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Department of Cardiology, Guy's, King's and St Thomas' School of Medicine, King's College London (Denmark Hill Campus), Bessemer Road, London SE5 9PJ, UK
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(Received 2 March 2004;
accepted after revision 3 June 2004; first published online 7 June 2004)
Corresponding author D. Grieve: Department of Cardiology, GKT School of Medicine, Bessemer Road, London SE5 9PJ, UK. Email: david.grieve{at}kcl.ac.uk
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Introduction |
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Over the last 20 years, the advent of technology which allows the simultaneous measurement of left ventricular (LV) pressure and volume has facilitated the detailed analysis of cardiac function (Baan et al. 1984; Kass et al. 1986). Pressure–volume relations assessed by the conductance technique have been firmly established as one of the most specific and reliable methods of measuring systolic and diastolic properties of the intact heart. Although initially employed mainly in humans and larger animals (Baan et al. 1984; Abe et al. 1995; Ito et al. 1996), recently the technique has also been applied to mice as small as 20 g using a microconductance catheter (Georgakopoulos et al. 1998; Yang et al. 1999; Reyes et al. 2003). Use of this method in gene-modified mice has significantly improved the characterization of contractile function in models of cardiac disease.
So far, the microconductance technique has only been applied to measure LV pressure–volume relations in the intact murine heart in vivo (Georgakopoulos et al. 1998; Yang et al. 1999). While this is an excellent integrated preparation, it does have a number of potential disadvantages. For example, assessment of intrinsic cardiac function may be confounded by the effects of anaesthesia, autonomic tone and circulating neurohumoral factors. Cardiac loading (in particular, preload) is also difficult to control in vivo. In this paper, we report the application of the microconductance technique to investigate LV pressure–volume relations in the murine isolated ejecting heart preparation. This ex vivo preparation provides a useful complementary model for the assessment of murine cardiac function under carefully controlled loading conditions, and is to a large extent free of the potentially confounding effects of anaesthesia and neurohumoral influences.
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Methods |
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Adult male C57BL6/J mice weighing 20–25 g (Harlan, Bicester, Oxon, UK) were used throughout the study. All procedures were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (Her Majesty's Stationery Office, London, UK). Experiments were performed using either normal animals (study 1, n= 10) or mice which had been subjected to aortic banding or sham surgery (study 2, n= 10).
In study 2, animals were anaesthetized by inhalation (2.5% isofluorane–97.5% oxygen) and underwent suprarenal abdominal aortic banding by tying an 8/0 nylon suture down onto a blunted 29 gauge needle between the coeliac and mesenteric arteries, producing a constriction of approximately 70% (Layland et al. 2004). Sham operation involved an identical procedure with the exception of band placement.
Isolated heart studies
Ejecting heart studies were undertaken as previously described (De Windt et al. 1999), with minor modifications. Animals were anaesthetized (sodium pentobarbitone; 60 mg kg–1I.P.) and heparinized (1000 IU kg–1I.P.) and hearts dissected into ice-cold Krebs–Henseleit buffer (KHB; mmol l–1: NaCl 118, KCl 3.8, KH2PO4 1.18, NaHCO3 25, MgSO4 1.19, CaCl2 2.0, glucose 10 and Na-pyruvate 5.0) gassed with 95% O2–5% CO2 and containing insulin (5 IU l–1) and indomethacin (10 µmol l–1; if necessary to inhibit prostanoid effects); the KHB composition (in particular calcium concentration) was found to be optimal in pilot experiments. The aorta was cannulated with a blunted 18 gauge needle (0.97 mm i.d), and hearts were initially perfused in Langendorff mode with KHB at a pressure of 50 mmHg and temperature of 38.5°C (found to result in optimal cardiac performance; De Windt et al. 1999). The left atrium was then canulated (19 gauge needle) via the largest pulmonary vein, and other pulmonary veins were tied off. Hearts were paced at 450 beats min–1 (found to be optimal and comparable with previous models; Bittner et al. 1996; Gauthier et al. 1998; De Windt et al. 1999) via a right atrial electrode and LV function was recorded via a modified high fidelity 1.4 F microconductance catheter (Millar Instruments, Houston, TX, USA) inserted into the LV cavity through a small hole in the apex made with a 25 gauge needle. Aortic pressure was measured using a transducer (Becton Dickinson, Oxford, Oxon, UK). The heart was then switched to the ejecting, recirculating mode (total volume 150 ml). Afterload was set by a constant fluid column of 60 mmHg. Compliance was provided by a syringe containing 1.5 ml of air. Steady-state analysis of LV function was performed by varying left atrial filling pressure (preload) between 10 and 25 cmH2O to generate Starling curves. Measurements were made 20 s after each change in preload. LV pressure–volume relations were also investigated under variable loading conditions by brief occlusion of the aortic outflow tract (resulting in beat-to-beat changes in cardiac loading as the heart adjusts to the occlusion), allowing analysis of both the end-systolic and end-diastolic pressure–volume relations (ESPVR and EDPVR, respectively). Pressure data were sampled at 1 kHz via a PowerLab module (ADInstruments, Chalgrove, Oxon, UK). Cardiac inflow (= cardiac output) and aortic flow were recorded continuously by in-line ultrasonic transit flow probes (1 N; Transonic, Ithaca, NY, USA). Coronary flow was calculated from the difference between the two values.
