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).

<ol>
	<li>For these experiments, two separate systems are utilized simultaneously. For acquisition of 31P spectra, a Bruker 14T magnet is interfaced with the Avance III console and a computer equipped with TopSpin V2.1 software. For assessment of cardiac function, a custom built heart perfusion system is interfaced with the PowerLab 4/30 data acquisition, equipped with the LabChartPro 6 software for data analysis.</li>
	<li>On the day of the experiment, 1 liter of Krebs-Henseleit buffer is prepared as follows: 0.5mM EDTA, 5.3mM KCl, 1.2mM MgSO4, 118mM NaCl, and 25mM NaHCO3. The mixture is then bubbled with 5% CO2/95% O2 for 10-15 minutes prior to the addition of 2mM CaCl2. Finally, substrates, in the form of 10 mM glucose and 0.5mM pyruvate, are added.</li>
 <li>Temperature regulation during the experiment is critical. Heated circulators are used to maintain the temperature between 37.0 - 37.5&deg;C while the heart is inside the magnet. Temperature is monitored for the duration of the experiment using a fiber optic temperature probe.</li>
	<li>Perfusion pressure and left ventricular pressure are monitored via pressure transducers attached to a data acquisition system and displayed using the included software. These are calibrated with a standard sphygmomanometer prior to the experiment. In addition, pressure lines are flushed adequately in order to remove all air bubbles.</li>
	<li>A standard sample of 150 mM sodium phosphate (which is equivalent to the ionic strength of the KH buffer) is used to "calibrate" the probe prior to the insertion of the heart. This facilitates the signal and decreases the time necessary to begin the acquisition period once the heart is positioned within the probe.</li>
	<li>To reduce coagulation, the mouse is injected with 200 Units of heparin I.P. After 5 minutes, sodium pentobarbital (175 mg/kg) I.P. is given.</li>
	<li>The heart is rapidly excised (with lungs and thymus intact) and arrested in ice cold KH buffer.</li>
	<li>While kept on ice, the lungs are quickly removed. The lobes of the thymus are identified and gently peeled back to expose the aorta. The thymus is removed. The aorta is then isolated by carefully removing any surrounding tissue. </li>
	<li>Micro suturing forceps are used to gently hold both walls of the aorta to expose the lumen. The aorta is carefully placed onto the cannula made from 0.965 mm OD polyethylene tubing (PE50). The aorta is held in place with a micro vessel clamp while sutures are quickly tied around the aorta. The clip is removed and the forceps are used to carefully check that the cannula is above aortic root. Additional ties are added as necessary to hold the heart in place.</li>
	<li>Any extra tissue is removed using forceps and microscissors. A small incision is made into the left auricle. A 0.61 mm OD polyethylene tubing (PE10) is carefully inserted through the left atrium, LV cavity, and out through the apex while gently holding the heart. The excess tubing is trimmed. </li>
	<li>A deflated water-filled balloon is inserted through the atrium into the LV and is held in place using adhesive tape or sutures. The peristaltic pump speed is gradually increased to provide sufficient flow to the heart. Until the heart is place into the the NMR probe, the heart will continue to be perfused with constant flow equivalent to approximately 2 ml/min. The LV balloon is inflated with a small volume using a micrometer syringe to verify that the LV pressure transducer is functioning.</li>
	<li>The heart is carefully inserted into a 10 mm NMR tube. A wide bore "spinner" is used to help guide the tube into the proper position within the probe. The entire apparatus is then firmly attached to the "umbilical cord" with adhesive tape.</li>
	<li>The umbilical cord is slowly lowered into the upper bore of magnet until the heart/NMR tube is inside the coil of the 10 mm NMR probe. </li>
	<li>Once the heart is in the proper position within the probe, the peristaltic pump flow is adjusted in order to achieve a perfusion pressure of 80mmHg. (Remember, that up to this point the heart was perfused with a constant flow of approximately 2 ml/min). The perfusion pressure is then maintained by enabling the "hold" mechanism on the pump controller. The heart is then allowed a 15-20 minute equilibration period. During that time, the volume of the LV balloon is adjusted to achieve an end-diastolic pressure of 8-10 mmHg.</li>
	<li>During the equilibration period, it is necessary to optimize the spectrometer parameters in order to obtain the best possible phosphorous signal. This is accomplished by setting the radio pulse at the frequency at which the phosphorous nucleus resonates ("tuning") and making the magnetic field homogenous ("shimming").</li>
	<li>After the equilibration period, multiple 31P NMR spectra can be obtained. The acquisition period for each spectrum is dependent on the field strength of the magnet, the size of the sample, and the signal to noise ratio required for a specific experiment. Spectra are obtained using a 14 Telsa magnet by averaging the signal obtained from 256 radio frequency pulses of 20 &mu;s with a 60 degree flip angle and delays of 2.0 seconds. This experiment will require approximately 10 min.</li>
</ol>
Representative Results
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 (&gamma;-ATP, &alpha;-ATP, and &beta;-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 &gamma;-ATP and &beta;-ATP areas. (The &alpha;-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
<img src="/files/ftp_upload/2069/2069tab1.jpg" alt="Table 1" /> 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.
<img src="/files/ftp_upload/2069/2069fig1.jpg" alt="Figure 1" /> Figure 1. Representative LV pressure waves from LabChart Pro software.
<img src="/files/ftp_upload/2069/2069fig2.jpg" alt="Figure 2" /> 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.
<img src="/files/ftp_upload/2069/2069fig3.jpg" alt="Figure 3" /> 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 &alpha;-ATP peak. This is the contribution of NAD molecules. Pi: inorganic phosphate; PCr: phosphocreatine; ATP: adenosine triphosphate.

