Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice
Summary
Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.
Abstract
Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.
Introduction
The hallmark of many cardiac diseases in human is a systolic and/or diastolic functional impairment of the left ventricle (LV). For the detection of structural abnormalities, the diagnosis, and the management of systolic heart failure as well as the evaluation of diastolic function in patients with symptoms of heart failure, echocardiography is used as a fundamental assessment modality.
Since the symptoms are unspecific and more than one third of patients with the clinical syndrome of heart failure may not suffer from the actual heart failure, it is important to find an objective echocardiographic correlate for the patient's clinical presentation1. Furthermore, some symptoms which are occult in the resting or static state may occur under conditions of activity or stress. In patients with coronary artery disease, already minor changes in coronary perfusion can lead to regional wall motion abnormalities. However, these subtle changes cannot be evaluated using conventional echocardiography as alterations of cardiac disease can be hidden in unstressed hearts. To gain a deeper understanding of the cardiac physiopathology, stress echocardiography provides a dynamic evaluation of myocardial structure and function under conditions of exercise or pharmacological induced stress, permitting matching symptoms with cardiac findings2. Also, in small animals, this method represents a non-invasive reliable in-vivo tool3,4,5. In-line with humans, stress reaction of the myocardium can be induced via pharmacological agents in mice and rats. Dobutamine is a frequently used drug and dobutamine stress echocardiography is widely performed in humans6,7 but only sometimes used in small animal models to assess cardiac stress reaction8,9,10,11. Dobutamine is a synthetical catecholamine with a predominantly β1-agonistic effect resulting in positive inotropy and chronotropy of the heart. To achieve a correct translation from human to mouse, the technology and the conceptual framework of echocardiography, technical limitations related to e.g., the small size and rapid heart rate in the mouse must be considered. The human target heart rate in dobutamine stress echocardiography is [(220-age) x 0.85] resulting in an average heart rate increase of about 150 ± 10% in healthy volunteers12,13. For mice, such a formula is missing. The ejection fraction (EF) is described to be increased by stress echocardiography in humans by 5-20%12,14. The EF in mice is, depending on the heart rate, reported between 58 ± 11% (< 450 bpm) and 71 ± 11% (≥ 450 bpm) and changes by nearly 20% with higher heart rates4. The main mechanism in mice to increase the cardiac output is an increase in the heart rate. Partly responsible for this mechanism is the Bowditch effect or staircase phenomenon, a frequency-dependent calcium-mediated positive-inotropic cardiac response, that is more pronounced in mice than in humans15,16. In addition, (stress) echocardiography underlies intra- and interobserver variability. Therefore, a highly standardized procedure is indispensable17,18.
Here we present the detailed procedure of dobutamine stress echocardiography to acquire standardized images to unravel subtle changes in cardiac function in mice in models of health and disease. Key components include adequate anesthesia, adequate heart rate monitoring and possible pitfalls in stress-induced imaging in mice. Key parameters are the evaluation of systolic and diastolic function including consideration of the LVEF. Because mice are resistant to afterload-induced cardiac dysfunction17, this protocol may add valuable information for the use in models of valvular heart disease as well.
Protocol
Representative Results
Discussion
Stress-induced evaluation of the cardiac function is widely used in humans in a clinical setting using exercise testing or pharmacological stress testing6,7. Because immediate post-exercise echocardiography of mice is very limited due to the need for sedation, dobutamine-induced stress echocardiography is likely to be the most translational method to assess stress-induced cardiac physiopathology. Reliable information on cardiac function can be obtained using real…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge the following funding sources: German Research Foundation (UMEA Junior Clinician Scientist, Stephan Settelmeier; RA 969/12-1, Tienush Rassaf; HE 6317/2-1, Ulrike Hendgen-Cotta), Else-Kroener-Fresenius-Stiftung (2014_A216, Tienush Rassaf).
