The authors thank Dorita Dekkers and Fatimah Al Darwish for assistance with the mouse measurements and data analysis.

<ol>
	<li>Vall&#233;e, J. P., Ivancevic, M. K., Nguyen, D., Morel, D. R., Jaconi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+cardiac+MRI+in+small+animals.">Current status of cardiac MRI in small animals.</a> Magnetic Resononance Materials in Physics, Biology and Medicine. 17 (3-6), 149-156 (2004).</li><li>Bakermans, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+cardiovascular+MR+imaging+and+spectroscopy.">Small animal cardiovascular MR imaging and spectroscopy.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 88-89, 1-47 (2015).</li><li>Wu, Y. L., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+application+of+MRI+for+mouse+phenotyping.">Diverse application of MRI for mouse phenotyping.</a> Birth Defects Research. 109 (10), 758-770 (2017).</li><li>Coolen, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frame+rate+retrospectively+triggered+Cine+MRI+for+assessment+of+murine+diastolic+function.">High frame rate retrospectively triggered Cine MRI for assessment of murine diastolic function.</a> Magnetic Resonance in Medicine. 69 (3), 648-656 (2013).</li><li>Lapinskas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+magnetic+resonance+feature+tracking+in+small+animals+-+a+preliminary+study+on+reproducibility+and+sample+size+calculation.">Cardiovascular magnetic resonance feature tracking in small animals - a preliminary study on reproducibility and sample size calculation.</a> BMC Medical Imaging. 17 (1), 51 (2017).</li><li>Pedrizzetti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+estimating+intraventricular+hemodynamic+forces+from+endocardial+dynamics:+A+comparative+study+with+4D+flow+MRI.">On estimating intraventricular hemodynamic forces from endocardial dynamics: A comparative study with 4D flow MRI.</a> Journal of Biomechanics. 60, 203-210 (2017).</li><li>Motaal, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+high-frame-rate+mouse+heart+cine-MRI+using+compressed+sensing+reconstruction.">Accelerated high-frame-rate mouse heart cine-MRI using compressed sensing reconstruction.</a> NMR in Biomedicine. 26 (4), 451-457 (2013).</li><li>Claus, P., Omar, A. M. S., Pedrizzetti, G., Sengupta, P. P., Nagel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+tracking+technology+for+assessing+cardiac+mechanics:+principles,+normal+values,+and+clinical+applications.">Tissue tracking technology for assessing cardiac mechanics: principles, normal values, and clinical applications.</a> JACC. Cardiovascular Imaging. 8 (12), 1444-1460 (2015).</li><li>Scatteia, A., Baritussio, A., Bucciarelli-Ducci, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+imaging+using+cardiac+magnetic+resonance.">Strain imaging using cardiac magnetic resonance.</a> Heart Failure Reviews. 22 (4), 465-476 (2017).</li><li>Modin, D., Andersen, D. M., Biering-S&#248;rensen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echo+and+heart+failure:+when+do+people+need+an+echo,+and+when+do+they+need+natriuretic+peptides.">Echo and heart failure: when do people need an echo, and when do they need natriuretic peptides.</a> Echo Research and Practice. 5 (2), 65-79 (2018).</li><li>Onishi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+strain+and+global+circumferential+strain+by+speckle-tracking+echocardiography+and+feature-tracking+cardiac+magnetic+resonance+imaging:+comparison+with+left+ventricular+ejection+fraction.">Longitudinal strain and global circumferential strain by speckle-tracking echocardiography and feature-tracking cardiac magnetic resonance imaging: comparison with left ventricular ejection fraction.