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 fi...

<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>

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...

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 border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Xlyzine 100mg/ml</td>
			<td style="width:187px;">Ana Sed</td>
			<td style="width:119px;">4821</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Katamin 50mg/ml</td>
			<td style="width:187px;">Ketalar</td>
			<td style="width:119px;">310006</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Heparin</td>
			<td style="width:187px;">APP Pharmaceuticals</td>
			<td style="width:119px;">6003922</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">4-0 silk thread</td>
			<td style="width:187px;">Surgical specialties</td>
			<td style="width:119px;">SP102</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">6-0 silk thread</td>
			<td style="width:187px;">Surgical specialties</td>
			<td style="width:119px;">MBKF270</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Forceps</td>
			<td style="width:187px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Curve forceps</td>
			<td style="width:187px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Scissors</td>
			<td style="width:187px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vascular clamp</td>
			<td style="width:187px;">Fine Science Tools</td>
			<td style="width:119px;">18555-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Microscope</td>
			<td style="width:187px;">World precision instruments</td>
			<td style="width:119px;">PZM-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pressure catheter</td>
			<td style="width:187px;">Millar instruments</td>
			<td style="width:119px;">SPR-839</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pressure and volume system</td>
			<td style="width:187px;">Millar instruments</td>
			<td style="width:119px;">MPVS-300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">PowerLab4/35</td>
			<td style="width:187px;">AD instruments</td>
			<td style="width:119px;">N12128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">LabchartPro 7</td>
			<td style="width:187px;">AD instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Temperature controller</td>
			<td style="width:187px;">CWE</td>
			<td style="width:119px;">TC-1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Stimulator</td>
			<td style="width:187px;">Grass</td>
			<td style="width:119px;">SD-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sterile glove</td>
			<td style="width:187px;">Micro-Touch</td>
			<td style="width:119px;">1305018821</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Hair remover lotion</td>
			<td style="width:187px;">Nair</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Betadine surgical scrub</td>
			<td style="width:187px;">Veterinary</td>
			<td style="width:119px;">NDC 6761815401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acohol</td>
			<td style="width:187px;">Decon Laboratories</td>
			<td style="width:119px;">2801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovie cautery</td>
			<td style="width:187px;">Bovie</td>
			<td style="width:119px;">AA29</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">1ml Syringe(26G needle)</td>
			<td style="width:187px;">BD</td>
			<td style="width:119px;">8017299</td>
			<td style="width:167px;"></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)...

Watch this Scientific Journal Video about Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect at JoVE.com

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

Research

JoVE Journal

Medicine

17.9K 조회수.  The Ohio State University. This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.