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 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., β-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 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’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., ± 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.
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
2. Catheterization
3. Data Acquisition
4. Bowditch Effect
5. Generating the ESPVR and PRSW
6. Volume Calibration
7. Data Processing
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 ± SD. * P <0.05 vs WT via ANOVA, n = 5 mice/group.
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 ± SD. * P <0.05 vs WT via unpaired t-test, n = 5 mice/group.
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 authors have nothing to disclose.
This study was supported by NIH grants HL091986 (JPD) and HL094692 (MTZ).
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Xlyzine 100mg/ml | Ana Sed | 4821 | |
Katamin 50mg/ml | Ketalar | 310006 | |
Heparin | APP Pharmaceuticals | 6003922 | |
4-0 silk thread | Surgical specialties | SP102 | |
6-0 silk thread | Surgical specialties | MBKF270 | |
Forceps | Fine Science Tools | 11251-10 | |
Curve forceps | Fine Science Tools | 11274-20 | |
Scissors | Fine Science Tools | 14090-09 | |
Vascular clamp | Fine Science Tools | 18555-03 | |
Microscope | World precision instruments | PZM-3 | |
Pressure catheter | Millar instruments | SPR-839 | |
Pressure and volume system | Millar instruments | MPVS-300 | |
PowerLab4/35 | AD instruments | N12128 | |
LabchartPro 7 | AD instruments | ||
Temperature controller | CWE | TC-1000 | |
Stimulator | Grass | SD-5 | |
Sterile glove | Micro-Touch | 1305018821 | |
Hair remover lotion | Nair | ||
Betadine surgical scrub | Veterinary | NDC 6761815401 | |
Acohol | Decon Laboratories | 2801 | |
Bovie cautery | Bovie | AA29 | |
1ml Syringe(26G needle) | BD | 8017299 |