare other time points in the experiment.
Return to the main menu. Save data and analysis in a spreadsheet report by typing in a desired name and selecting Save Data for further analysis with other programs. Shown here is the spectrum of the catheter and the raw spectrum of the transmitted light from the rabbit heart free wall.
These data reveal a very large attenuation of light in the blue region of the spectrum. However, the bands of absorbance from myoglobin and the mitochondrial cytochromes can be directly observed between 490 and 580 nanometers in the insert. The difference spectrum of the control and cyanide-treated heart is shown here.
The fit spectrum is calculated from the reference spectra. The Residuals spectrum is the subtraction of the fit from the raw data. Shown here are spectra amplitudes of the reference spectra.
Strong increases in absorbance of most of the cytochromes are observed as the flow of electrons down the cytochrome chain was blocked by cyanide in the steady state. In addition, the absorbance of oxygenated myoglobin increased as the consumption of oxygen was eliminated by cyanide. The difference spectrum from the cyanide washout versus cyanide injection reveals a partial reversal of the cyanide effect.
Shown here is the difference spectrum of the no-flow ischemia state versus control. This spectrum represents the fully deoxygenated and reduced state of the cytosolent mitochondria verus the control condition. We are currently studying the effects of nitric oxide on cardiac metabolism.
In addition to the aforementioned chromophores we can also track other optically-detectable species included metmyoglobin generated by nitric oxide. Additionally, the catheter can be connected to a laser to study lipid metabolism using raman spectroscopy. Calcium cardiac metabolism can also be studied by absorption of calcium probes incorporated exogenously or genetically.
