Name: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice (Video) | JoVE Journal
Uploaded: 2015-09-17T14:00:00.000+00:00
Duration: 8 min 15 s
Description: Learn cardiac pressure-volume loop analysis using conductance catheters in mice to assess load-dependent and independent ventricular function. This method enables rigorous evaluation of systolic and diastolic cardiac performance.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work is supported by the American Heart Association 14FTF20370058 (DMA) and NIH T32 HL007101-35 (DMA).

1. Conductance Catheter Preparations and Pressure Calibration
<ol>
	<li>Connect conductance catheter to the hemodynamic catheter module. Electronically calibrate pressure and volume measurements by recording preset pressure and volume set on the catheter module. Record a tracing of 0 mm Hg and 25 mm Hg (Figure 1A) and assign voltages to both pressure tracings (Figure&#160;1B and 1C). Similarly, record a volume tracing of 5 RVU and 25 RVU (Figure 1D) and assign voltages to both volume tracings (1E and 1F).</li>
	<li>Confirm the electronic pressure calibration with a manual pressure calibration, using a mercury column sphygmomanometer. Fit the sphygmomanometer cuff port with a 3-way stopcock. Fill a 3-port hemostasis valve system, often used for coronary angioplasty, with RT water using the side port.
	<ol>
		<li>Place the tip of a conductance catheter into the fluid filled hemostasis valve and gently secure without kinking catheter. Connect the hemostasis valve to the sphygmomanometer and inflate to 200 mmHg and lock 3-way stopcock. Examine whether the measured pressure corresponds with the pressure inflated on the sphygmomanometer.</li>
	</ol>
	</li>
	<li>Place catheter in saline heated to 37 &#176;C that is at the level of the operating field and measure pressure. Adjust pressure control until the recorded pressures is at zero.</li>
</ol>
2. Anesthesia/Intubation
<ol>
	<li>Administer ketamine/xylazine (80-100/10 mg kg-1) as an intraperitoneal injection. 
	Note: Alternative anesthetic agents can be used. An extensive list of anesthetics are provided in previous reviews of this technique 11,12. Proper anesthetization can be confirmed by gentle tail pinch.&#160;</li>
	<li>Once anesthetized, shave the neck and chest with hair clippers and place on a heated pad. Maintain the mouse rectal temperature at 36.5-37.5 &#186;C. Low body temperatures will result in depressed heart rates. Apply ointment to the eyes to prevent dryness</li>
	<li>Make a midline incision in the neck and dissect the tracheal muscles away to expose the trachea. Place an endotracheal tube through the mouth, while visualizing the trachea to ensure intubation, and connect to the respirator.</li>
	<li>Maintain mouse on the ventilator during the procedure. Set ventilator settings based on animal weight as previously described 11. Tidal Volume (ml)= 6.2 x (animal weight in kilograms) 1.01 and Respiratory rate= 53.5 x (animal weight in kilograms)-0.26.</li>
</ol>
3. Placement of Conductance Catheter in LV chamber
<ol>
	<li>Carotid approach
	<ol>
		<li>To ensure sterility, two sets of sterile surgical instruments are used- one for initial skin incision and one to operate in the thoracotomy. Instruments should be decontaminated using a dry sterilizer between animals during an individual surgical session and autoclaved at the end of each surgical day.</li>
		<li>&#160;After the skin has been decontaminated with three cycles of a Chlorhexidine + alcohol skin scrub (0.5% chlorhexidine/70% alcohol solution), make an incision over the right carotid from mandible to sternum. Dissect surrounding tissue to expose the right carotid, and cut the vagus nerve which runs adjacent to the carotid.</li>
		<li>Place a sterile 6-0 silk suture around the distal end (away from the chest) of the carotid artery, tie and secure. Place two additional sutures beneath the carotid artery proximal (closer to the chest) to the first suture, loosely tying the middle suture. Gently pull the proximal suture and secure by clamping it to the skin. Ensure that the carotid artery has been clamped both proximally and distally before proceeding.</li>