Materials
| Activated Charcoal Filter | UNO BV | 180000140 | http://www.unobv.com/Rest%20Gas%20Filters.html |
| Aquasonic 100 Ultrasound Transmission Gel | Parker Laboratories | 001-02 | https://www.parkerlabs.com/aquasonic-100.asp |
| Chemical Hair removal lotion | General Supply | – | |
| Cotton Swaps | General Supply | – | |
| ddH2O | General Supply | – | |
| Dobutamine | Carinopharm | 71685.00.00 | https://www.carinopharm.de/stammsortiment/#103 |
| Flowmeter for laboratory animal anesthesia | UNO BV | SF3 | http://www.unobv.com/Flowmeters.html |
| Gas Exhaust Unit | UNO BV | – | http://www.unobv.com/Gas%20Exhaust%20Unit.html |
| Heating Lamp | Philips | – | |
| Induction Box | UNO BV | – | http://www.unobv.com/Induction%20box.html |
| Medical Sharps Container | BD | 305626 | https://legacy.bd.com/europe/safety/de/products/sharps/ |
| MX400 ultrasound transducer (20-46 Mhz) | VisualSonics | MX400 | https://www.visualsonics.com/product/transducers/mx-series-transducers |
| Octenisept disinfectant | Schuelke | 173711 | https://www.schuelke.com/de-de/produkte/octenisept.php |
| Omnican F syringe with needle 1ml | B. Braun | 9161502S | https://www.bbraun.de/de/products/b60/omnican-f.html |
| Paper Towels | General Supply | – | |
| Signacreme Electrode Cream | Parker Laboratories | 017-05 | https://www.parkerlabs.com/Signacreme.asp |
| Standard Gauze Pads | BeeSana Meditrade | 4852728 | https://www.meditrade.de/de/wundversorgung/verbandstoffe/beesana-mullkompresse/ |
| Thermasonic Gel Warmer | Parker Laboratories | 82-03-20 CE | https://www.parkerlabs.com/thermasonic_apta_sbp.asp |
| Transpore Tape | 3M | 1527NP-0 | https://www.3mdeutschland.de/3M/de_DE/unternehmen-de/produkte/~/3M-Transpore-Fixierpflaster/ |
| Vaporizer Sigma Delta | UNO BV | – | http://www.unobv.com/Vaporizers.html |
| Vevo 3100 high-frequency preclinical ultrasound imaging system | VisualSonics | Vevo3100 | https://www.visualsonics.com/product/imaging-systems/vevo-3100 * required software package: Cardiovascular package; B-mode, M-mode, pulsed-wave doppler mode |
| Vevo Imaging Station with integrated rail system, heated platform and physiological monitoring unit | VisualSonics | – | https://www.visualsonics.com/product/accessories/imaging-stations |
| VevoLab Analysis Software | VisualSonics | Vers. 3.2.5 | https://www.visualsonics.com/product/software/vevo-lab *required software package: Vevo Strain, LV analysis |
Riferimenti
- Oh, J. Echocardiography in heart failure: Beyond diagnosis. European Journal of Echocardiography. 8 (1), 4-14 (2007).
- Lancellotti, P., et al. The clinical use of stress echocardiography in non-ischaemic heart disease: Recommendations from the european association of cardiovascular imaging and the american society of echocardiography. Journal of the American Society of Echocardiography. 30 (2), 101-138 (2017).
- Lindsey, M. L., Kassiri, Z., Virag, J. A. I., de Castro Bras, L. E., Scherrer-Crosbie, M. Guidelines for measuring cardiac physiology in mice. American Journal of Physiology-Heart and Circulatory Physiology. 314 (4), 733-752 (2018).
- Zacchigna, S., et al. Toward standardization of echocardiography for the evaluation of left ventricular function in adult rodents: a position paper of the ESC Working Group on myocardial function. Cardiovascular Research. , 110 (2020).
- Hendgen-Cotta, U. B., et al. A novel physiological role for cardiac myoglobin in lipid metabolism. Scientific Reports. 7, 43219 (2017).
- Al-Lamee, R. K., et al. Dobutamine stress echocardiography ischemia as a predictor of the placebo-controlled efficacy of percutaneous coronary intervention in stable coronary artery disease: The stress echocardiography-stratified analysis of ORBITA. Circulation. 140 (24), 1971-1980 (2019).
- Cadeddu Dessalvi, C., Deidda, M., Farci, S., Longu, G., Mercuro, G. Early ischemia identification employing 2D speckle tracking selective layers analysis during dobutamine stress echocardiography. Echocardiography. 36 (12), 2202-2208 (2019).
- Li, Z., et al. Reduced myocardial reserve in young x-linked muscular dystrophy mice diagnosed by two-dimensional strain analysis combined with stress echocardiography. Journal of the American Society of Echocardiography. 30 (8), 815-827 (2017).
- Puhl, S. L., Weeks, K. L., Ranieri, A., Avkiran, M. Assessing structural and functional responses of murine hearts to acute and sustained beta-adrenergic stimulation in vivo. Journal of Pharmacological and Toxicological Methods. 79, 60-71 (2016).
- Ferferieva, V., et al. Assessment of strain and strain rate by two-dimensional speckle tracking in mice: comparison with tissue Doppler echocardiography and conductance catheter measurements. European Heart Journal Cardiovascular Imaging. 14 (8), 765-773 (2013).
- Wiesmann, F., et al. Dobutamine-stress magnetic resonance microimaging in mice : acute changes of cardiac geometry and function in normal and failing murine hearts. Circulation Research. 88 (6), 563-569 (2001).
- Pellikka, P. A., et al. Guidelines for performance, interpretation, and application of stress echocardiography in ischemic heart disease: From the American Society of Echocardiography. Journal of the American Society of Echocardiography. 33 (1), 1-41 (2020).
- Ahonen, J., et al. Pharmacokinetic-pharmacodynamic relationship of dobutamine and heart rate, stroke volume and cardiac output in healthy volunteers. Clinical Drug Investigation. 28 (2), 121-127 (2008).
- Nath Das, R. Determinants of cardiac ejection fraction for the patients with dobutamine stress echocardiography. Epidemiology. 07 (03), (2017).
- Balcazar, D., et al. SERCA is critical to control the Bowditch effect in the heart. Scientific Reports. 8 (1), 12447 (2018).
