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1Cardiology Division, Department of Medicine, University of California; and 2Veterans Affairs Medical Center, San Francisco, California
Submitted 3 July 2006 ; accepted in final form 7 August 2006
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ABSTRACT |
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ventricular function; mouse physiology
Recently, we developed a method to measure LV pressure in conscious mice with an implanted LV polyethylene (PE)-50 tube (4). This method allowed us to assess hemodynamic variables repeatedly. To evaluate the LV pressure-volume relationship using a conductance catheter, we applied our new method to conscious mice. An externally controlled inferior vena cava snare was also implanted to induce a transient reduction of LV preload. We confirmed that these load-independent parameters of contractility were reproducible and changed reasonably by three different interventions. These findings suggested that this approach was feasible to assess repeatedly the LV pressure-volume relationship in conscious mice. This method may be useful to recognize cardiac function modified by genetic intervention without the influence of any anesthesia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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This study was approved and performed in accordance with the guidelines of the Committee on Animal Research, University of California, San Francisco. Male C57BL/6 mice (11–20 wk old), mean body weight 25.2 g (20–28 g), were included in this study.
Surgical Procedure
PE-50 implantation into LV. The surgical procedure utilized has been described in detail previously (4) and is illustrated in Fig. 1. Briefly, the LV apex was exposed via a subdiaphragmatic incision, leaving the chest wall and sternum intact. A 6-0 proline suture was placed in the LV apex through a small incision in the diaphragm. The LV apex suture was gently pulled, and an apical stab wound was made with a 25-gauge needle, and PE-50 tubing (Intramedics, Becton-Dickinson; Sparks, MD), filled with heparinized saline, was inserted into the LV cavity. The tubing was fixed with a drop of cyanoacrylate adhesive (3M Vetbond Tissue Adhesive, 3M Animal Care products, St. Paul, MN). Through a subcutaneous tunnel, the tubing curved leftward from the cardiac apex on the ventral surface of the mouse around the left flank and on to the dorsal surface. The end of the tubing was exteriorized, and a metallic pin was inserted in the end of the tubing, acting as a plug.
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LV Pressure-Volume Measurements
LV pressure-volume measurements were done on an average of 6 days after surgical instrumentation to allow time for the recovery from the surgical procedure (4). During these 6 days following instrumentation, the mice were trained to lie quietly head first in a soft plastic cone (Fig. 1). The experiments were carried out in a silent air-conditioned room (24°C). A 1.4-Fr pressure-volume catheter (SPR-719, Millar) was calibrated with a mercury manometer at the beginning of each experiment. The baseline zero reference was obtained by placing the sensor in normal saline before insertion.
The mice were laid prone head first in the soft cone, placed on a warm cushion (37°C), and fixed with adhesive tape (Fig. 1, B and C). The end of the PE-50 tubing and snare were carefully exteriorized via a slit made in the plastic cone. The 1.4-Fr pressure-volume catheter was advanced into the LV through the implanted tubing. The catheter length was monitored as the catheter was advanced. After insertion of the catheter, baseline LV pressure and conductance signals were acquired at 2,000 Hz and stored for off-line conversion to the pressure-volume relationship. Data were also acquired when the exteriorized snare was pulled gently, changing the loading conditions by producing transient occlusion of the inferior vena cava. Particular attention was given to avoid ambient noise and stimulation throughout the study.
