This study was supported by NIH grants HL091986 (JPD) and HL094692 (MTZ).

NOTE: This animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University. This procedure can be used on any mouse in which the inner diameter of the carotid artery is large enough to insert the catheter. Use mice that are above 16 g (older than ~2 months).
1. Preparing Mouse for Catheterization
<ol>
	<li>Seal all surgical instruments and supplies in a sterilization pouch. Sterilize the pouch in an autoclave machine. Maintain a sterile field throughout the procedure and wear sterile gloves.</li>
	<li>Anesthetize mice with ketamine (55 mg/kg) plus xylazine (15 mg/kg) by intraperitoneal injection. 
	NOTE: The whole procedure measuring both pressure/volume at various heart rates and ESPRV takes less than 20 min. If additional time is required (i.e., more than 30 min), give an additional &#188; dose of anesthesia every 30 min.</li>
	<li>Remove the hair in the anterior region of neck and chest area using hair removing lotion (e.g., Nair) and tape the limbs of the mouse onto the foam platform. Confirm a state of deep anesthesia by a toe pinch.</li>
	<li>Insert a rectal probe to monitor the body temperature (37 &#177; 1 &#176;C), and maintain using a thermo-regulated heating pad (located between the surgical drape and platform).</li>
	<li>Prepare a length of 4-0 suture (~10 cm). Loop suture around the upper incisors and tape to platform. This will keep the neck straight.</li>
	<li>Sterilize the surgical area by swabbing the area with Betadine and 75% alcohol three times.</li>
</ol>
2. Catheterization
<ol>
	<li>Prepare the catheter by presoaking the tip in saline or distilled water (37 &#176;C) for at least 30 min prior to use (according to manufacturer&#8217;s instructions) to acclimate the pressure sensor diaphragm for the wet biological environment and to prevent pressure signal drift and negative pressure recordings.</li>
	<li>Make a longitudinal 0.8 cm incision between the lower jaw and sternum in the anterior region of the neck. With the fine scissors, separate the skin-muscular connective tissue to expose the trachea located under the stemohyoideus muscle.</li>
	<li>Separate the fat and muscular tissue at the right side of the trachea with curved forceps to expose the right carotid artery. 
	NOTE: The carotid artery is the biggest artery in the anterior region of the neck, contains bright red blood, and is pulsatile. Do not confuse with the jugular vein that runs parallel to the carotid artery. The jugular vein is dark red and non-pulsatile. In addition, during the isolation of the carotid artery, the user should be aware of not damaging the pneumogastric nerve.</li>
	<li>Remove the fat from the right carotid artery with the curved forceps. If there exists branching of the vessel which will impede this operational technique, cut them with a Bovie cautery to dissociate the carotid artery. Separate as much of the tissue as possible under the carotid artery using curved forceps.</li>
	<li>Cut two 5 cm 6-0 silk threads. Pass each silk thread under the right carotid artery.</li>
	<li>Position one thread near the proximal part and the other near the distal part of the artery. Make a tight knot on the thread at the distal part, and make a loose knot on the thread at the proximal part.</li>
	<li>Block blood flow by clamping the proximal part of the artery using a small hemostat vascular clamp (place the clamp below the proximal thread). The sealed region of the artery will be filled with blood making it easy to perform step 2.8.</li>
	<li>Puncture a small hole in the right carotid artery between the two threads (but closer to the distal thread) with a 26 G needle. Insert the catheter into the carotid artery. Slightly tighten the loose knot at the proximal part of the carotid artery onto the catheter to keep in place. 
	NOTE: Using the needle puncture is preferred compared to scissors incision. By making a tight knot in the distal part of the artery first, and then clamping the proximal part, the artery will be fully filled with blood. This makes it very easy to poke through the blood vessel. Furthermore, the size of the needle (26 G) punctures the artery with a hole that nicely fits the size of the catheter. When using the scissors incision method, it was more difficult to control the size of the incision. However, the method chosen should be dependent upon on which one the surgeon feels more comfortable with.</li>
	<li>Start recording pressure signals as in step 3.</li>