</a> Journal of American Society of Echocardiography. 28 (5), 587-596 (2015).</li><li>Faganello, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+integrated+approach+to+cardiac+mechanics:+reference+values+for+normal+left+ventricle.">A new integrated approach to cardiac mechanics: reference values for normal left ventricle.</a> The International Journal of Cardiovascular Imaging. 36, 2173-2185 (2020).</li><li>Lapinskas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraventricular+hemodynamic+forces+estimated+using+routine+CMR+Cine+images:+a+new+marker+of+the+failing+heart.">The intraventricular hemodynamic forces estimated using routine CMR Cine images: a new marker of the failing heart.</a> JACC. Cardiovascular Imaging. 12 (2), 377-379 (2019).</li><li>T&#246;ger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+hemodynamic+forces+using+4D+flow:+a+new+reproducible+method+applied+to+healthy+controls,+elite+athletes+and+heart+failure+patients.">Intracardiac hemodynamic forces using 4D flow: a new reproducible method applied to healthy controls, elite athletes and heart failure patients.</a> Journal of Cardiovascular Magnetic Resonance. 18, 61 (2016).</li><li>Eriksson, J., Bolger, A. F., Ebbers, T., Carlh&#228;ll, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+left+ventricular+hemodynamic+forces+in+healthy+subjects+and+patients+with+dilated+cardiomyopathy+using+4D+flow+MRI.">Assessment of left ventricular hemodynamic forces in healthy subjects and patients with dilated cardiomyopathy using 4D flow MRI.</a> Physiological Reports. 4 (3), 12685 (2016).</li><li>Coolen, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+T1+mapping+of+the+mouse+heart+using+variable+flip+angle+steady-state+MR+imaging.">Three-dimensional T1 mapping of the mouse heart using variable flip angle steady-state MR imaging.</a> NMR in Biomedicine. 24 (2), 154-162 (2011).</li><li>Nijm, G. M., Swiryn, S., Larson, A. C., Sahakian, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+magnetohydrodynamic+effect+as+a+signal+from+the+surface+electrocardiogram+during+cardiac+magnetic+resonance+imaging.">Characterization of the magnetohydrodynamic effect as a signal from the surface electrocardiogram during cardiac magnetic resonance imaging.</a> Computers in Cardiology. 33, 269-272 (2006).</li><li>Domenichini, F., Pedrizzetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+forces+in+a+model+left+ventricle.">Hemodynamic forces in a model left ventricle.</a> Physical Review Fluids. 1, 083201 (2016).</li><li>Voigt, J. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitions+for+a+common+standard+for+2D+speckle+tracking+echocardiography:+consensus+document+of+the+EACVI/ASE/Industry+Task+Force+to+standardize+deformation+imaging.">Definitions for a common standard for 2D speckle tracking echocardiography: consensus document of the EACVI/ASE/Industry Task Force to standardize deformation imaging.</a> European Heart Journal - Cardiovascular Imaging. 16 (1), 1-11 (2015).</li><li>Pedrizzetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+computation+of+hemodynamic+forces+in+the+heart+chambers.">On the computation of hemodynamic forces in the heart chambers.</a> Journal of Biomechanics. 95, 109323 (2019).</li><li>Hoffman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+strain+and+cardiac+output+are+preferable+measurements+for+cardiac+dysfunction+and+can+predict+mortality+in+septic+mice.">Myocardial strain and cardiac output are preferable measurements for cardiac dysfunction and can predict mortality in septic mice.</a> Journal of American Heart Association. 8 (10), 012260 (2019).</li></ol>