Echocardiography
To calibrate the LV volume signal obtained by the conductance catheter, we used the estimated LV volume derived from echocardiography. Echocardiography was performed with a system (Acuson Sequoia c256; Mountain View, CA) using a 15-MHz linear array transducer (15L8) in both conscious and anesthetized conditions [1.5 vol% isoflurane (ISF) with 1 l/min oxygen] the day before surgery, as described previously (4). Two-dimensional long-axis images of the LV were obtained in parasternal long- and short-axis views with M-mode recordings at the midventricular level in both views. LV internal dimensions at end-diastole and end-systole (LVDd and LVDs) were measured. The LV EDV and end-systolic volume (ESV) were calculated using the following two-dimensional area-length method: LV EDV = (5/6)
(LVDd/2)2 x Ld, where Ld is the parasternal long-axis length at end diastole; LV ESV = (5/6)(LVDs/2)2 x Ls, where Ls is the parasternal long-axis length at end systole. To confirm the feasibility of using this equation for estimation of LV volume, we measured LV mass at necropsy soon after echocardiography in 28 additional mice with and without cardiac hypertrophy and compared it with LV mass calculated by echo. LV mass was calculated by echo as: LV mass = 1.05({(5/6)[(LVDd + IVSd + PWd)/2]2 x (Ld + WTad)} – [(5/6)(LVDd/2)2 x Ld]), where IVSd is the wall thickness of interventricular septum at end diastole, PWd is the wall thickness of posterior wall at end diastole, WTad is the wall thickness at the apex at diastole. We found that the LV mass by echo (Y) correlated well with necropsy LV mass (X) (Y = 0.94X – 4, R = 0.96, n = 28, range of necropsy LV mass, 64–191 mg) (Fig. 2).
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Protocol 1: determination of reproducibility in conscious state. Five mice were included in this protocol. To assess reproducibility, the LV pressure-volume relationship was evaluated by inferior vena caval occlusion on two different days (n = 5; mean time interval = 3 days).
Protocol 2: isoproterenol study in conscious state. Eight mice were entered into this protocol. Both echocardiography and LV pressure-volume measurements were performed in each mouse separately. After baseline measurements, isoproterenol (3 µg/kg) was administered intraperitoneally. In preliminary studies, dP/dtmax and heart rate increased 1 min after the injection and were maximal at 2 to 5 min after the injection. Therefore, the pressure-volume loops were obtained 2 to 5 min after the injection.
Protocol 3: atenolol study in conscious state. Ten mice were entered into this protocol. Both echocardiography and LV pressure-volume measurements were performed separately in each mouse. After baseline measurements, atenolol (2.5 mg/kg) was administered intraperitoneally. In preliminary studies, dP/dtmax and the heart rate began to decrease gradually 1 min after injection, and maximum effect was seen at 10 to 15 min after the injection. Therefore, the measurements were performed at 10 to 15 min after the injection.
Protocol 4: ISF study. Seven mice were entered into this protocol.
For pressure-volume measurements, mice anesthetized with the ISF were placed in a box filled with 2 vol% ISF with 1 l/min oxygen flow for induction of the anesthesia. After induction, the mice were intubated with PE-50 tubing and were laid prone on a thermal controlled table. Anesthesia was maintained with 1.5 vol% ISF with 1 l/min oxygen flow. Mechanical ventilation was performed with a tidal volume of 200 µl at 140 breaths/min. The pressure-volume catheter was advanced into the LV through the implant tubing. After insertion of the catheter, stable hemodynamics variables were obtained. The exteriorized snare was pulled gently to change the loading conditions.
For echocardiography, after completion of the baseline echo measurements in the conscious mice, gas anesthesia with ISF (1.5 vol% at 1 l/min oxygen flow) was induced and maintained via a facial mask. In the preliminary studies, dP/dtmax and the heart rate measured during anesthesia with mechanical ventilation were found to be comparable with those from 5 to 10 min after the induction of the anesthesia without mechanical ventilation. Therefore, the echo measurements were performed during this time period.
Data Analysis
The pressure-volume data were analyzed with software (PVAN 3.2) licensed to and modified by Millar Instruments.
Volume calibration. Because acquired volume signals do not represent absolute volume, we calibrated them with the calculated LV volume by echo. In the clinical setting, conductance volumes are often calibrated to a contrast left ventriculogram, using a two-point calibration based on matching EDV and ESV (7, 13). In the same way, the LV maximal and minimal volume signals obtained with the conductance catheter and their associated volume estimated by echocardiography were used for a two-point calibration in this study. To confirm the accuracy of LV volume measurements during vena caval occlusion, we also measured echo and LV pressure-volume relationship simultaneously during vena caval occlusion (n = 5). The EDV at the end of vena caval occlusion measured by echo was similar to conductance-derived EDV (Fig. 3).
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After completion of the study, the animals were euthanized with an overdose of pentobarbital sodium, and the hearts were examined to confirm that the PE-50 tube was properly implanted. No clots were observed around the ventricular tube, and the inferior vena cavae showed no stenosis near the snare.