- Zhang, B., Davis, J. P., Ziolo, M. T. Cardiac catheterization in mice to measure the pressure volume relationship: Investigating the Bowditch Effect. Journal of Visualized Experiments. (100), e52618 (2015).
- Casaclang-Verzosa, G., Enriquez-Sarano, M., Villaraga, H. R., Miller, J. D. Echocardiographic approaches and protocols for comprehensive phenotypic characterization of valvular heart disease in mice. Journal of Visualized Experiments. (120), e54110 (2017).
- Respress, J. L., Wehrens, X. H. Transthoracic echocardiography in mice. Journal of Visualized Experiments. (39), e1738 (2010).
- Rea, D., et al. Strain analysis in the assessment of a mouse model of cardiotoxicity due to chemotherapy: Sample for preclinical research. In Vivo. 30 (3), 279-290 (2016).
- Beyhoff, N., et al. Application of speckle-tracking echocardiography in an experimental model of isolated subendocardial damage. Journal of the American Society of Echocardiography. 30 (12), 1239-1250 (2017).
- Pappritz, K., et al. Speckle-tracking echocardiography combined with imaging mass spectrometry assesses region-dependent alterations. Scientific Reports. 10 (1), 3629 (2020).
- Krahwinkel, W., et al. Dobutamine stress echocardiography. European Heart Journal. 18, 9-15 (1997).
- Michel, L., et al. Real-time pressure-volume analysis of acute myocardial infarction in mice. Journal of Visualized Experiments. (137), e57621 (2018).
- Baumgartner, H., et al. ESC/EACTS Guidelines for the management of valvular heart disease. European Heart Journal. 38 (36), 2739-2791 (2017).
- Knuuti, J., et al. 2019 ESC Guidelines for the diagnosis and management of chronic coronary syndromes. European Heart Journal. 41 (3), 407-477 (2020).
- Schoensiegel, F., et al. High throughput echocardiography in conscious mice: Training and primary screens. European Journal of Ultrasound. 32, 124-129 (2011).
- Gao, S., Ho, D., Vatner, D. E., Vatner, S. F. Echocardiography in Mice. Current Protocols in Mouse Biology. 1, 71-83 (2011).
- Scherrer-Crosbie, M., Thibault, H. B. Echocardiography in translational research: of mice and men. Journal of the American Society of Echocardiography. 21 (10), 1083-1092 (2008).
- Tanaka, N., et al. Transthoracic echocardiography in models of cardiac disease in the mouse. Circulation. 94 (5), 1109-1117 (1996).
- Roth, D. M., et al. Cardiac-directed adenylyl cyclase expression improves heart function in murine cardiomyopathy. Circulation. 99 (24), 3099-3102 (1999).
- Castle, P. E., et al. Anatomical location, sex, and age influence murine arterial circumferential cyclic strain before and during dobutamine infusion. Journal of Magnetic Resonance Imaging. 49 (1), 69-80 (2019).
- Ren, S., et al. Implantation of an isoproterenol mini-pump to induce heart failure in mice. Journal of Visualized Experiments. (152), e59646 (2019).
- Carillion, A., Biais, M., Riou, B., Amour, J. Comparison of Dobutamine with Isoproterenol in echocardiographic evaluation of cardiac β-adrenergic response in rats: 4AP8-9. European Journal of Anaesthesiology. 29, (2012).
- Lang, R. M., et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. European Heart Journal Cardiovascular Imaging. 16 (3), 233-270 (2015).
- Lindsey, M. L., et al. Guidelines for experimental models of myocardial ischemia and infarction. American Journal of Physiology-Heart and Circulatory Physiology. 314 (4), 812-838 (2018).
- Pieske, B., et al. How to diagnose heart failure with preserved ejection fraction: the HFA-PEFF diagnostic algorithm: a consensus recommendation from the Heart Failure Association (HFA) of the European Society of Cardiology (ESC). European Heart Journal. 40 (40), 3297-3317 (2019).
- Riehle, C., Bauersachs, J. Small animal models of heart failure. Cardiovascular Research. 115 (13), 1838-1849 (2019).
- Rammos, C., et al. Impact of dietary nitrate on age-related diastolic dysfunction. European Journal of Heart Failure. 18 (6), 599-610 (2016).
- Koshizuka, R., et al. Longitudinal strain impairment as a marker of the progression of heart failure with preserved ejection fraction in a rat model. Journal of the American Society of Echocardiography. 26 (3), 316-323 (2013).
- Bunting, K. V., et al. A practical guide to assess the reproducibility of echocardiographic measurements. Journal of the American Society of Echocardiography. 32 (12), 1505-1515 (2019).
- Grune, J., et al. Accurate assessment of LV function using the first automated 2D-border detection algorithm for small animals – evaluation and application to models of LV dysfunction. Cardiovascular Ultrasound. 17 (1), 7 (2019).
- Lau, E. M. T., et al. Dobutamine stress echocardiography for the assessment of pressure-flow relationships of the pulmonary circulation. Chest. 146 (4), 959-966 (2014).
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