Ruslan Garipov is an employee of MR Solutions Ltd., Guildford, United Kingdom. David Hautemann is an employee of Medis medical imaging systems B.V., Leiden, The Netherlands.

The presented protocol describes the use of CMR imaging for longitudinal, non-invasive, in vivo experiments to analyze heart function in mice. These results are examples of healthy animals to demonstrate the feasibility of using CINE images to quantify the cardiac parameters. However, the methods described can be used for various animal models. Although specific disease models might require small alterations to the protocol, its basic structure to assess the different cardiac functional parameters will be very similar. One particular case worth mentioning is a myocardial infarction model where part of the heart has significant loss in contractility. This can cause low quality of the cardiac navigator signal within this slice. In this case, an alternative option would be acquiring the navigator from a separate slice, as described in a previous study by Coolen et al.16. CINE images in different views are reconstructed from retrospectively gated data using CS algorithms and are analyzed using image analysis software to calculate the strain and HDF values.
The quality of the acquired images naturally depends on all preparation steps, which need to be carefully performed before starting the cardiac MRI protocol. For instance, if no clear ECG and respiratory signals are seen when placing the animal inside the MRI scanner, this will likely result in suboptimal acquisitions and even increased scan times due to the added effect of magnetohydrodynamic distortions17. It is important to realize that due to the sequential planning of the slice orientations, the animals cannot just be repositioned in between scans. It is therefore not possible to re-adjust the ECG leads in between scans, as this will alter the position of the mouse in the scanner. During scanning, temperature control is crucial for maintaining a constant cardiac and respiratory interval, which especially benefits the quality of the retrospectively gated scans that are acquired over a longer period of time. During this high-duty-cycle scan, the temperature of the animal might steadily increase, causing the heart rate and respiratory rate to increase. Adjusting the temperature of the heating system and the anesthesia could greatly contribute to stabilizing the respiratory rate prior to or during scanning.
A critical step during the analysis is the consistency in contour drawing. While automatic segmentation works well for clinical data, it does not perform robustly in the case of mouse cardiac data (not tested for rats). The high heart rate and high blood flow during specific cardiac phases, especially at the start of LV filling, may cause intravoxel dephasing and signal voids, compromising myocardial wall delineation. It is therefore not advised to analyze each frame independently, but visually inspect the motion of the myocardial wall between frames and take this into account when drawing the contours across all frames. It is advised to copy and adjust the endocardial contour between two consecutive frames to maintain a more natural contractile motion in the analysis. In this protocol, papillary muscles are excluded from the ventricular lumen volume in the SA images for systolic and diastolic function assessment, while they are included in the 2CH, 3CH, and 4CH views for strain and HDF analysis because the latter relies on knowledge of the precise motion of the myocardial wall, rather than the precise volume of the ventricular lumen.
Whereas systolic and diastolic function parameters are based on measuring LV volumes throughout the cardiac cycle, strain and HDF parameters depend on motion patterns within the myocardial wall as well. For this, feature-tracking techniques are used where the displacement of the myocardial segment can be assessed by recognizing distinct anatomical features and signal intensities between subsequent CINE phases. The strong contrast between blood pool and myocardium in CMR images facilitates the use of feature-tracking for subsequent strain and HDF analysis8. Prior to CMR feature-tracking, the myocardial strain was determined with speckle tracking echography and CMR tissue-tagging. CMR feature-tracking does not require additional scanning time compared to CMR tissue-tagging. However, despite the use of retrospective triggering, CMR still has a limited temporal resolution, which could make it difficult to correctly evaluate fast deformations within the cardiac cycle.
Assessment of HDF throughout the cardiac cycle requires measurements of the diameters of the mitral and aortic valves to calculate the HDF in apex-base and inferolateral-anteroseptal directions using previously described equations18. This method has shown consistent estimates of the HDF compared to the reference standard 4D-flow MRI, which has a limited availability in clinical use due to its complexity6. It is important to know that robust estimation of the valve diameters is difficult, and therefore, the valve diameters should be kept constant for a group of animals and across repeated measurements in a longitudinal study, as variations in this parameter by incorrect estimations could easily overshadow subtle changes in HDF parameters. The specific software used to calculate GLS and HDF parameters might not be available to all users. Therefore, one can refer to Voigt et al.19&#160;(GLS) as well as Pedrizzetti et al.6,20 (HDF), which contain all mathematical descriptions that form the basis of the respective calculations as performed by the analysis software.
For the purpose of this study, the protocol was evaluated in healthy animals (N = 6). A representative set of time curves for LV volume, dV/dt, endoGLS, and HDF are shown in Figure 5A-C. Mean values of multiple cardiac functional parameters (EF, E&#39;/A&#39;-ratio, peak GLS, and HDF) are shown in Figure 5D. These agree well with comparable protocols used in the literature21. Literature on GLS and HDF data in mice is scarce. A mean GLS value of -22.8% was measured, which is in the same range as clinical data8, indicating that GLS measurements obtained with the described method are feasible in mice. HDF curves obtained in mice also show the same distinct phases as seen in human data, showing the successful translation of this technique to preclinical research. While HDF parameters are hypothesized to serve as early biomarkers of cardiac dysfunction, more studies are warranted to investigate the diagnostic and predictive value of this new parameter. The results in this protocol do show that HDF and GLS outcomes are expected to be more variable across animals, which needs to be taken into account when subtle differences in animal models or treatment effects are expected.