Statistics
Data are expressed as means ± SD. Hemodynamic data were compared between baseline and during interventions in each protocol using a paired t-test. P < 0.05 was considered statistically significant.
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RESULTS |
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Both linear and nonlinear regression analyses were performed for Ees in all studies, as shown in Fig. 6, A and B, because Ees has been shown to display contractility-dependent curvilinearity (7). R values for nonlinear regression were slightly higher than for linear regression (0.974 ± 0.025 vs. 0.958 ± 0.036, P < 0.0001).
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As shown in Table 1, hemodynamics variables were almost the same on two different days (3 days apart). Likewise, load-independent parameters were also similar as shown in Fig. 7. Ees' of the first day and that of the second day were 11.5 ± 0.7 and 11.3 ± 1.2 mmHg/µl. Likewise, dP/dtmax-EDV relation slopes were 534 ± 140 and 515 ± 149 mmHg·s–1·µl–1, and pressure-recruitable stroke work was 111 ± 18 and 117 ± 19 mmHg.
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Fig. 6 shows an example of the acute effects of isoproterenol and atenolol in the same mouse. The Ees and Ees' lines were shifted leftward and became steeper with isoproterenol and shifted rightward and became less steep after atenolol. Similar to Ees, the dP/dtmax-EDV relation and pressure-recruitable stroke work were also shifted leftward by isoproterenol and shifted rightward with atenolol (Fig. 8).
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Hemodynamic parameters at baseline and after injection of isoproterenol are shown in Table 2 and Fig. 9A. Heart rate and dP/dtmax increased significantly. Ejection fraction also increased, but the change did not reach statistical significance (P = 0.06). Although three different parameters of contractility were all increased by isoproterenol, the dP/dtmax-EDV relation was the most sensitive (+66%) to change of contractility among them, as reported in a previous study (11) with dobutamine. Because of enhanced contractility and heart rate with isoproterenol, cardiac size tended to be smaller than at baseline.
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Hemodynamic parameters at baseline and after injection of atenolol were shown in Table 3 and Fig. 9B. Atenolol significantly decreased not only load-independent parameters but also ejection fraction.
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Hemodynamic parameters in conscious and anesthetized conditions using 1.5 vol% of ISF are shown in Table 4 and Fig. 9C. Ees in the anesthetized condition was significantly lower than in the conscious state. The dP/dtmax-EDV relation and preload-recruitable stroke work slope were also much steeper in conscious versus anesthetized mice (dP/dtmax-EDV relation, 527 ± 172 vs. 164 ± 68 mmHg·s–1·µl–1, P < 0.001; and pressure-recruitable stroke work, 124 ± 19 vs. 48 ± 10 mmHg, P < 0.001).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Comparison to Previous Reports
The methods for calibrating the LV volume signal obtained with a conductance catheter have varied in previous reports. Some investigators (2, 14) adopted a method to calibrate the LV volume signal using flow-probe derived aortic flow. Others (8, 13, 18, 21) used a correlation between the conductance signal and cylinder-derived actual blood volume. These methods are all necessary to determine parallel conductance. We chose to calibrate by using an estimated LV volume derived by echocardiography. This method allowed us to measure LV volume even in conscious mice.
Table 5 compares our findings in conscious and anesthetized mice with prior reports of LV pressure-volume relations in anesthetized mice. In our study, we found that the slope of the end-systolic pressure-volume relationship in conscious mice was 
13 mmHg/µl, slightly larger than that reported in previous pressure-volume studies using a pressure-volume conductance catheter approach or a micromanometer with implanted piezoelectric crystals in closed-chest experiments (Table 5) (11, 12, 16, 21). Similarly, preload-recruitable stroke works was reported in previous studies as 80–110 mmHg in normal mice and 45–60 mmHg in heart failure mice: in our study, it is about 110–120 mmHg in conscious/baseline and 50–70 mmHg in mice with negative inotropic agents (atenolol, anesthesia). Thus, load-independent parameters were all higher than those in previous reports. This suggests that cardiac function may be enhanced in mice, at least in part, by the conscious state itself.