		<li>Make a small incision in the R. carotid artery, proximal to the first suture, and extend longitudinally toward the chest.</li>
		<li>Insert the conductance catheter tip, previously soaked in warm saline for 30 min, into the vessel through the incision and secure the catheter using the middle suture.</li>
		<li>Gently advance the catheter in the left ventricle through the carotid, while watching the pressure-volume loop tracing to ensure correct placement. 
		Note: Optimal placement of the catheter should yield pressure-volume loops that appear rectangular (See Figure 2). If any resistance to the passage of the catheter is encountered, gently pull back and advance again with gentle pressure. Gentle rotation of the catheter may help with placement into the left ventricle. Forcing the conductance catheter can lead to serious cardiovascular complications or damage to the catheter.</li>
		<li>Record baseline pressure-volume loops ~10 min&#160;after catheter placement and achieving a steady state (Figure 2).</li>
	</ol>
	</li>
	<li>Apical Approach
	<ol>
		<li>In an anesthetized and ventilated mouse, make an incision from the xiphoid process and cut through the chest wall laterally until the diaphragm is visible.</li>
		<li>Cut though the diaphragm and visualize the apex of the heart.</li>
		<li>Insert the conductance catheter into the apex of the left ventricle through a needle stab wound (using a 25-30 G needle), until the proximal electrode is just inside the ventricle.</li>
		<li>Record baseline pressure-volume loops ~10 min&#160;after catheter placement and achieving a steady state.</li>
	</ol>
	</li>
</ol>
4. Varying Afterload Using Transient Aortic Occlusion
<ol>
	<li>To perform transient aortic occlusion, make a small horizontal incision in the upper chest and dissect surrounding tissue to expose the transverse aorta.</li>
	<li>Place a 6-0 silk ligature underneath the transverse aorta. After pressure-volume loops have returned to baseline, clasp both ends of the suture with a needle clamp, gently and slowly raise the suture over 1-2 sec, and slowly release tension.</li>
	<li>Repeat this procedure, until three separate optimal recordings are made from the same animal. 
	Note: Optimal recordings should have at least 5 pressure volume loop cycles and a steady rise in end systolic pressures during the application of tension on the suture (Figure 3A and 3C).</li>
</ol>
5. Varying Preload Using Transient Inferior Vena Cava Occlusion (IVC)
<ol>
	<li>To perform transient inferior vena cava occlusion, make a horizontal incision below the xiphoid process, underneath the diaphragm to expose the IVC.</li>
	<li>Place a 6-0 silk ligature underneath the IVC. After pressure-volume loops have returned to baseline, clasp both ends of the suture with a needle clamp; gently and slowly raise the suture over 1-2 sec, and slowly release tension. IVC occlusion can also be performed by the gentle application of pressure using a cotton tipped swab.</li>
	<li>Repeat this procedure, until three separate optimal recordings are made from the same animal. 
	Note: Optimal recordings should have at least 5 pressure volume loop cycles and a steady drop in left ventricular end-diastolic pressures during the application of tension on the suture (Figure 4A and 4B).</li>
</ol>
6. Saline Calibration
<ol>
	<li>At the conclusion of the study, a parallel conductance (Vp) value can be obtain by injecting a 10 &#956;l bolus of hypertonic saline (15%) into the animal through the jugular vein (Figure 5A and 5B). 
	Note: This bolus will cause an apparent increase in volume with no change in pressure. This apparent change in volume is the result of a change in blood pool conductance rather than due to an actual increase in volume. A transient decline in dP/dtmax can be observed, as hypertonic saline has a negative inotropic effect 13. The calculated Vp can be entered into pressure volume loop analysis software along with the cuvette calibration parameters to and convert from RVUs in microliters.</li>