Cardiovascular magnetic resonance (CMR) in small animals provides an accurate in vivo measurement of myocardial function, making CMR an optimal tool for preclinical research in cardiovascular disease. Due to the high spatial resolution and high contrast between blood and myocardium in CMR images, it is possible to delineate the endo- and epicardial contours and calculate myocardial mass and ventricular volumes1,2. Despite the high heart rates of up to 600 beats/min, the use of electrocardiogram (ECG) and respiratory triggering allows high-quality measurements of different cardiac phases (also called CINE images) without respiratory motion artifacts. In this way, multiple slices can be used to cover the heart from apex to base to extract systolic function parameters such as ejection fraction (EF), end-systolic volume (ESV), end-diastolic volume (EDV), and cardiac output (CO)3. Aside from basic systolic function assessment, additional CMR techniques have been recently developed to assess diastolic dysfunction4, myocardial strain5, and hemodynamic forces (HDF)6.
ECG gating allows synchronization to the cardiac cycle by starting MR signal acquisition after detection of the R-peak and recording a defined number of cardiac phases during the R-R interval. However, the number of cardiac phases (frame rate) that can be acquired this way depends on the lowest possible repetition time (TR) the system can reach while maintaining an acceptable signal-to-noise ratio (SNR) and spatial resolution4. Furthermore, because the use of high magnetic field gradients can temporarily distort the ECG signal, the acquisition is usually stopped before the end-diastolic phase. Both factors limit the use of such scans to systolic function assessments, as the calculation of other cardiac functional parameters requires a better definition of the left ventricular (LV) volume-time curve.
High-frame-rate CINE images can be acquired by retrospective gating, whereby MR signal is continuously acquired during scanning, and an incorporated navigator echo after radiofrequency (RF) excitation detects cardiac and respiratory motion. Because the CMR acquisition is performed asynchronously with the cardiac motion, the acquired MR signals can then be assigned to a retrospectively chosen number of cardiac frames. In this way, if sufficient data is collected, high-frame-rate CINE images can be reconstructed4,7. This then allows for diastolic function assessment, represented by the ratio between the peak early filling rate (E&#39;) and peak late filling rate from atrial contraction (A&#39;).
In clinical research, CINE images can be analyzed with CMR feature-tracking to assess the myocardial strain and HDF6,8. Myocardial strain is a cardiac deformation parameter that measures the difference in percentages between the initial length (usually in end-diastolic length) and maximum length (usually in end-systole) of a myocardial segment9. Myocardial strain measurements can be of incremental value for assessing the LV function as strain values quantify myocardial wall shortening and thickening. A reduction in shortening function might be an indication of subendocardial fiber damage10. Alterations in myocardial strain can occur independent of EF and could be a precursor for underlying complications.
Specifically, the global longitudinal strain (GLS) and global circumferential strain (GCS) have been shown to be of added value in characterizing heart disease10,11,12. Similarly, HDF has been suggested to be a potential novel parameter to indicate altered cardiac function6,13. These HDF or interventricular pressure gradients (IVPG) drive the blood motion during ejection and filling of the heart and are affected by the momentum exchange between blood and myocardium, including the aortic and mitral valve14,15.