	<li>Loosen the hemostat clamp and continue inserting the catheter forward into the left ventricle. If some resistance is experienced when advancing the catheter, gently pull it back and try advancing again. For a mouse weighing ~18-25 g, the estimated length of the catheter that is inserted is 18 mm. 
	NOTE: The arterial pressure signal will fluctuate from 70 to 120 mm Hg. Once the catheter is in the left ventricle the shape of the pressure signal changes and the pressure will fluctuate from 0 to 120 mm Hg (shown in Figure 1). Heart function will stabilize within 2-3 min after insertion of the catheter.</li>
	<li>Continuously monitor body temperature, anesthesia level, and breathing rate.</li>
</ol>
3. Data Acquisition
<ol>
	<li>Use LabChartPro 7 software (or similar software). Use the WorkFlow option of the PV Loop LabChart Module. Using this module, select the Pressure and Volume Loops default setting.</li>
	<li>Set-up three channels: one channel for pressure, one channel for volume, and one channel for heart rate. Set scale ranges of above parameters as 0-150 mm Hg, 0-100 &#181;l and 0-800 beat/min, respectively.</li>
	<li>Press start key to record.</li>
</ol>
4. Bowditch Effect
<ol>
	<li>Make a 1 cm incision in the precordium area parallel to the manubrium. Cut the layer of muscle and expose the intercostal space using scissors.</li>
	<li>Using a square pulse stimulator, set the following parameters: Voltage of 2 V, duration of 2 msec, and enable repeat mode.</li>
	<li>Hold the negative electrode with forceps and insert it through the fourth intercostal space to the apical region of the heart. Hold the positive electrode with forceps and insert it through the second intercostal space to the right atrium region of the heart.</li>
	<li>Turn on the stimulator and change the frequency to pace the heart from 4 Hz (240 beats/min) up to 10 Hz (600 beats/min). At each new heart rate, stimulate the heart for 1 min before data collection.</li>
</ol>
5. Generating the ESPVR and PRSW
<ol>
	<li>Cut the skin and muscle tissue perpendicular to the manubrium in the abdominal area with scissors. Open the enterocoelia and expose the liver.</li>
	<li>Drag the arcus costarum towards the head using metallic traction.</li>
	<li>Gently push the liver downwards with a cotton swab. Be careful not to push too much to affect the chest cavity. This will change heart function.</li>
	<li>Cut the falciform ligament of the liver with scissors to expose the suprahepatic inferior vena cava (IVC).</li>
	<li>Use curved forceps to rapidly squeeze the IVC for 5 sec to block the return of blood to the right atrium. The left ventricular pressure and volume will fall due to the reduced inflow to the heart. In generating these values, do not use loops below 60 mm Hg. The 60 mm Hg is in reference to systolic pressure. 
	NOTE: This value is set at 60 mm Hg because this will cause a significant drop in perfusion pressure to considerably decrease coronary perfusion and affect contractility.</li>
</ol>
6. Volume Calibration
<ol>
	<li>Heparinize the mouse with 0.1 ml of 1:5,000 heparin solution (diluted with normal saline) by intraperitoneal injection.</li>
	<li>Remove the catheter from the carotid artery. When the catheter is pulled out from the carotid artery, heparinized blood will seep from the hole where the catheter was inserted.</li>
	<li>Collect this blood for volume calibration using a 1 ml syringe. Fill each well in calibration cuvette.</li>
	<li>Remove heart to euthanize the mouse via exsanguination.</li>
	<li>Position the catheter in each well and get a steady relative volume unit (RVU) value. Generate a standard curve using the various standard volumes and RVU values from each well.</li>
	<li>Convert the recorded RVU to &#181;l.</li>
</ol>
7. Data Processing
<ol>
	<li>To examine the Bowditch effect, select steady-state pressure/volume traces from each heart rate. Click the baseline analysis to obtain data.</li>
	<li>For ESPVR and PRSW data, select the first ~15 pressure/volume traces, click occlusion analysis in the software to generate the ESPVR (slope of the pressure developed by the LV at end-diastolic volume) and PRSW (the linear regression of stroke work with the end-diastolic volume ) slopes.</li>
	<li>Give attention to the shape of the loops. Make sure the loop is closed with no angular points or twists. This is a sign of improper catheter placement or excess noise. Periodically check loops during the experiment to ensure proper pressure and volume data are being generated.</li>
</ol>