</ol>
7. Cuvette Calibration
<ol>
	<li>To perform a cuvette calibration, place a cuvette with wells of known diameters provided by manufacturer on a heating pad or a water bath warmed to 37 &#186;C. Fill the first 4&#8211;5 holes with fresh warm heparinized blood from mice undergoing hemodynamic assessments. 
	Note: A cuvette calibration allows for accurate assessment of left ventricular blood pool using the mouse blood and allows for conversion of volume data from RVUs to microliters.</li>
	<li>Insert the conductance catheter into the first well, until all the electrodes are submerged. Gently move the catheter in the well, which will generate varying RVUs.</li>
	<li>Record the conductance changes in the volume channel in RVUs. Select the highest RVU for the calibration. 
	Note: The volume of the wells can either be calculated by 1) using the equation for the volume of a cylinder, where the radius is that of the cuvette well and the length is based on the length between the inner two sensing electrodes or 2) checking in the manufacturer&#39;s instructions. The conductance output can be correlated with the known volumes to develop a calibration equation that converts the data from RVUs into microliters 11.</li>
</ol>
8. Euthanasia
<ol>
	<li>&#160;At the conclusion of the protocol, mice are euthanized using cervical dislocation while under anesthesia.</li>
	<li>&#160;In order to ensure death, mice having undergone cervical dislocation must undergo a secondary method of euthanasia. We use exsanguination under anesthesia, with harvesting of cardiac tissue for experimentation, or bilateral thoracotomy under anesthesia.</li>
</ol>
9.&#160;Data Analysis using Pressure Volume Loop Analysis Software
<ol>
	<li>Calculate Vp from Saline Calibration
	<ol>
		<li>Select pressure and volume loops obtained during the ejection of hypertonic saline (Figure 5A and 5B)</li>
		<li>Export loops to pressure volume analysis software. Choose option for Saline calibration (Figure 5C)</li>
		<li>Record the calculated Vp value (Figure 5D). 
		Note: The Vp value is calculated by identifying the intersection of the 1) End-diastolic volume vs. End-systolic volume from the saline calibration and 2) End-diastolic volume = End-systolic volume line. The intersection of these lines provides the Vp, which is calculated by the pressure volume loop analysis software.</li>
	</ol>
	</li>
	<li>Enter conductance to volume (RVU) relation in volume channel options (Figure 1F)</li>
	<li>Measure Baseline Pressure-Volume loop relation
	<ol>
		<li>Select 8-10 cardiac cycles from pressure and volume channels once steady state has been achieved (Figure 2A) and export to analysis software. Identify 5-6 end-expiratory loops (Figure 2B)</li>
		<li>Use the Vp value to correct for parallel conductance. Choose the &#8220;Steady State&#8221; option and generate a hemodynamic summary table (Figure 2C)</li>
	</ol>
	</li>
	<li>Measure Pressure-Volume loop Relation during Aortic Constriction
	<ol>
		<li>Select 8-10 cardiac cycles from pressure and volume channels that correspond to aortic constriction prior to the increase of end-diastolic pressures (Figure 3A) and export to analysis software. Identify 5-6 end-expiratory loops (Figure 3C)</li>
		<li>Select &#8220;Contractility&#8221; analysis (Figure 3B), which will calculate the End-Systolic Pressure Volume Relation (ESPVR)</li>
	</ol>
	</li>
	<li>Measure Pressure-Volume loop Relation during IVC Constriction
	<ol>
		<li>Select 8-10 cardiac cycles from pressure and volume channels that correspond to IVC constriction (Figure 4A) and export to analysis software. Identify 5-6 end-expiratory loops (Figure 4B)</li>
		<li>Select &#8220;Contractility&#8221; analysis (Figure 3B), which will calculate Preload Recruitable Stroke Work (PRSW) (Figure 4C), Maximum dP/dt vs EDV (Figure 4D), as well as the ESPVR and End-diastolic pressure volume relation (Figure 4E)</li>
	</ol>
	</li>
</ol>