<ol>
	<li>Nascimben, L., Ingwall, J. S., Lorell, B. H., Pinz, I., Schultz, V., Tornheim, K., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+increased+glycolysis+in+the+hypertrophied+rat+heart.">Mechanisms for increased glycolysis in the hypertrophied rat heart.</a> Hypertension. 44, 662-667 (2004).</li><li>Spindler, M., Saupe, K. W., Tian, R., Ahmed, S., Matlib, M. A., Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+creatine+kinase+enzyme+kinetics+in+diabetic+cardiomyopathy.+A(31)P+NMR+magnetization+transfer+study+of+the+intact+beating+rat+heart.">Altered creatine kinase enzyme kinetics in diabetic cardiomyopathy. A(31)P NMR magnetization transfer study of the intact beating rat heart.</a> J Mol Cell Cardiol. 31, 2175-2189 (1999).</li><li>Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+nuclear+magnetic+resonance+spectroscopy+of+cardiac+and+skeletal+muscles.">Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles.</a> Am J Physiol. 242, H729-H744 (1982).</li><li>Ingwall, J. S., Javadpour, M. M., Miao, W., Hoit, B. D., Walsh, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=31P+NMR+spectroscopy+of+the+mouse+heart.">31P NMR spectroscopy of the mouse heart.</a> Cardiovascular physiology in the genetically engineered. , 151-163 (2002).</li><li>Luptak, I., Balschi, J. A., Xing, Y., Leone, T. C., Kelly, D. P., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+contractile+and+metabolic+reserve+in+peroxisome+proliferator-activated+receptor-alpha-null+hearts+can+be+rescued+by+increasing+glucose+transport+and+utilization.">Decreased contractile and metabolic reserve in peroxisome proliferator-activated receptor-alpha-null hearts can be rescued by increasing glucose transport and utilization.</a> Circulation. 112, 2339-2346 (2005).</li><li>Yan, J., Young, M. E., Cui, L., Lopaschuk, G. D., Liao, R., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glucose+uptake+and+oxidation+in+mouse+hearts+prevent+high+fatty+acid+oxidation+but+cause+cardiac+dysfunction+in+diet-induced+obesity.">Increased glucose uptake and oxidation in mouse hearts prevent high fatty acid oxidation but cause cardiac dysfunction in diet-induced obesity.</a> Circulation. 119, 2818-2828 (2009).</li><li>Luptak, I., Shen, M., He, H., Hirshman, M. F., Musi, N., Goodyear, L. J., Yan, J., Wakimoto, H., Morita, H., Arad, M., Seidman, C. E., Seidman, J. G., Ingwall, J. S., Balschi, J. A., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+activation+of+AMP-activated+protein+kinase+remodels+metabolic+network+in+favor+of+cardiac+glycogen+storage.">Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage.</a> J Clin Invest. 117, 1432-1439 (2007).</li><li>Xing, Y., Musi, N., Fujii, N., Zou, L., Luptak, I., Hirshman, M. F., Goodyear, L. J., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+and+energy+homeostasis+in+mouse+hearts+overexpressing+dominant+negative+alpha2+subunit+of+AMP-activated+protein+kinase.">Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase.</a> J Biol Chem. 278, 28372-28377 (2003).</li><li>Luptak, I., Yan, J., Cui, L., Jain, M., Liao, R., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+increased+glucose+entry+on+mouse+hearts+during+normal+aging+and+ischemic+stress.">Long-term effects of increased glucose entry on mouse hearts during normal aging and ischemic stress.</a> Circulation. 116, 901-909 (2007).</li><li>Tian, R., Abel, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+GLUT4-deficient+hearts+to+ischemia+underscore+the+importance+of+glycolysis.">Responses of GLUT4-deficient hearts to ischemia underscore the importance of glycolysis.</a> Circulation. 103, 2961-2966 (2001).</li><li>Liao, R., Jain, M., Cui, L., D'Agostino, J., Aiello, F., Luptak, I., Ngoy, S., Mortensen, R. M., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac-specific+overexpression+of+GLUT1+prevents+the+development+of+heart+failure+attributable+to+pressure+overload+in+mice.">Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice.</a> Circulation. 106, 2125-2131 (2002).</li><li>Tian, R., Musi, N., D'Agostino, J., Hirshman, M. F., Goodyear, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+adenosine+monophosphate-activated+protein+kinase+activity+in+rat+hearts+with+pressure-overload+hypertrophy.">Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy.</a> Circulation. 104, 1664-1669 (2001).</li></ol>

