&#65279;This work was supported by research grants from the Fukuda Foundation for Medical Technology (to E.T. and G. N.) and the JSPS KAKENHI Scientific Research Grant-in-Aid 21K08047 (to E.T.).

This animal protocol was approved by the Institutional Animal Care and Use Committee and followed the regulations for animal experiments and related activities at the University of Tokyo. For the present study, 8-12 week old male C57/Bl6J mice were used. The animals were obtained from a commercial source (see the Table of Materials). A model of HFpEF was established by administering a high-fat diet for 15 weeks in conjunction with NG-nitro-L-arginine methyl ester, as described previously10.
1. Catheter preparations and pressure/volume calibration
<ol>
	<li>Place a conductance catheter in normal saline, and attach it to a module consisting of the PowerLab 8/35 and a pressure-volume unit (MPVS module, see the Table of Materials).</li>
	<li>Electronically calibrate the pressure and volume through the recording of predetermined pressure (0 mmHg and 100 mmHg) and volume parameters (these vary between MPVS modules) on the MPVS module3,11&#160;(see also the manufacturer&#39;s instructions).</li>
</ol>
2. Preparing an animal for catheterization
<ol>
	<li>Anesthesia and ventilation
	<ol>
		<li>Administer an intraperitoneal injection of 5 mg/kg of etomidate and 500 mg/kg of urethane (see the Table of Materials) 5-10 min prior to intubation. 
		NOTE: Urethane, while effective as an anesthetic agent in animal studies, is suspected to be carcinogenic to humans. Therefore, when urethane is necessary for the achievement of experimental objectives and no alternative agents suffice, it must be handled with caution. Appropriate protective measures, such as wearing gloves and masks and utilizing a fume hood during preparation, are mandated. As a possible alternative, ketamine (80 mg/kg, ip) might be employed.</li>
		<li>Place the mouse in an anesthesia chamber previously saturated with 2% isoflurane, and transfer the animal to a pre-warmed heating pad maintained between 38 &#176;C and 40 &#176;C upon the induction of anesthesia.</li>
		<li>Shave the surgical area. Then, disinfect the surgical site with three alternating rounds of betadine and alcohol.</li>
		<li>Make a horizontal incision (1-2 cm) in the neck, excise the tracheal muscle, and expose the trachea. Pass a surgical 2-0 silk suture beneath the trachea, elevate it, and make a small incision (1-2 mm) to open it.</li>
		<li>Insert an endotracheal tube into the trachea, and connect it to a ventilator that delivers a mixture of 100% oxygen and 2% isoflurane (reduced to 0.5% to 1% later).</li>
	</ol>
	</li>
	<li>Central venous (CV) catheter insertion and fluid injection
	<ol>
		<li>Locate the internal jugular vein beneath the sternocleidomastoid muscle3.</li>
		<li>Insert the central venous catheter, consisting of PE-10 silastic tubing (see the Table of Materials) attached to a 30 G needle, into the jugular vein.</li>
		<li>Administer a bolus infusion of 5-6 &#181;L/g of body weight of 10% albumin/NaCl over 3 min, followed by a constant infusion rate of 5-10 &#181;L/min. 
		&#8203;NOTE: This step is crucial for preventing hypotension resulting from the peripheral vasodilation caused by the anesthesia. The internal jugular vein is located between the sternocleidomastoid muscle and the carotid artery, and it appears darker in color than the artery.</li>
	</ol>
	</li>
</ol>
3. Surgical procedure for left ventricular catheterization (open chest approach)
<ol>
	<li>Shave the surgical area of the anesthetized mouse. Then, disinfect the surgical site with three alternating rounds of betadine and alcohol.</li>
	<li>Confirm the depth of anesthesia by performing a toe pinch. Then, make a horizontal incision (2-3 cm) below the xiphoid process, and separate the skin from the chest wall using blunt scissors.</li>