<ol><li>Clark, J. E., Marber, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advancements+in+pressure-volume+catheter+technology+-+stress+remodelling+after+infarction%5BTitle%5D)+AND+%22Exp+Physiol%22%5BJournal%5D)">Advancements in pressure-volume catheter technology - stress remodelling after infarction.</a> Exp Physiol. 98 (3), 614-621 (2013).</li>
<li>Sunagawa, K., Maughan, W. L., Burkhoff, D., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Left+ventricular+interaction+with+arterial+load+studied+in+isolated+canine+ventricle%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Left ventricular interaction with arterial load studied in isolated canine ventricle.</a> Am J Physiol. 245 (Pt 1), H773-H780 (1983).</li>
<li>Suga, H., Sagawa, K., Demer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determinants+of+instantaneous+pressure+in+canine+left+ventricle.+Time+and+volume+specification%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Determinants of instantaneous pressure in canine left ventricle. Time and volume specification.</a> Circ Res. 46 (2), 256-263 (1980).</li>
<li>Suga, H., Sagawa, K., Shoukas, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Load+independence+of+the+instantaneous+pressure-volume+ratio+of+the+canine+left+ventricle+and+effects+of+epinephrine+and+heart+rate+on+the+ratio%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio.</a> Circ Res. 32 (3), 314-322 (1973).</li>
<li>Kass, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+left+ventricular+mechanics+from+acute+VDD+pacing+in+patients+with+dilated+cardiomyopathy+and+ventricular+conduction+delay%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Improved left ventricular mechanics from acute VDD pacing in patients with dilated cardiomyopathy and ventricular conduction delay.</a> Circulation. 99 (12), 1567-1573 (1999).</li>
<li>Kass, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diastolic+Compliance+of+Hypertrophied+Ventricle+Is+Not+Acutely+Altered+by+Pharmacological+Agents+Influencing+Active+Processes%5BTitle%5D)+AND+%22Annals+of+Internal+Medicine%22%5BJournal%5D)">Diastolic Compliance of Hypertrophied Ventricle Is Not Acutely Altered by Pharmacological Agents Influencing Active Processes.</a> Annals of Internal Medicine. 119 (6), 466-473 (1993).</li>
<li>Georgakopoulos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+murine+left+ventricular+pressure-volume+relations+by+miniaturized+conductance+micromanometry%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry.</a> Am J Physiol. 274 (4 pt 2), H1416-H1422 (1998).</li>
<li>Cingolani, O. H., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pressure-volume+relation+analysis+of+mouse+ventricular+function%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Pressure-volume relation analysis of mouse ventricular function.</a> Am J Physiol Heart Circ Physiol. 301 (6), H2198-H2206 (2011).</li>
<li>Baan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+Measurement+of+Left-Ventricular+Volume+in+Animals+and+Humans+by+Conductance+Catheter%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Continuous Measurement of Left-Ventricular Volume in Animals and Humans by Conductance Catheter.</a> Circulation. 70 (5), 812-823 (1984).</li>
<li>Pearce, J. A., Porterfield, J. E., Larson, E. R., Valvano, J. W., Feldman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accuracy+considerations+in+catheter+based+estimation+of+left+ventricular+volume%5BTitle%5D)+AND+%22Conf+Proc+IEEE+Eng+Med+Biol+Soc%22%5BJournal%5D)">Accuracy considerations in catheter based estimation of left ventricular volume.</a> Conf Proc IEEE Eng Med Biol Soc. 2010, 3556-3558 (2010).</li>
<li>Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nature Protocols. 3 (9), 1422-1434 (1038).</li>
<li>Hanusch, C., Hoeger, S., Beck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anaesthesia+of+small+rodents+during+magnetic+resonance+imaging%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Anaesthesia of small rodents during magnetic resonance imaging.</a> Methods. 43 (1), 68-78 (2007).</li>
<li>Georgakopoulos, D., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+parallel+conductance+by+dual-frequency+conductance+catheter+in+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Estimation of parallel conductance by dual-frequency conductance catheter in mice.</a> Am J Physiol Heart Circ Physiol. 279 (1), H443-H450 (2000).</li>