No conflicts of interest declared.

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

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>
<tr>
<td>Magnesium Sulfate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>M7506</td>
</tr>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>E1644</td>
</tr>
<tr>
<td>Potassium chloride</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P4505</td>
</tr>
<tr>
<td>Sodium bicarbonate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S6297</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S7653</td>
</tr>
<tr>
<td>Calcium chloride dihydrate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>C5080</td>
</tr><tr>
<td>D-Glucose</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>G7528</td>
</tr><tr>
<td>Sodium Pyruvate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P2256</td>
</tr><tr>
<td>Bruker Ultrashield 600WB Plus</td>
<td>Equipment</td>
<td>Bruker</td>
<td>&nbsp;</td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab 4/30</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">ML866/P</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 6 Pro</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">MLS260/6</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/FE224/" target="_blank">Quad Bridge Amp</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/FE224/" target="_blank">ML224</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/research/product/ML175/" target="_blank">STH Pump Controller</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/research/product/ML175/" target="_blank">ML175</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML172/" target="_blank">Minipuls 3 Peristaltic Pump</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML172/" target="_blank">ML172</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0699/" target="_blank">Disposable BP Transducer</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0699/" target="_blank">MLT0699</a></td>
</tr><tr>
<td>10mm NMR Sample Tube</td>
<td>Equipment</td>
<td>Wilmad LabGlass</td>
<td>513-7PP-7</td>
</tr><tr>
<td>Polyethylene tubing PE10</td>
<td>Equipment</td>
<td>Becton-Dickinson</td>
<td>427401</td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT844/" target="_blank">Physiological Pressure Transducer</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT844/" target="_blank">MLT844</a></td>
</tr><tr>
<td>Polyethylene tubing PE50</td>
<td>Equipment</td>
<td>Becton-Dickinson</td>
<td>427411</td>
</tr><tr>
<td>Micrometer syringe</td>
<td>Equipment</td>
<td>Gilmont Instruments</td>
<td>GS-1101</td>
</tr><tr>
<td>McPherson Forceps</td>
<td>Equipment</td>
<td>Miltex Inc.</td>
<td>18-949</td>
</tr><tr>
<td>Castraviejo microscissors</td>
<td>Equipment</td>
<td>Roboz Surgical Instruments</td>
<td>RS-5650</td>
</tr><tr>
<td>Neoptix Signal Conditioner</td>
<td>Equipment</td>
<td>Neoptix, Inc.</td>
<td>Reflex - 1</td>
</tr>	
</tbody>
</table>

assessment of cardiac function and energetics in isolated mouse hearts using 31p nmr spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Watch this Scientific Journal Video about Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy at JoVE.com

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

assessment-cardiac-function-energetics-isolated-mouse-hearts-using

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Medicine

26.0K Views.  University of Washington School of Medicine. Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Video: Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...