Department of Anesthesiology & Pain Medicine, University of Washington School of Medicine
Kolwicz Jr., S. C., Tian, R. Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy. J. Vis. Exp. (42), e2069, doi:10.3791/2069 (2010).
From the data acquisition hardware and the LabChart software, several parameters of cardiac function can be measured throughout the experimental protocol. The typical measure of cardiac function, the left ventricular developed pressure (LVDevP), is obtained by subtracting the end-diastolic pressure (EDP) from the systolic pressure (Figure 1). This measure can vary depending on the strain of the mouse and the condition of the heart (i.e., pressure overload). However, in a normal C57BL6 mouse heart LVDevP is typically between 100-110 mmHg at fixed end-diastolic pressure of 8-10 mmHg. In addition, the LabChart program allows for the measurement of heart rate based on the cyclic measurements of the LV pressure waves. Again, this measure can vary but typical values are 350-400 bpm when hearts are allowed to beat at intrinsic rates. However, heart can be standardized using a pacing system where heart rate is kept at 420 bpm. In addition, measures of contractility (+dP/dt) and relaxation (-dP/dt) can be estimated using the first derivative of the LV pressure wave. During the experimental protocol it is easy to assess the Starling mechanism by incorporating a pressure-volume relationship. This is accomplished by making gradual increases in the LV balloon volume and noting the LVDevP as well as the EDP. These values can then be plotted as depicted in Figure 2. While the Starling curve is optimal, noting the volume necessary to achieve an EDP of 8-10 mmHg can give an indirect idea of LV chamber dimension. This can be used in models of aortic banding as hypertrophied hearts typically will require a smaller balloon volume while dilated hearts require a larger volume when compared to controls. Table 1 displays representative cardiac function data as acquired during the perfusion protocol.
The 31P NMR spectrometer will provide signals of phosphocreatine (PCr) and the three phosphates from ATP (γ-ATP, α-ATP, and β-ATP) as well as inorganic phosphate (Pi) as shown in Figure 2. Analysis of each of these peaks provides a value for the area under the curve. The amount of ATP is estimated by averaging the γ-ATP and β-ATP areas. (The α-ATP is not used because NAD molecules contribute to a unknown portion of the total signal). The energetic status of the heart is determined by the quotient of the PCr and ATP areas (PCr:ATP ratio). This value is typically 1.5 - 1.7 in a mouse heart supplied with glucose as the primary substrate. Although 31P NMR does not provide direct measures of ATP or PCr, the area of the peaks is proportional to the amount of the phosphorous containing compounds in the sample. Values for these signals can be estimated by using other methods. For example, direct measures of ATP by high-performance liquid chromatography (HPLC) in a cohort of hearts can yield an average concentration. This value can then be used to calibrate the average ATP areas observed in the spectra. The PCr concentration can be calculated based on the PCr area relative to the ATP area. It is also possible to estimate pH by analyzing the relative chemical shift of the inorganic phosphate (Pi) signal to the PCr signal.1 Using different radio pulse sequences, the creatine kinase reaction velocity or the ATP synthesis reaction velocity can also be measured.2
Table 1. Baseline cardiac function from isolated perfused hearts. LVDevP: left ventricular developed pressure; LVEDP: left ventricular end-diastolic pressure; HR: heart rate; RPP: rate pressure product; +dP/dt: first derivative LV pressure positive; -dP/dt: first derivative LV pressure negative; PP: perfusion pressure; CF: coronary flow.
Figure 1. Representative LV pressure waves from LabChart Pro software.
Figure 2. Representative Starling curves from control (solid line) and aortic banded (dotted-line) mice. A) Systolic function as represented by LVDevP over increasing LV volumes as determined by the volume of the LV balloon. B) Diastolic function as represented by EDP over increasing LV volumes as determined by volume of the LV balloon. LVDevP: left ventricular developed pressure (systolic minus diastolic pressure); EDP: end-diastolic pressure.