<li>Esposito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+myocardial+contractility+and+enhanced+exercise+function+in+transgenic+mice+overexpressing+either+adenylyl+cyclase+5+or+8%5BTitle%5D)+AND+%22Basic+Res+Cardiol%22%5BJournal%5D)">Increased myocardial contractility and enhanced exercise function in transgenic mice overexpressing either adenylyl cyclase 5 or 8.</a> Basic Res Cardiol. 103 (1), 22-30 (2008).</li>
<li>Kohout, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Augmentation+of+cardiac+contractility+mediated+by+the+human+beta%283%29-adrenergic+receptor+overexpressed+in+the+hearts+of+transgenic+mice%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.</a> Circulation. 104 (20), 2485-2491 (2001).</li>
<li>Kim, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-Arrestin-biased+AT1R+stimulation+promotes+cell+survival+during+acute+cardiac+injury%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">beta-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury.</a> Am J Physiol Heart Circ Physiol. 303 (8), H1001-H1010 (2012).</li>
<li>Suga, H., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mathematical+Interrelationship+between+Instantaneous+Ventricular+Pressure-Volume+Ratio+and+Myocardial+Force-Velocity+Relation%5BTitle%5D)+AND+%22Annals+of+Biomedical+Engineering%22%5BJournal%5D)">Mathematical Interrelationship between Instantaneous Ventricular Pressure-Volume Ratio and Myocardial Force-Velocity Relation.</a> Annals of Biomedical Engineering. 1 (2), 160-181 (1972).</li>
<li>Suga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventricular+energetics%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Ventricular energetics.</a> Physiol Rev. 70 (2), 247-277 (1990).</li>
<li>Kass, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+contractile+state+on+curvilinearity+of+in+situ+end-systolic+pressure-volume+relations%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Influence of contractile state on curvilinearity of in situ end-systolic pressure-volume relations.</a> Circulation. 79 (1), 167-178 (1989).</li>
<li>Little, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+left+ventricular+dP%2Fdtmax-end-diastolic+volume+relation+in+closed-chest+dogs%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">The left ventricular dP/dtmax-end-diastolic volume relation in closed-chest dogs.</a> Circ Res. 56 (6), 808-815 (1985).</li>
<li>Glower, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linearity+of+the+Frank-Starling+relationship+in+the+intact+heart%3A+the+concept+of+preload+recruitable+stroke+work%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Linearity of the Frank-Starling relationship in the intact heart: the concept of preload recruitable stroke work.</a> Circulation. 71 (5), 994-1009 (1985).</li>
<li>Sharir, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventricular+systolic+assessment+in+patients+with+dilated+cardiomyopathy+by+preload-adjusted+maximal+power.+Validation+and+noninvasive+application%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Ventricular systolic assessment in patients with dilated cardiomyopathy by preload-adjusted maximal power. Validation and noninvasive application.</a> Circulation. 89 (5), 2045-2053 (1994).</li>
<li>Baan, J., Van der Velde, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitivity+of+left+ventricular+end-systolic+pressure-volume+relation+to+type+of+loading+intervention+in+dogs%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Sensitivity of left ventricular end-systolic pressure-volume relation to type of loading intervention in dogs.</a> Circ Res. 62 (6), 1247-1258 (1988).</li>
<li>Wei, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volume+catheter+parallel+conductance+varies+between+end-systole+and+end-diastole%5BTitle%5D)+AND+%22IEEE+Trans+Biomed+Eng%22%5BJournal%5D)">Volume catheter parallel conductance varies between end-systole and end-diastole.</a> IEEE Trans Biomed Eng. 54 (8), 1480-1489 (2007).</li>
<li>Porterfield, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+correction+for+parallel+conductance%2C+GP%2C+and+gain+factor%2C+alpha%2C+in+invasive+murine+left+ventricular+volume+measurements%5BTitle%5D)+AND+%22J+Appl+Physiol+%281985%29%22%5BJournal%5D)">Dynamic correction for parallel conductance, GP, and gain factor, alpha, in invasive murine left ventricular volume measurements.</a> J Appl Physiol (1985). 107 (6), 1693-1703 (2009).</li>
</ol>