<ol>
	<li>Janssen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+contraction-relaxation+coupling.">Myocardial contraction-relaxation coupling.</a> Am J Physiol Heart Circ Physiol. 299, H1741-H1749 (2010).</li><li>Roman, M. J., Devereux, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+noninvasive+measures+of+contractility+in+dilated+cardiomyopathy.">Comparison of noninvasive measures of contractility in dilated cardiomyopathy.</a> Echocardiography. 8, 139-150 (1991).</li><li>Hamlin, R. L., del Rio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dP/dt(max)--a+measure+of+'baroinometry.">dP/dt(max)--a measure of 'baroinometry.</a> J Pharmacol Toxicol Methods. 66, 63-65 (2012).</li><li>Feneley, M. P., 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=Comparison+of+preload+recruitable+stroke+work,+end-systolic+pressure-volume+and+dP/dtmax-end-diastolic+volume+relations+as+indexes+of+left+ventricular+contractile+performance+in+patients+undergoing+routine+cardiac+catheterization.">Comparison of preload recruitable stroke work, end-systolic pressure-volume and dP/dtmax-end-diastolic volume relations as indexes of left ventricular contractile performance in patients undergoing routine cardiac catheterization.</a> J Am Coll Cardiol. 19, 1522-1530 (1992).</li><li>Kass, D. A., 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=Comparative+influence+of+load+versus+inotropic+states+on+indexes+of+ventricular+contractility:+experimental+and+theoretical+analysis+based+on+pressure-volume+relationships.">Comparative influence of load versus inotropic states on indexes of ventricular contractility: experimental and theoretical analysis based on pressure-volume relationships.</a> Circulation. 76, 1422-1436 (1987).</li><li>Nemoto, S., DeFreitas, G., Mann, D. L., Carabello, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+changes+in+left+ventricular+contractility+on+indexes+of+contractility+in+mice.">Effects of changes in left ventricular contractility on indexes of contractility in mice.</a> Am J Physiol Heart Circ Physiol. 283, H2504-H2510 (2002).</li><li>Ziolo, M. T., Kohr, M. J., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+signaling+and+the+regulation+of+myocardial+function.">Nitric oxide signaling and the regulation of myocardial function.</a> J Mol Cell Cardiol. 45, 625-632 (2008).</li><li>Barouch, L. A., 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=Nitric+oxide+regulates+the+heart+by+spatial+confinement+of+nitric+oxide+synthase+isoforms.">Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms.</a> Nature. 416, 337-339 (2002).</li><li>Wang, H., 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=Neuronal+nitric+oxide+synthase+signaling+within+cardiac+myocytes+targets+phospholamban.">Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban.</a> Am J Physiol Cell Physiol. 294, C1566-C1575 (2008).</li><li>Georgakopoulos, D., 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=In+vivo+murine+left+ventricular+pressure-volume+relations+by+miniaturized+conductance+micromanometry.">In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry.</a> Am J Physiol. 274, H1416-H1422 (1998).</li></ol>

There are no competing financial interests.