	<li>Cut through the chest wall laterally on both sides using electrical cautery (see the Table of Materials).</li>
	<li>Expose the heart by cutting through the diaphragm, and remove the pericardium gently from the heart using forceps.</li>
	<li>Insert a 27 G needle into the apex of the left ventricle (LV), and retrogradely insert a conductance catheter into the LV via the puncture hole.</li>
	<li>Adjust the catheter position so that a square-shaped pressure-volume loop is obtained.</li>
	<li>Verify that the catheter does not contact the papillary muscle when changes in loading conditions occur by checking the shape of the PV loop during inferior vena cava (IVC) occlusion. 
	&#8203;NOTE: Adequate heart exposure facilitates the procedure and helps to obtain a clear view.</li>
</ol>
4. Recording PV loop data and determining the end-systolic pressure-volume relationship (ESPVR) and the end-diastolic pressure-volume relationship (EDPVR)
NOTE: Reducing the preload by IVC occlusion enables the determination of the ESPVR and EDPVR.
<ol>
	<li>Record and analyze the baseline pressure-volume (PV) loop with LabChart software (see the Table of Materials), PowerLab, and the MPVS module after signal stabilization (5-10 min after canulation).</li>
	<li>Perform IVC occlusion by compressing the IVC with forceps, and record the PV loop for at least 20 cardiac cycles during the IVC occlusion. Determine the ESPVR by fitting a linear regression line through the end-systolic points of the PV loop and the EDPVR by fitting a curvilinear line through the end-diastolic points of the PV loop using LabChart software. 
	&#8203;NOTE: Stop the ventilator during the IVC occlusion to prevent lung motion artifacts. A paralytic agent like pancuronium (0.5-1 mg/kg) may be helpful when lung motion is excessive and should be used only after a stable anesthetic plane is confirmed.</li>
</ol>
5. Transesophageal pacing
<ol>
	<li>Insert a 2-Fr tetrapolar electrode catheter into the esophagus, connect the catheter to a pulse stimulator (see the Table of Materials), and determine the atrial capture threshold (normally, the stimulus amplitude is 3 mA, and the pulse width is 1 ms).</li>
	<li>Slow the HR below 400 beats/min using 20 mg/kg of ivabradine (see the Table of Materials) administered intraperitoneally.</li>
	<li>Following stabilization, acquire 20 continuous cardiac cycles of PV loops at different pacing rates from 400 beats/min to 700 beats/min, with an increment of 100 beats/min; acquire the cycles over 5 min at each pacing rate.</li>
</ol>
6. Saline calibration and aortic flow calibration
<ol>
	<li>Inactivate the ventilator, and administer a 5-10 &#181;L of hypertonic saline solution intravenously through the CV catheter.</li>
	<li>Document the fluctuations in pressure and volume during the saline injection, and calculate the Vp value using PowerLab3,11.</li>
	<li>Repeat the saline calibration to enhance the accuracy and reproducibility.</li>
	<li>Turn the mouse onto its left side in order not to disturb the volume signal.</li>
	<li>Make a lateral thoracotomy between Th3 to Th5 toward the spine, and gently dissect a small part of the descending aorta with forceps.</li>
	<li>Place a vascular flow probe (see the Table of Materials) over the aorta to measure the cardiac output. 
	&#8203;NOTE: The accurate calculation of the absolute volume requires the use of two types of calibration: saline calibration and aortic flow calibration. It is important to recognize the potential risks associated with a hypertonic saline infusion in animal subjects, as excessive salt loading can result in a decline in contractility.</li>
</ol>
7. Euthanasia
<ol>
	<li>After the study, euthanize the mice under an anesthetic overdose via cervical dislocation. 
	NOTE: To ensure the complete cessation of vital function, a secondary method of euthanasia is employed, such as exsanguination under anesthesia with subsequent cardiac tissue harvesting.</li>
</ol>