The authors have nothing to disclose.

We describe a method for perfoming pressure-volume loop analysis using a conductance catheter in mice, to derive comprehensive analyses of both cardiac contractility and relaxation. Suga, Sagawa and colleagues utilized pressure-volume loops to define measures of cardiac contractility, specifically the slope of the ESPVR, or the end-systolic elastance (Ees), and Emax. Elastance, defined by the ratio of pressure to volume (P/V), varies over the duration of systole. During each systole, the instantaneo...

Cardiac pressure volume loop analysis provides detailed information of cardiac function and are the gold standard for functional assessment 1. While imaging techniques such as echocardiography or cardiac MRI provide functional measures, these measures are highly dependent on loading conditions. Load-independent measures of cardiac contractility and relaxation require dynamic measurements of the ventricular pressure and volume relation over a range of preload and afterload. This understanding of the pressure-volume relation arises from the groundbreaking work of Sagawa and colleagues 2,3. They demonstrated in ex vivo perfused canine hearts that the pressure-volume loop derived contractility measures were independent of loading conditions 4.
In vivo application of these analyses became possible with the development of conductance catheters in the 1980s. This technical advance allowed Kass and colleagues to perform pressure-volume loop analysis in humans 5,6. Miniaturization of conductance catheters and improvements in surgical techniques in the late 1990&#8217;s 7 made analysis of rodent cardiac function feasible, allowing for genetic and pharmacologic studies to be performed. This advance has since lead to the widespread use of pressure-volume loop analysis and has generated a great deal of insight into mammalian cardiac physiology.
A key concept in the use of conductance catheters and the interpretation of data obtained from it is the relationship between volume and conductance. Conductance is inversely related to voltage, which is measured using a catheter with electrodes placed proximally, usually placed below the aortic valve, and distally, at the LV apex 8. Changes in voltage or conductance are measured by changes in current flowing from proximal to distal electrode. Although the blood pool contributes significantly to conductance, the contribution of the ventricular wall, termed parallel conductance (Vp), to measured conductance must be subtracted to obtain absolute LV volume measurements.
The methods to perform this correction, called a saline calibration, are discussed in the protocol below. The mathematical relationship between conductance and volume, described by Baan and colleagues, is that volume=1/&#945;; (&#961;L2)(G-Gp), where &#945;=uniform field correction factor, &#961;=blood resistivity, L=distance between the electrodes, G=conductance and Gp=non-blood conductance 9. Of note, the uniform field correction factor in mice approaches 1.0 due to small chamber volumes10. Coupled with pressure transducers, the conductance catheter provides real time simultaneous pressure and volume data.
Cardiac pressure-volume analysis presents particular advantages over other measures of cardiac function, as they allow for measurement of ventricular function independent of loading conditions and of heart rate. Specific load-independent cardiac indices of contractility include: end-systolic pressure volume relation (ESPVR), dP/dtmax&#8211;end-diastolic volume relation, maximal elastance (Emax) and preload recruitable stroke work (PRSW). A load-independent measure of diastolic function is the end-diastolic pressure volume relationship (EDPVR) 11. The following protocol describes the conduct of cardiac pressure volume loop analysis, using both a carotid and an apical approach. While the methodology to perform these studies have been described in detail previously 8,11, we will review key steps to obtain precise pressure-volume measurements, including both saline and cuvette calibration correction, and provide a visual demonstration of these procedures. &#160;&#160;Research with animals carried out for this study was handled according to approved protocols and animal welfare regulations of Duke University Medical Center&#8217;s Institutional Animal Care and Use Committee.