A critical step for this technique to obtain a reliable measure of contractility is proper catheter placement into the LV. If the catheter is not placed correctly, when the LV contracts the walls may contact the catheter resulting in very high, and not physiological, pressure values causing irregular shaped PV loops. If needed, the catheter can be rotated to achieve the correct placement. Another key step for this technique is to make sure the mouse received proper anesthesia. If the mouse is over anesthetized, this will greatly decrease heart function. A mouse with a heart rate less than ~250 beats/min can be considered over anesthetized. Additionally, the volume of blood that the mouse heart pumps is small making it difficult to get accurate volumes. It is important to calibrate the volume for each mouse. For volume calibration, we described the cuvette calibration technique. There are additional methods that are also used to calibrate volumes (i.e., stroke volume calculation using a flow probe in the descending thoracic aorta)10.
There are many limitations of using this method in the mouse; all due to its small body size. For example, this technique requires accurate microsurgical skill. Furthermore, this technique results in some blood loss, which could potentially change heart function. Massive blood loss can be avoided by advancing the catheter through the vasculature compared to other methods (e.g., puncturing the left ventricle). Here, we describe the whole procedure in detail with critical details to avoid these issues.
Pressure/volume analysis is an important approach to investigate in vivo contractility. Performing this technique in mice is significant since there are many benefits over other species (cost, genetic manipulation, etc.). Since heart rate is an important determinant of heart function1 and affected by anesthesia, we presented additional steps to ascertain that comparable heart rates are achieved to allow a valid comparison between groups. Furthermore, using this modified PV loop technique, an investigator is able to directly test the Bowditch effect. For these reasons, we describe how to perform this technique in the mouse to obtain accurate measurements of in vivo contractility at different heart rates.

The purpose of the heart is to pump blood throughout the body to meet the metabolic demands of the organism. Since these demands are constantly fluctuating (e.g., during exercise), the heart must adapt (i.e., increase cardiac output). The heart has devised numerous pathways to accomplish this feat. The prime manner the heart achieves this is via an increase in heart rate (i.e., Bowditch effect)1. That is, as one&#8217;s heart rate increases, this results in an increase in contractility and an increase in cardiac output. Thus, heart function is exceedingly dependent upon heart rate. Unfortunately, heart disease (e.g., myocardial infarction, hypertrophy, etc.) results in poor heart function in which the heart consequently will not be able to meet the metabolic demands of the body. Heart disease is the main cause of morbidity and mortality in Western society. Animal models that recapitulate many human cardiomyopathies are used to investigate molecular mechanisms and to test potential therapies. To discern these mechanisms and determine if a therapy may be viable, investigators must assess heart function in vivo.
There are several ways to assess heart function in vivo (e.g., echocardiography, MRI, etc.), which routinely measure ejection fraction, fractional shortening, cardiac output, etc. However, these parameters are highly dependent upon afterload, preload, and heart rate in addition to contractility2. Measuring contractility is indispensable to comprehend the intrinsic properties of the heart in its native environment. The maximum (dP/dtmax) rate of pressure development brings us a step closer to understanding contractility. Unfortunately, dP/dt is also dependent upon heart rate and loading conditions3. Therefore techniques have been developed to measure load (and heart rate, see below) independent indices of myocardial contractility (i.e., end systolic pressure volume relationship (ESPVR) and preload recruitable stroke work (PRSW))4-6. ESPVR describes the maximal pressure that can be developed by the ventricle at any given LV volume. The slope of ESPVR represents the end-systolic elastance (Ees). PRSW is the linear regression of stroke work (area enclosed by the PV loop) with the end-diastolic volume. These procedures are a more accurate and precise measurement of contractility compared to hemodynamic parameters such as ejection fraction, cardiac output, and stroke volume. ESPVR and PRSW can be obtained via the temporary blocking of the inferior vena cava (IVC). Blocking the IVC can be performed with a closed chest to avoid the effect of changing intrapleural pressure on heart function.
Increasing heart rate also enhances contraction and relaxation1. Thus, when comparing heart function between experimental groups (e.g., &#177; dP/dt), heart rates need to be similar. However, similar heart rates usually do not occur in each animal due to various conditions (disease, research intervention, etc.). It should be noted that anesthesia (injectable and inhaled) lowers heart rate. As heart rate is a major determinant of contractility, anesthesia will considerably affect contractility. For this reason, we are describing our procedure. Additionally, a hallmark of many cardiomyopathies is a decreased contractile reserve (i.e., a decreased Bowditch effect). Therefore, heart function should be measured over a range of heart rates. Here we describe how to use a stimulator (with a closed chest) to achieve these effects.
In addition to heart rate, nitric oxide (NO) is also an important modulator of contractility7. NO is produced via enzymes termed NO synthase (NOS). We and others have shown that mice with knockout of neuronal NOS (NOS1-/-) have blunted myocyte contraction and in vivo cardiac hemodynamics8,9. This mouse will be used to demonstrate the measurement of left ventricular contractility via the LV pressure/volume analysis procedure performed at various heart rates.