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Comparison of Different Parameters of Cardiac Function in This Study
Ejection fraction versus Ees. In this study, LV ejection fraction tended to increase with isoproterenol, whereas load-independent parameters changed significantly. With atenolol or ISF, however, LV ejection fraction and load-independent parameters fell significantly. These changes are consistent with data obtained in humans where there was an exponential correlation between the end-systolic pressure-volume relation and ejection fraction, as reported by Mehmel et al. (10). Considering this relation, we can expect that Ees should be increased, but ejection fraction increased mildly even when contractility was enhanced. On the other hand, Ees and ejection fraction would be decreased almost equally when contractility was depressed by several agents. These expectations are consistent with the findings of our study.
Load-independent parameters of contractility. Among the three load-independent parameters of contractility, we found that the dP/dtmax-EDV relation changed the most (+66%) in response to isoproterenol. On the other hand, the dP/dtmax-EDV relation responded similarly to Ees with either atenolol or ISF. Similar to our study, a differential response of the dP/dtmax-EDV relation between positive and negative inotropic state was found in some previous studies. Nemoto et al. (11) reported that the dP/dtmax-EDV relation was the most changed parameter in response to dobutamine but not in response to esmolol. Nishio et al. (12) studied time-varying cardiac function using a mouse model of viral myocarditis and found that contractility was enhanced one day after viral infection but depressed severely 7 days after infection. At the stage of enhanced contractility, the dP/dtmax-EDV relation was the most changed parameter (+103%). However, changes in various parameters were similar at the stage of depressed contractility. Tao et al. (18) examined the time course of cardiac function in mice with septic shock. Although contractility was depressed progressively in this model, the dP/dtmax-EDV relation failed to detect changes in contractility more sensitively than three other parameters. Thus, the dP/dtmax-EDV relation may be more sensitive to a positive inotropic state than a negative inotropic one.
Advantages of This Method
So far, hemodynamic studies involving conscious and anesthetized mice have utilized varied techniques (4, 5, 19). Our method demonstrates a way to evaluate conscious LV pressure-volume relations using a miniaturized conductance catheter and has several advantages. First, pressure-volume relations can be obtained with minimal disturbance of the mouse just before and during measurement. Pressure-volume evaluations in previous reports were all done in anesthetized mice and performed soon after surgical preparation, including insertion of a catheter from the carotid artery or LV apex. These surgical procedures could influence hemodynamics. In contrast, we obtained the pressure-volume relation several days after recovery from the operation.
Secondly, with our approach, it is not necessary to inject hypertonic saline to obtain parallel conductance. Third, as shown in this manuscript, our approach lends itself to serial observations and could be used in experimental protocols where frequent (e.g., daily) measurements of LV pressure-volume relations were required. We also found that this system was fully functional even after 3 wk.
Limitations
Limitations of this study need to be emphasized. One limitation is related to the volume measurement with inferior vena caval occlusion, which changes right ventricular (RV) as well as LV volumes and geometry. Although the examination was limited to a few mice, we recorded echocardiography during vena caval occlusion to measure LV long-axis length and LV dimensions. During vena caval occlusion, LV size decreased substantially, but at the end of vena caval occlusion, the minimal EDV-measured echocardiographically was similar to conductance-derived volume. Thus, it is likely that the conductance catheter-derived LV volume is reliable even with changed RV and LV volumes during vena caval occlusion.
Second, this study utilized only male mice. We also used only one strain of mice (C57BL/6), the ages of which were limited from 11 to 20 wk for this study. In larger mice, the Ees may be lower because LV weight should be greater. Yang et al. (22) reported that the load-independent parameters were lower in old mice (16 mo old) than in young mice (6 mo old). Thus, we cannot exclude the possibility that the values we measured would be different among mice of different strain, age, or sex.
Finally, although we could show depressed contractility resulting from atenolol or ISF, we did not test the ability of our method to detect differences between normal mice and those with LV dysfunction due to specific forms of heart disease. Further studies of several kinds of diseased mice are warranted to test more reliably the applicability of this method.
Although limited for these reasons, this study allowed us to gain additional information about the influence of anesthesia in mice. Moreover, the technique described provides a potentially important methodological advance for the study of physiology in conscious mice.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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