<table><tbody><tr><td>AnaSed (xylazine)&amp;nbsp;</td><td>Lloyd Laboratories</td><td>NADA no. 139-236</td><td>Anesthetic</td></tr><tr><td>Ketaset (ketamine)</td><td>Pfizer</td><td>440842</td><td>Anesthetic</td></tr><tr><td>VIP3000</td><td>Matrx Medical Inc.</td><td></td><td>Anesthesia machine</td></tr><tr><td>Ventilator</td><td>Harvard Apparatus</td><td>Model 683</td><td>Surgical Equipment</td></tr><tr><td>Tubing kit</td><td>Harvard Apparatus</td><td>72-1049</td><td>Surgical Equipment</td></tr><tr><td>Homeothermic Blanket&amp;nbsp;</td><td>Kaz Inc.</td><td>5628</td><td>Surgical Equipment</td></tr><tr><td>Stereo microscope</td><td>Carl Zeiss Optical Inc.</td><td>Stemi 2000</td><td>Surgical Equipment</td></tr><tr><td>Illuminator</td><td>Cole&amp;ndash;Parmer</td><td>41720</td><td>Surgical Equipment</td></tr><tr><td>Dumont no. 55 Dumostar Forceps&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>11295-51</td><td>Surgical Instruments</td></tr><tr><td>Graefe forceps, curved&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>11052-10</td><td>Surgical Instruments</td></tr><tr><td>Moria MC31 forceps&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>11370-31</td><td>Surgical Instruments</td></tr><tr><td>Mayo scissors&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>14512-15</td><td>Surgical Instruments</td></tr><tr><td>Iris scissors&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>14041-10</td><td>Surgical Instruments</td></tr><tr><td>Halsey needle holder&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>12501-13</td><td>Surgical Instruments</td></tr><tr><td>Olsen&amp;ndash;Hegar needle holder&amp;nbsp;</td><td>Fine Science Tools Inc</td><td>12002-12</td><td>Surgical Instruments</td></tr><tr><td>spring scissors</td><td>Fine Science Tools Inc</td><td>15610-08</td><td>Surgical Instruments</td></tr><tr><td>disposable underpads</td><td>Kendall/Tyco Healthcare</td><td>1038</td><td>Surgical Supplies</td></tr><tr><td>Sterile gauze sponges, sterile&amp;nbsp;</td><td>Dukal</td><td>62208</td><td>Surgical Supplies</td></tr><tr><td>Cotton-tipped applicators, sterile&amp;nbsp;</td><td>Solon</td><td>368</td><td>Surgical Supplies</td></tr><tr><td>Surgical suture,&amp;nbsp; silk, 6-0&amp;nbsp;</td><td>DemeTECH</td><td>FT-639-1</td><td>Surgical Supplies</td></tr><tr><td>1 cc Insulin syringes&amp;nbsp;</td><td>Becton Dickenson</td><td>329412</td><td>Surgical Supplies</td></tr><tr><td>Access 9 Hemostasis Valve</td><td>Merit Medical&amp;nbsp;</td><td>MAP111</td><td>Hemodynamic equipment</td></tr><tr><td>Sphygmomanometer</td><td>Baumanometer</td><td>320</td><td>Hemodynamic equipment</td></tr><tr><td>Millar PV system MPVS-300/400 or MPVS Ultra (includes calibration cuvette)</td><td>ADInstruments Inc</td><td></td><td>Hemodynamic equipment</td></tr><tr><td>1.4F conductance catheter&amp;nbsp;</td><td>ADInstruments Inc</td><td>SPR-839</td><td>Hemodynamic equipment</td></tr><tr><td>PowerLab 4/30 with Chart Pro</td><td>ADInstruments Inc.</td><td>ML866/P</td><td>Hemodynamic software</td></tr><tr><td>animal clipper</td><td>Wahl</td><td>8787-450A</td><td>Miscellaneous</td></tr><tr><td>Intradermic tubing PE-10</td><td>Becton Dickenson</td><td>427401</td><td>Miscellaneous</td></tr><tr><td>Intradermic tubing PE-50</td><td>Becton Dickenson</td><td>427411</td><td>Miscellaneous</td></tr><tr><td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td><td></td><td></td><td>Miscellaneous</td></tr><tr><td>0.9% (wt/vol) sodium chloride injection, USP)</td><td>Hospira</td><td>NDC no. 0409-4888-50</td><td>Miscellaneous</td></tr><tr><td>Surgical tape</td><td></td><td></td><td>Miscellaneous</td></tr><tr><td>Alconox (Alconox Inc.) for catheter cleaning</td><td>ADInstruments Inc.</td><td></td><td>Miscellaneous</td></tr></tbody></table>

cardiac pressure-volume loop analysis using conductance catheters in mice

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.

Pressure-volume loop analysis can be used to measure cardiac function in genetically modified mice 14,15 or mice undergoing drug studies16. Representative pressure volume loops are provided from previously published work 16 investigating the effect of ß-Arrestin biased AT1R ligand, TRV120023. To test whether TRV120023 affects cardiac function in vivo, pressure-volume loop analysis was performed on wild type mice receiving conventional and novel angiotensin receptor blockers. Intr...

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 ...