<table><tbody><tr><td>Xlyzine 100&amp;nbsp;mg/ml</td><td>Ana Sed</td><td>4821</td><td></td></tr><tr><td>Katamin 50&amp;nbsp;mg/ml</td><td>Ketalar</td><td>310006</td><td></td></tr><tr><td>Heparin</td><td>APP Pharmaceuticals</td><td>6003922</td><td></td></tr><tr><td>4-0 silk thread</td><td>Surgical specialties</td><td>SP102</td><td></td></tr><tr><td>6-0 silk thread</td><td>Surgical specialties</td><td>MBKF270</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Curve forceps</td><td>Fine Science Tools</td><td>11274-20</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>Vascular clamp</td><td>Fine Science Tools</td><td>18555-03</td><td></td></tr><tr><td>Microscope</td><td>World precision instruments</td><td>PZM-3</td><td></td></tr><tr><td>Pressure catheter</td><td>Millar instruments</td><td>SPR-839</td><td></td></tr><tr><td>Pressure and volume system</td><td>Millar instruments</td><td>MPVS-300</td><td></td></tr><tr><td>PowerLab4/35</td><td>AD instruments</td><td>N12128</td><td></td></tr><tr><td>LabchartPro 7</td><td>AD instruments</td><td></td><td></td></tr><tr><td>Temperature controller</td><td>CWE</td><td>TC-1000</td><td></td></tr><tr><td>Stimulator</td><td>Grass</td><td>SD-5</td><td></td></tr><tr><td>Sterile glove</td><td>Micro-Touch</td><td>1305018821</td><td></td></tr><tr><td>Hair remover lotion</td><td>Nair</td><td></td><td></td></tr><tr><td>Betadine surgical scrub</td><td>Veterinary</td><td>NDC 6761815401</td><td></td></tr><tr><td>Alcohol</td><td>Decon Laboratories</td><td>2801</td><td></td></tr><tr><td>Bovie cautery</td><td>Bovie</td><td>AA29</td><td></td></tr><tr><td>1&amp;nbsp;ml Syringe (26&amp;nbsp;G needle)</td><td>BD</td><td>8017299</td><td></td></tr></tbody></table>

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.