In this study, a comprehensive protocol is described for performing robust small animal CMR measurements to quantify LV function, myocardial strain, and HDF of mouse hearts. It contains the necessary steps for animal preparation, data acquisition using both prospectively and retrospectively gated CINE images of the heart, as well as analysis with dedicated software capable of calculating the volumetric measurements, E&#39;/A&#39; ratio, myocardial strain, and HDF of the heart. This protocol can be used for the extensive assessment of LV function in various mouse models of cardiovascular disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AccuSens single and multi-channel signal conditioner</td>
			<td>Opsens solutions inc., Canada</td>
			<td>&#160;ACS-P4-N-62SC</td>
			<td>Used with fiber optic temperature sensor to monitor body temperature</td>
		</tr>
		<tr>
			<td>Duratears eye ointment</td>
			<td>Alcon Nederland B.V., Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse cell</td>
			<td>&#201;quipment V&#233;t&#233;rinaire Minerve, France</td>
			<td></td>
			<td>referred to as mouse cradle</td>
		</tr>
		<tr>
			<td>MR-compatible Monitoring &#38; Gating System for Small animals</td>
			<td>SA Intuments, Inc., United States</td>
			<td>Model 1030</td>
			<td>ERT Module (ECG/respiratory interface module) , ERT Control/Gating Module, battery pack and subdermal ECG Electrode Set</td>
		</tr>
		<tr>
			<td>MRI scanner</td>
			<td>MR Solutions Ltd., United Kingdom</td>
			<td>Model: MRS-7024&#160;&#160;</td>
			<td>Preclinical MRI System 7.0T/24 cm</td>
		</tr>
		<tr>
			<td>Multistation temperature control unit and High Flow PCA</td>
			<td>&#201;quipment V&#233;t&#233;rinaire Minerve, France</td>
			<td>Model: URT Multipostes</td>
			<td>animal heating system</td>
		</tr>
		<tr>
			<td>Respiration Sensor</td>
			<td>Graseby Medical Limited, United Kingdom</td>
			<td>Ref 2005100</td>
			<td></td>
		</tr>
		<tr>
			<td>RF coil</td>
			<td>MR Solutions Ltd., United Kingdom</td>
			<td>MRS-MVC</td>
			<td>38mm mouse volume RF coil for mouse body studies</td>
		</tr>
		<tr>
			<td>SF flowmeter</td>
			<td>flow-meter, Italy</td>
			<td>SF 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer sigma delta Intermed</td>
			<td>Penlon Ltd., United Kingdom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>AST farma, Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vaseline petroleum jelly</td>
			<td>Unilever, United Kingdom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BART toolbox</td>
			<td></td>
			<td></td>
			<td>https://mrirecon.github.io/bart/</td>
		</tr>
		<tr>
			<td>Mathematica 12.0</td>
			<td>Wolfram Research, Inc., United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB 2019a</td>
			<td>The MathWorks,Inc., United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEDIS Suite MR</td>
			<td>Medis Medical Imaging Systems B.V. ,Netherlands</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>PC-SAM</td>
			<td>SA Intuments, Inc., United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preclinical Scan</td>
			<td>MR Solutions Ltd., United Kingdom</td>
			<td></td>
			<td>Scanning software</td>
		</tr>
		<tr>
			<td>Retrospective version 7.0</td>
			<td>Amsterdam UMC, the Netherlands</td>
			<td></td>
			<td>Reconstuction software: https://github.com/Moby1971?tab=repositories</td>
		</tr>
	</tbody>
</table>