The overall goal of this procedure is to assess the load dependent and independent measures of ventricular, systolic and diastolic function. This is accomplished first by the placement of a conductance catheter into the left ventricle of a mouse heart. In the second step, the aorta is constricted to change afterload, followed by the alteration of preload via the constriction of the inferior vena cva.In the final step, the conductance catheter is carefully calibrated to accurately assess the changes in the cardiac chamber volume. Ultimately, cardiac pressure volume analysis is used to obtain the load dependent and independent measures of cardiac function. The implications of this technique extend towards the therapy of cardiac dysfunction, as it allows both a rigorous evaluation of cardiac function and the potential in masking of functional phenotypes missed by non-invasive testing.Demonstrating this procedure will be Dr.Lan Mao, a mouse neurosurgeon from our laboratory 30 minutes before placing a conductance catheter into the left ventricle chamber. Soak the tip of the catheter in warm saline while the tip is soaking. Shave the neck and chest of an anesthetized adult mouse and place the animal on a heated pad.After applying ointment to the animal's eyes, make a midline incision in the neck and dissect the tracheal muscles to expose the trachea. Then place an endotracheal tube through the mouth, visualizing the trachea for the correct placement of the tube, and connect the tube to the respirator. Set the ventilator settings based on the animal's weight as indicated.Then to place the catheter by a carotid approach, make an incision over the right carotid from the mandible to the sternum, and dissect the surrounding tissue to expose the right carotid. Next, cut the vagus nerve that runs adjacent to the carotid and secure a six oh silk suture around the distal end of the carotid artery. Place two additional sutures beneath the carotid artery proximal to the first suture loosely tying the middle suture.Then gently pull the proximal suture and use a clamp to secure the suture to the skin. When the carotid artery has been clamped, both proximally and distally make a small incision in the right carotid artery proximal to the first suture and extending longitudinally toward the chest. Now insert the saline soaked conductance catheter tip into the vessel through the incision using the middle suture to secure the catheter.Then gently advance the catheter into the left ventricle through the carotid. Watching the pressure volume loop tracing to ensure a correct placement, optimal placement of the catheter will yield pressure volume loops that appear rectangular for five to 10 minutes after placement record Baseline pressure volume loops until a steady state is achieved to place the catheter by an apical approach. Make the starting incision from the xiphoid process through the chest wall laterally until the diaphragm is visible.Cut through the diaphragm to visualize the apex of the heart and use a 25 to 30 gauge needle to make a stab wound in the apex of the left ventricle. Then insert the conductance catheter through the stab wound until the proximal electrode is just inside the ventricle. And record the baseline pressure volume loops as just demonstrated to vary the afterload with the transient aortic occlusion.Begin by making a small horizontal incision in the upper chest and dissect the surrounding tissue to expose the transverse aorta. Place a 6.0 silk ligature underneath the transverse aorta. Then when the pressure volume loops have returned to baseline, place both ends of the ligature in a small segment of PE 50 tubing class both ends of the suture with a needle clamp and gently and slowly raise the suture over a period of one to two seconds, followed by a slow release of the tension on the vessel.Optimal recordings should have at least five pressure volume loop cycles and exhibit a steady rise in the end systolic pressures during the application of the tension on the suture. To vary the preload with the transient inferior vena CVA occlusion. Make a vertical incision below the xiphoid process and underneath the diaphragm to expose the inferior vena cva.Place a six oh silk ligature underneath the inferior vena cva. Then after the pressure volume loops have returned to baseline, clasp both ends of the suture with a needle clamp and gently and slowly raise the suture over a period of one to two seconds, followed by a slow release of the tension on the vessel. Optimal recordings should have at least five pressure volume loop cycles and exhibit a steady drop in the left ventricular and diastolic pressures during the application of the tension on the suture to perform a saline calibration.At the conclusion of the study, inject a 10 microliter bolus of hypertonic saline into the animal through the jugular vein to obtain a parallel conductance value. An apparent increase in volume will be observed with no change in the pressure as a result of the change in blood pool conductance. To perform a vete calibration, warm a vete with wells of known diameter to 37 degrees Celsius and fill the first three holes with fresh, warm heparinized.Blood from mice undergoing hemodynamic assessments fit a plastic adapter provided by the manufacturer to the vete. This will hold the conductance catheter stationary during the calibration. Then insert the conductance catheter into the first well until all of the electrodes are submerged.Finally, record the conductance in the volume channel to test whether the beta arrestin biased angiotensin two receptor type one ligand TR RV 1 2 0 0 2 3 affects cardiac function. In vivo pressure volume loop analysis was performed on wild type mice receiving conventional or novel angiotensin receptor blockers. Indeed, in this experiment, intravenous infusion of TR RV 1 2 0 0 2 3 increased the cardiac contractility significantly compared to conventional and control treatments.After watching this video, you should have a good understanding of how to perform pressure volume loop analysis using both aortic and inferior vena cava constriction to l obtain both load dependent and independent measures of cardiac function.

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

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

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

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

Measuring Pressure Volume Loops in the Mouse

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.

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

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the β-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.

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

Here we present a closed chest approach to admittance-based bi-ventricular pressure-volume loop recordings in pigs with acute right ventricular dysfunction.

Closed Chest Biventricular Pressure-Volume Loop Recordings with Admittance Catheters in a Porcine Model

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.

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

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.

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

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioengineering Human Microvascular Networks in Immunodeficient Mice

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Learn cardiac pressure-volume loop analysis using conductance catheters in mice to assess load-dependent and independent ventricular function. This method enables rigorous evaluation of systolic and diastolic cardiac performance.

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Explore More Videos