הערכה של תפקוד הלב Energetics בלבבות עכבר בודדות שימוש 31 P NMR ספקטרוסקופיה
Summary
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.
Abstract
מודלים העכבר bioengineered הפכו כלי מחקר רב עוצמה בקביעת קשרים סיבתיים בין שינויים ומודלים המולקולרי של מחלות לב וכלי דם. למרות הביולוגיה המולקולרית יש צורך בזיהוי שינויים מרכזיים מסלול איתות, זה לא פונדקאית עבור משמעות תפקודית. בעוד הפיזיולוגיה יכולה לספק תשובות לשאלה של פונקציה, תוך שילוב עם הפיזיולוגיה הערכה ביוכימיים של מטבוליטים של שלם, לב פועם מאפשר לקבל תמונה שלמה של תפקוד אנרגטיקה לב. במשך שנים במעבדה שלנו ניצלה perfusions לב מבודדים בשילוב עם ספקטרוסקופית תהודה מגנטית גרעינית (NMR) כדי לבצע משימה זו. תפקוד חדר שמאל נבחנת על ידי Langendorff מצב perfusions לב מבודדים תוך אנרגטיקה הלב נמדד על ידי ביצוע<sup> 31</sup> P ספקטרוסקופיית תהודה מגנטית של הלב perfused. בעזרת טכניקות אלו, מדדי תפקוד הלב בשילוב עם רמות phosphocreatine ו ATP ניתן למדוד בו זמנית לבבות פועמים. יתר על כן, פרמטרים אלה ניתן לנטר בזמן דחק פיזיולוגי או פתולוגי הם הנהיגו. לדוגמה, איסכמיה / reperfusion או גבוה אתגר פרוטוקולים עומס יכול להיות מאומץ. השימוש העורקים banding או דגמים אחרים של הלב הפתולוגיה נוטים גם כן. משנה גרסאות בתוך הפרוטוקול, משמעות תפקודית האנרגטי של שינויים מולקולריים של מודלים עכבר מהונדס ניתן לתאר באופן הולם, המוביל תובנות חדשות על מסלולים מטבוליים הקשורים האנזימטית. לכן,<sup> 31</sup> P NMR ספקטרוסקופיה בלב perfused מבודד היא טכניקה ערך מחקר בבעלי חיים למחלות לב וכלי דם.
Protocol
Discussion
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
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Material Name | Type | Company | Catalogue Number |
| Magnesium Sulfate | Reagent | Sigma Aldrich | M7506 |
| EDTA | Reagent | Sigma Aldrich | E1644 |
| Potassium chloride | Reagent | Sigma Aldrich | P4505 |
| Sodium bicarbonate | Reagent | Sigma Aldrich | S6297 |
| Sodium chloride | Reagent | Sigma Aldrich | S7653 |
| Calcium chloride dihydrate | Reagent | Sigma Aldrich | C5080 |
| D-Glucose | Reagent | Sigma Aldrich | G7528 |
| Sodium Pyruvate | Reagent | Sigma Aldrich | P2256 |
| Bruker Ultrashield 600WB Plus | Equipment | Bruker | |
| PowerLab 4/30 | Equipment | ADInstruments | ML866/P |
| 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 | Becton-Dickinson | 427401 |
| Physiological Pressure Transducer | Equipment | ADInstruments | MLT844 |
| Polyethylene tubing PE50 | Equipment | Becton-Dickinson | 427411 |
| Micrometer syringe | Equipment | Gilmont Instruments | GS-1101 |
| McPherson Forceps | Equipment | Miltex Inc. | 18-949 |
| Castraviejo microscissors | Equipment | Roboz Surgical Instruments | RS-5650 |
| Neoptix Signal Conditioner | Equipment | Neoptix, Inc. | Reflex – 1 |
References
- Nascimben, L., Ingwall, J. S., Lorell, B. H., Pinz, I., Schultz, V., Tornheim, K., Tian, R. Mechanisms for increased glycolysis in the hypertrophied rat heart. Hypertension. 44, 662-667 (2004).
- Spindler, M., Saupe, K. W., Tian, R., Ahmed, S., Matlib, M. A., Ingwall, J. S. Altered creatine kinase enzyme kinetics in diabetic cardiomyopathy. A(31)P NMR magnetization transfer study of the intact beating rat heart. J Mol Cell Cardiol. 31, 2175-2189 (1999).
- Ingwall, J. S. Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles. Am J Physiol. 242, H729-H744 (1982).
- Ingwall, J. S., Javadpour, M. M., Miao, W., Hoit, B. D., Walsh, R. A. 31P NMR spectroscopy of the mouse heart. Cardiovascular physiology in the genetically engineered. , 151-163 (2002).
- Luptak, I., Balschi, J. A., Xing, Y., Leone, T. C., Kelly, D. P., Tian, R. Decreased contractile and metabolic reserve in peroxisome proliferator-activated receptor-alpha-null hearts can be rescued by increasing glucose transport and utilization. Circulation. 112, 2339-2346 (2005).
- Yan, J., Young, M. E., Cui, L., Lopaschuk, G. D., Liao, R., Tian, R. Increased glucose uptake and oxidation in mouse hearts prevent high fatty acid oxidation but cause cardiac dysfunction in diet-induced obesity. Circulation. 119, 2818-2828 (2009).
- 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. Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage. J Clin Invest. 117, 1432-1439 (2007).
- Xing, Y., Musi, N., Fujii, N., Zou, L., Luptak, I., Hirshman, M. F., Goodyear, L. J., Tian, R. Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase. J Biol Chem. 278, 28372-28377 (2003).
- Luptak, I., Yan, J., Cui, L., Jain, M., Liao, R., Tian, R. Long-term effects of increased glucose entry on mouse hearts during normal aging and ischemic stress. Circulation. 116, 901-909 (2007).
- Tian, R., Abel, E. D. Responses of GLUT4-deficient hearts to ischemia underscore the importance of glycolysis. Circulation. 103, 2961-2966 (2001).
- Liao, R., Jain, M., Cui, L., D’Agostino, J., Aiello, F., Luptak, I., Ngoy, S., Mortensen, R. M., Tian, R. Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice. Circulation. 106, 2125-2131 (2002).
- Tian, R., Musi, N., D’Agostino, J., Hirshman, M. F., Goodyear, L. J. Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy. Circulation. 104, 1664-1669 (2001).