quantification of mouse heart left ventricular function, myocardial strain, and hemodynamic forces by cardiovascular magnetic resonance imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

Using the previously described protocol, a group of healthy C57BL/6 wildtype mice (n = 6, age 14 weeks) was scanned using a 7-Tesla MRI scanner using a 38-mm-diameter birdcage coil. During each scan session, the multi-slice CINE SA images were acquired using prospectively gated GRE sequences, while the single-slice midventricular SA, 2CH, 3CH, and 4CH views CINE images were acquired using retrospective gating. Representative high-frame rate reconstructions of retrospectively gated scans using a custom-built, post-processing software can be seen in Supplemental Video 1. From the resulting images, volume-time curves during the cardiac cycle (Figure 5A) were determined as well as the corresponding first-derivative curves (dV/dt) for calculation of systolic (EF = 72.4 &#177; 2.8%) and diastolic function parameters (E&#39;/A&#39; ratio = 1.5 &#177; 0.3) respectively.
The 2CH, 3CH, and 4CH view CINE images were analyzed using image analysis software to determine endocardial GLS (endoGLS) changes across the cardiac cycle (Figure 5B) and corresponding peak GLS values (-22.8 &#177; 2.4%) as a measure for myocardial strain. Additionally, the software calculates the root mean square (RMS) HDF in longitudinal (apex-base) (135.2 &#177; 31.7%) and transversal (inferolateral-anteroseptal) (12.9 &#177; 5.0%) directions. For each animal, it is also possible to produce an HDF time profile, which follows a consistent pattern of positive and negative peaks that represent the magnitude and direction of the HDF during the cardiac cycle (Figure 5C). Descriptive results of all outcome parameters are summarized in Figure 5D.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62595/62595fig05.jpg" /> 
Figure 5: Quantification of LV functional parameters based on the mouse heart. (A)&#160;Representative volume-time curve and corresponding dV/dt curve. The latter depicts the flow velocity with distinct early filling peak (E&#39;) and atrial contraction (A&#39;) peak. (B) Representative GLS curve indicating strain deformation in the longitudinal direction throughout the cardiac cycle. (C) Representative HDF curve with distinct force peaks in the apex-base direction, starting with the systolic ejection force and followed by a downward force at the transition between systole and diastole, E-wave deceleration force, A-wave acceleration, and deceleration force. (D) Descriptive results of all animals for values of EF, E&#39;/A&#39; ratio, peak GLS, and root mean square of the HDF in apex-base and inferolateral-anteroseptal directions. Values are expressed as mean &#177; SD. Abbreviations: LV = left ventricle; V = volume; t = time; GLS = global longitudinal strain; HDF = hemodynamic forces; EF = ejection fraction. <a href="https://www.jove.com/files/ftp_upload/62595/62595fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video 1:&#160;Representative reconstruction of retrospectively gated CINE images in SA, 2CH, 3CH, and 4CH views. Abbreviations: SA = short axis; CH = chamber. <a href="https://www.jove.com/files/ftp_upload/62595/Supplemental_Material_movie.mp4" target="_blank">Please click here to download this Video.</a>
Supplemental Material:&#160;<a href="https://www.jove.com/files/ftp_upload/62595/Supplemental_Material_script.nb" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging at JoVE.com

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

quantification-mouse-heart-left-ventricular-function-myocardial

Research

JoVE Journal

Bioengineering

6.0K Views.  University of Amsterdam. This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Video: Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk stratification in various cardiac disorders. Recently, 3D echocardiography became available in routine cardiology practice; however, high-quality image acquisition and subsequent analysis have a steep learning curve. The present article aims to guide the reader through a detailed 3D protocol by presenting tips and tricks and also by highlighting the potential pitfalls to facilitate the widespread but technically sound use of this important technique concerning the LV. First and foremost, we show the acquisition of a high-quality 3D dataset with optimal spatial and temporal resolution. Then, we present the analytical steps toward a detailed quantification of the LV by using one of the most widely applied built-in software. We will quantify LV volumes, sphericity, mass and also systolic function by measuring ejection fraction and myocardial deformation (longitudinal and circumferential strain). We will discuss and provide clinical examples about the essential scenarios where the transition from a conventional echocardiographic approach to a 3D-based quantification is highly recommended.

In this article, we provide a step-by-step acquisition and analysis protocol for the volumetric assessment and speckle-tracking analysis of the left ventricle by 3D echocardiography, particularly focusing on practical aspects that maximize the feasibility of this technique.

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...