Exploring HR-Dependent Cardiac Performance Using an Atrial-Pacing Protocol

<ol>
	<li>Backhaus, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+stress+real-time+cardiac+magnetic+resonance+imaging+for+noninvasive+characterization+of+heart+failure+with+preserved+ejection+fraction.">Exercise stress real-time cardiac magnetic resonance imaging for noninvasive characterization of heart failure with preserved ejection fraction.</a> Circulation. 143 (15), 1484-1498 (2021).</li><li>Borlaug, B. A., Nishimura, R. A., Sorajja, P., Lam, C. S. P., Redfield, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+hemodynamics+enhance+diagnosis+of+early+heart+failure+with+preserved+ejection+fraction.+Circulation.">Exercise hemodynamics enhance diagnosis of early heart failure with preserved ejection fraction. Circulation.</a> Heart Failure. 3 (5), 588-595 (2010).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., B&#225;tkai, S., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nature Protocols. 3 (9), 1422-1434 (2008).</li><li>Cingolani, O. H., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-volume+relation+analysis+of+mouse+ventricular+function.">Pressure-volume relation analysis of mouse ventricular function.</a> American Journal of Physiology - Heart and Circulatory Physiology. 301 (6), 2198-2206 (2011).</li><li>Numata, 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+pacing-controlled+protocol+for+frequency-diastolic+relations+distinguishes+diastolic+dysfunction+specific+to+a+mouse+HFpEF+model.">A pacing-controlled protocol for frequency-diastolic relations distinguishes diastolic dysfunction specific to a mouse HFpEF model.</a> American Journal of Physiology - Heart and Circulatory Physiology. 323 (3), H523-H527 (2022).</li><li>Pi&#241;a, I. L., 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=Exercise+and+heart+failure.">Exercise and heart failure.</a> Circulation. 107 (8), 1210-1225 (2003).</li><li>Georgakopoulos, D., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+force-frequency+modulation+of+inotropy+and+relaxation+of+in+situ+murine+heart.">Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart.</a> Journal of Physiology. 534 (2), 535-545 (2001).</li><li>Takimoto, E., 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=Frequency-+and+afterload-dependent+cardiac+modulation+in+vivo+by+troponin+I+with+constitutively+active+protein+kinase+A+phosphorylation+sites.">Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites.</a> Circulation Research. 94 (4), 496-504 (2004).</li><li>Meyer, M., Lewinter, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+and+heart+failure+with+preserved+ejection+fraction:+Time+to+slow+&#946;-blocker+use?+Circulation.">Heart rate and heart failure with preserved ejection fraction: Time to slow &#946;-blocker use? Circulation.</a> Heart Failure. 12 (8), 006213 (2019).</li><li>Schiattarella, G. 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=Nitrosative+stress+drives+heart+failure+with+preserved+ejection+fraction.">Nitrosative stress drives heart failure with preserved ejection fraction.</a> Nature. 568 (7752), 351-356 (2019).</li><li>Abraham, D., Mao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+pressure-volume+loop+analysis+using+conductance+catheters+in+mice.">Cardiac pressure-volume loop analysis using conductance catheters in mice.</a> Journal of Visualized Experiments. (103), e52942 (2015).</li><li>Zhang, B., Davis, J. P., Ziolo, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+catheterization+in+mice+to+measure+the+pressure+volume+relationship:+Investigating+the+Bowditch+effect.">Cardiac catheterization in mice to measure the pressure volume relationship: Investigating the Bowditch effect.</a> Journal of Visualized Experiments. (100), e52618 (2015).</li><li>Townsend, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+pressure+volume+loops+in+the+mouse.">Measuring pressure volume loops in the mouse.</a> Journal of Visualized Experiments. (111), e53810 (2016).</li><li>Georgakopoulos, D., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+parallel+conductance+by+dual-frequency+conductance+catheter+in+mice.">Estimation of parallel conductance by dual-frequency conductance catheter in mice.</a> American Journal of Physiology - Heart and Circulatory Physiology. 279 (1), H47 (2000).</li></ol>

There are no competing financial interests.