Figure 3. Representative 31P NMR spectra of isolated perfused mouse heart. Notice the relatively small Pi peak. In an aerobically perfused heart supplied with pyruvate or fatty acids in addition to glucose, this peak should be minimal. During periods of ischemia, this peak increases while the PCr peak decreases. Notice the shoulder to the right of the α-ATP peak. This is the contribution of NAD molecules. Pi: inorganic phosphate; PCr: phosphocreatine; ATP: adenosine triphosphate.
31P NMR spectroscopy in the Langendorff-perfused isolated mouse heart provides reliable and reproducible data.3, 4 However, it is imperative that cannulation of the aorta and insertion of the LV balloon are done properly such to allow stable cardiac performance while inside the NMR tube. In addition, temperature regulation is paramount in order to achieve proper baseline function. One important factor in obtaining good, analyzable NMR spectra is increasing the signal to noise ratio. This can be achieved by ensuring optimal "tuning" and "shimming" on the sample. As mentioned in the protocol text, the use of a standard sample prior to the insertion of the heart can facilitate this. It is also helpful to have an adequate sized "sample". Hearts weighing less than 100 mg typically provide lower PCr and ATP signals so increases in acquisition time will be necessary to obtain good phosphorous spectra.
There are several ways to modify the existing protocol to garner additional information regarding cardiac function and energetics. In our laboratory, we have perfused hearts with mixed substrate buffers which can include the presence of different combinations of fatty acids (in low and high concentrations), lactate, ketones, and insulin. With the use of stable isotopes in the perfusion buffer (i.e., 13C labeled substrates), we possess the ability to estimate substrate utilization by the relative contribution of labeled acetyl CoA to the TCA cycle.5-7 For this application, we perform isotopomer analysis of 13C3- and 13C4- glutamate with 13C NMR spectroscopy. This requires freeze-clamping the heart at the end of the perfusion protocol and performing an extraction of the frozen tissue. This will be an additional experiment as the analysis requires the use of a different probe with separate setup parameters. Other applications include the substitution of glucose with deoxyglucose in the buffer while monitoring the time-dependent accumulation of 2-deoxyglucose phosphate in the heart using 31P NMR spectroscopy. This method allows for the measurement of myocardial glucose uptake.7, 8 In addition, our laboratory has analyzed cardiac function and energetics in perfusion protocols consisting of ischemia/reperfusion and high workload challenge.6, 8-10
In summary, 31P NMR spectroscopy in isolated mouse hearts is a technically challenging procedure requiring the use of sophisticated equipment. However, the data that it yields is invaluable to the researcher who wishes to analyze the function and energetics of bioengineered mouse models. For our laboratory, these techniques have been vital in our understanding of the consequences of a variety of stressors on cardiac function, energetics, and metabolism.1, 11, 12
No conflicts of interest declared.
The authors would like to thank Lynne Spencer for her support during the NMR spectroscopy portion of the experiment. This work was supported by grants from the National Institutes of Health fund R01 HL059246, R01 HL067970, R01 HL088634 (to Dr. Tian) and F32 HL096284 (to Dr. Kolwicz).
|Calcium chloride dihydrate||Reagent||Sigma-Aldrich||C5080|
|Bruker Ultrashield 600WB Plus||Equipment||Bruker Corporation||Â|
|LabChart 6 Pro||Equipment||ADInstruments||MLS260/6|
|Quad Bridge Amp||Equipment||ADInstruments||ML224|
|STH Pump Controller||Equipment||ADInstruments||ML175|
|Minipuls 3 Peristaltic Pump||Equipment||ADInstruments||ML172|
|Disposable BP Transducer||Equipment||ADInstruments||MLT0699|
|10mm NMR Sample Tube||Equipment||Wilmad LabGlass||513-7PP-7|
|Polyethylene tubing PE10||Equipment||BD Biosciences||427401|
|Physiological Pressure Transducer||Equipment||ADInstruments||MLT844|
|Polyethylene tubing PE50||Equipment||BD Biosciences||427411|
|Micrometer syringe||Equipment||Gilmont Instruments||GS-1101|
|McPherson Forceps||Equipment||Miltex Inc.||18-949|
|Castraviejo microscissors||Equipment||Roboz Surgical Instruments Co.||RS-5650|
|Neoptix Signal Conditioner||Equipment||Neoptix, Inc.||Reflex - 1|