The proper insertion of the catheter into the left ventricle is an important step to attain appropriate pressure and volume values. Shown in Figure 1, using LabChart Pro 7, is the changing of the pressure waveform (shape and values) as the catheter goes from the artery into the ventricle.
After proper insertion of the catheter into the left ventricle, the pressure (P) and volume (V) values obtained will then be used to generate the PV loops (shown in Figure 2).
Using these pressure values, mechanisms that alter contractility can be investigated. Shown in Figure 3 is an example of how pressure and volume changes as heart rate increases. In this example, increasing the heart rate from 300 to 600 beats/min, the LV pressure increased from 80 to 100 mm Hg, while the diastolic and systolic LV volumes decreased. Shown in Figure 4 is the heart rate dependence of the maximum and minimum rates of pressure development (dP/dt). As heart rate increases, so does the maximum and minimum rates of pressure development. This type of data analysis can also be used to investigate regulators of contractility. Our data demonstrates that NOS1-/- mice have decreased maximum and minimum rates of pressure development compared to wildtype (WT) mice (Figure 4). Hence, knockout of NOS1 results in a decreased Bowditch effect.
Using the pressure and volume values obtained during IVC occlusion, we can also obtain a measure of load-independent contractility. Shown in Figure 5, are calculated Ees and PRSW values (measured at 420 beats/min) for WT and NOS1-/- mice. These data suggest that NOS1-/- mice have decreased contractility compared to WT mice.
<img alt="Figure 1" src="/files/ftp_upload/52618/52618fig1highres.jpg" width="700" /> 
Figure 1: The pressure and volume signals as detected using the PV Loop LabChart Module. The pressure signal changes when the catheter is positioned in the LV. The arterial pressure signal (top) fluctuated from 80 to 120 mm Hg. Once the catheter was placed in the left ventricle, the shape of the pressure signal changes and the pressure fluctuated from 0 to 120 mm Hg. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52618/52618fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52618/52618fig2highres.jpg" width="700" /> 
Figure 2: Generation of PV loops using the PV Loop LabChart Module. LEFT) Representative data of pressure (top), volume (middle) and heart rate (bottom). These LV pressure/volume values are used to generate the PV loop by plotting pressure (P) against volume (V). (Right) Illustration of loops generated by the data on the left. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52618/52618fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52618/52618fig3highres.jpg" width="700" /> 
Figure 3: Effect of heart rate on LV pressure and volume. Representative data demonstrating LV pressure and volume changes with increasing heart rate using the PV Loop LabChart Module. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52618/52618fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52618/52618fig4highres.jpg" width="600" /> 
Figure 4: NOS1 knockout (NOS1-/-) mice have decreased in vivo heart function and contractile reserve. Compared to wildtype (WT) mice, NOS1-/- mice have significantly lower maximum (dP/dtmax) and minimum (dP/dtmin) rate of pressure development with increasing heart rate. Data are presented as mean &#177; SD. * P &#60;0.05 vs WT via ANOVA, n = 5 mice/group.
<img alt="Figure 5" src="/files/ftp_upload/52618/52618fig5highres.jpg" width="600" /> 
Figure 5: NOS1 knockout (NOS1-/-) mice have decreased contractility. LEFT) representative pressure/volume loops obtained during inferior vena cava occlusion. Note the proper shape of each loop. The thick lines are the end systolic pressure volume relationship (ESPVR). RIGHT) Compared to wildtype (WT) mice, NOS1-/- mice have a decreased end systolic elastance (Ees) and preload recruitable stroke work (PRSW). Data are presented as mean &#177; SD. * P &#60;0.05 vs WT via unpaired t-test, n = 5 mice/group.

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

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 ...

cardiac-catheterization-mice-to-measure-pressure-volume-relationship

Nanyang Technological University

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JoVE Journal

Medicine

18.0K Views.  The Ohio State University. This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

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

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles.
Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using&#160;admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...

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 ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Biology

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

Monitoring Cardiac Function of Rats Using the Pressure-Volume Conductance Technique

Real-Time Detection of Ferulic Acid Effects on Rat Left Ventricle Using Pressure-Volume Conductivity Catheter

Decreased cardiac function can have a negative impact on other organs. The left ventricular pressure-volume relationship is considered to be a valid method for evaluating cardiac function. Real-time monitoring of cardiac function is important for drug evaluation. Under closed-chest conditions, the miniature transducer, which is an important component of the pressure-volume catheter, enters the left ventricle of the rat through the right carotid artery. The device visualizes the changes in cardiac function during the experiment in the form of a pressure-volume loop. The actual volume of the ventricle is calculated by altering the conductivity of the blood by injecting 50 &#181;L of a 20% sodium chloride solution into the rat's left jugular vein. The actual volume of the rat's ventricular cavity is calculated by measuring the conductivity of the blood in a known volume using a pressure-volume conductance catheter. This protocol allows for continuous observation of the effects of drugs on the heart and will promote the rationale for the use of specialty ethnic drugs in cardiovascular disease.

Author Spotlight: Advantages of Pressure-Volume Loop Measurement Method in Cardiovascular Research

This protocol describes a method for measuring left ventricular pressure and volume using the pressure-volume conductance technique. This method enables continuous real-time monitoring of the effects of drugs on the heart.

Decreased cardiac function can have a negative impact on other organs. The left ventricular pressure-volume relationship is considered to be a valid ...

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

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