We present a methodology to assess pressure-volume relationships with the application of transesophageal pacing. Exercise intolerance is one of the key characteristics of HFpEF, yet there are no techniques available for the evaluation of cardiac function in mice during exercise. Our pacing protocol offers a valuable tool for detecting diastolic dysfunction, which may not be apparent under resting conditions.
To achieve a PV loop of accurate and consistent quality, the following steps must be m...

Heart failure represents a leading cause of hospitalization and death across the globe, and heart failure with preserved ejection fraction (HFpEF) accounts for around 50% of all heart failure diagnoses. HFpEF is characterized by diastolic dysfunction and impaired exercise tolerance, and the associated hemodynamic abnormalities, such as diastolic dysfunction, can be clearly detected through exercise-stressed hemodynamic testing or MRI scans1,2.
In experimental models, however, available modalities for assessing the physiological abnormalities related to HFpEF are limited3,4. Treadmill exercise testing (TMT) is used to determine running time and distance, which might reflect exercise-stress cardiac hemodynamics; however, this method is susceptible to interference from extraneous variables such as the body weight, skeletal muscle strength, and mental status.
To circumvent these limitations, we have devised an atrial-pacing protocol that detects subtle but crucial changes in diastolic performance based on the heart rate (HR) and have validated its usefulness in a mouse model of HFpEF5. Several physiological factors contribute to exercise-related cardiac function, including the sympathetic nerve and catecholamine response, peripheral vasodilation, the endothelial response, and the heart rate6. Among these, however, the HR-pressure relationship (also called the Bowditch effect) is known as a critical determinant of cardiac physiological features7,8,9.
The protocol involves performing a conventional pressure-volume analysis at baseline to assess the systolic and diastolic function, including parameters such as the rate of pressure development (dp/dt), the end-systolic pressure-volume relationship (ESPVR), and the end-diastolic pressure-volume relationship (EDPVR). However, it should be noted that these parameters are influenced by the HR, which can vary between animals due to differences in their intrinsic heart rate. Additionally, the effects of anesthesia on the HR should also be considered. To address this, the HR was standardized by administering atrial pacing concomitantly with ivabradine, and cardiac parameter measurements were performed at incremental heart rates. Notably, the HR-dependent cardiac response distinguished HFpEF mice from the control group mice, while no significant differences were observed in the baseline PV loop measurements (using the intrinsic heart rate)5.
While this pacing protocol may seem relatively complicated, its success rate exceeds 90% when it is well understood. This protocol would provide a useful way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and help in the development of new treatments for this condition.

<table><tbody><tr><td>2-0 silk suture, sterlie</td><td>Alfresa Pharma Corporation, Osaka, Japan</td><td>62-9965-57</td><td>Surgical Supplies</td></tr><tr><td>2-Fr tetrapolar electrode catheter</td><td>Fukuda Denshi, Japan and UNIQUE MEDICAL, Japan</td><td>custom-made</td><td>Surgical Supplies</td></tr><tr><td>Albumin Bovine Serum</td><td>Nacalai Tesque, Inc., Kyoto, Japan</td><td>01859-47</td><td>Miscellaneous</td></tr><tr><td>C57/BI6J mouse</td><td>Jackson Laboratory</td><td></td><td>animals</td></tr><tr><td>Conductance catheter</td><td>Millar Instruments, Houston, TX</td><td>PVR 1035</td><td></td></tr><tr><td>Electrical cautery, Electrocautery Knife Kit</td><td>ellman-Japan,Osaka, Japan</td><td>1-1861-21</td><td>Surgical Supplies</td></tr><tr><td>Etomidate</td><td>Tokyo Chemical Industory Co., Ltd., Tokyo Japan</td><td>E0897</td><td>Anesthetic</td></tr><tr><td>Grass Instrument S44G Square Pulse Stimulator</td><td>Astro-Med, West Warwick, RI</td><td></td><td>Pacing equipment</td></tr><tr><td>Isoflurane</td><td>Viatris Inc., Tokyo, Japan</td><td>8803998</td><td>Anesthetic</td></tr><tr><td>Ivabradine</td><td>Tokyo Chemical Industory Co., Ltd., Tokyo Japan</td><td>I0847</td><td>Miscellaneous</td></tr><tr><td>LabChart software</td><td>ADInstruments, Sydney, Australia</td><td>LabChart 7</td><td>Hemodynamic equipment</td></tr><tr><td>MPVS Ultra</td><td>Millar Instruments, Houston, TX</td><td>PL3516B49</td><td>Hemodynamic equipment</td></tr><tr><td>Pancronium bromide</td><td>Sigma Aldrich Co., St. Louis, MO</td><td>15500-66-0</td><td>Anesthetic</td></tr><tr><td>PE10 polyethylene tube</td><td>Bio Research Center&amp;nbsp; Co. Ltd., Tokyo, Japan</td><td>62101010</td><td>Surgical Supplies</td></tr><tr><td>PowerLab 8/35</td><td>ADInstruments, Sydney, Australia</td><td>PL3508/P</td><td>Hemodynamic equipment</td></tr><tr><td>PVR 1035</td><td>Millar Instruments, Houston, TX</td><td>842-0002</td><td>Hemodynamic equipment</td></tr><tr><td>Urethane (Ethyl Carbamate)</td><td>Wako Pure Chemical Industries, Ltd., Osaka, Japan</td><td>050-05821</td><td>Anesthetic</td></tr><tr><td>Vascular Flow Probe</td><td>Transonic, Ithaca, NY</td><td>MA1PRB</td><td>Surgical Supplies</td></tr></tbody></table>

a pacing-controlled procedure for the assessment of heart rate-dependent diastolic functions in murine heart failure models

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While exercise-stressed hemodynamic tests or MRI can be used to detect diastolic dysfunction and diagnose HFpEF in humans, such modalities are limited in basic research using mouse models. A treadmill exercise test is commonly used for this purpose in mice, but its results can be influenced by body weight, skeletal muscle strength, and mental state. Here, we describe an atrial-pacing protocol to detect heart rate (HR)-dependent changes in diastolic performance and validate its usefulness in a mouse model of HFpEF. The method involves anesthetizing, intubating, and performing pressure-volume (PV) loop analysis concomitant with atrial pacing. In this work, a conductance catheter was inserted via a left ventricular apical approach, and an atrial pacing catheter was placed in the esophagus. Baseline PV loops were collected before the HR was slowed with ivabradine. PV loops were collected and analyzed at HR increments ranging from 400 bpm to 700 bpm via atrial pacing. Using this protocol, we clearly demonstrated HR-dependent diastolic impairment in a metabolically induced HFpEF model. Both the relaxation time constant (Tau) and the end-diastolic pressure-volume relationship (EDPVR) worsened as the HR increased compared to control mice. In conclusion, this atrial pacing-controlled protocol is useful for detecting HR-dependent cardiac dysfunctions. It provides a new way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and may help develop new treatments for this condition.

The baseline PV loop data are displayed in Figure 1 and Table 1. At baseline (in the absence of pacing), there were no significant differences in diastolic parameters such as the relaxation time constant (Tau), the minimum rate of pressure change (dP/dt min), and EDPVR between the control and HFpEF mice. However, the HFpEF mice exhibited higher blood pressure and arterial elastance (Ea), as shown in Figure 1, and demonstrated a typical mountain-...

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Author Spotlight: Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach 

The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function&#160;in mouse models of heart failure.

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While ...

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1.2K Views.  The University of Tokyo Hospital. The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function in mouse models of heart failure.

Video: Author Spotlight: Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach 

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Medicine

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

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.

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.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

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