Once optimal conditions had been established for the model, our success rate for this technically demanding procedure was approximately 80%. For individuals who are reasonably skilled with the practical aspects of the isovolumic mouse Langendorff preparation, we estimate that the learning curve would be in the region of 40–50 hearts.
LV volume measurement by conductance
A custom-made miniaturized 1.4 F conductance catheter (SPR-853; Millar Instruments, Houston, TX, USA) was used in these studies. The catheter consists of a high-fidelity pressure transducer flanked by four platinum electrodes. The two outermost excitation electrodes are each separated from a sensing electrode by a distance of 0.5 mm with a space between the two inner sensing electrodes of 4.0 mm. The catheter also contains a rigid extension of approximately 2 cm in length, proximal to the first electrode to allow easy introduction into the LV via the apex of the heart. A constant current was applied to the excitation electrodes and the instantaneous voltage signal then measured by the sensing electrodes, thus allowing the calculation of volume (AriaTM 1 pressure–volume conductance system, Millar Instruments).
The measured volume signal includes an offset term (Vp), due to parallel conductance of the myocardium and surrounding structures, which is caused by extension of the electric current beyond the LV cavity (Baan et al. 1984). Parallel conductance was estimated by the saline dilution method (Baan et al. 1984; Yang et al. 1999), which involved injecting a 1 µl bolus of hypertonic (10 N) saline into the left atrial cannula, causing a transient change in the conductivity of the KHB in the LV. This is demonstrated by the rightward shift in the pressure–volume loops shown in Fig. 1A. A fundamental assumption of this method is that cardiac haemodynamics remain unchanged; LV pressure (see Fig. 1A), the maximal rate of LV pressure rise dP/dtmax (not shown) and coronary/aortic flow were unaffected by hypertonic saline in our model. Vp is calculated by plotting the linear relation between maximum and minimum volume from each loop (Fig. 1B). The intercept between this line and the line of identity (when maximum and minimum volumes are equal, i.e. LV is empty) is Vp. This is based on the assumption that when the conductivity of the perfusate is zero, all current is conducted through surrounding structures. Relative volume measurements were then converted to absolute volumes by submerging the catheter in a series of KHB-filled cylindrical holes of known diameter housed within a Plexiglass block. An interelectrode distance of 4.0 mm was used to calculate the absolute volume using the equation for the volume (V) of a cylinder, V=
r2l, where r is the radius and l the length of the cylinder. This method has the advantage that both perfusate resistivity and electrode spacing are accounted for in the volume calibration. In order to allow for the differences in the density of the electrical field within Plexiglass and heart tissue, absolute volume measurements were divided by 
, which was defined as the ratio of stroke volume recorded by the flow probe to the stroke volume measured by conductance (Reyes et al. 2003).
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In study 1, hearts from normal mice were used to evaluate the LV response to ß-adrenergic stimulation and ß-blockade. The pacing rate was increased to 500 beats min–1 in these experiments, in order that heart rate would remain stable during pharmacological intervention. Starling curves and aortic occlusions were performed before and after incubation with 10 nmol l–1 isoproterenol. This was followed by treatment with 100 nmol l–1 propranolol before a final Starling curve and aortic occlusion were performed. Stable responses to isoproterenol and propranolol were achieved after 5 and 8 min, respectively.
In study 2, we compared steady-state and beat-to-beat LV pressure–volume relations in hearts isolated from banded and sham-operated mice 2 weeks after surgery, a point at which significant LV systolic and diastolic dysfunction has previously been demonstrated in banded hearts in this model (Grieve et al. 2002).
Drugs and reagents
Sodium pyruvate, indomethacin, isoproterenol hemisulphate and propranolol hydrochloride were purchased from Sigma Chemical Co. All other chemicals were obtained from BDH Laboratory Supplies. Indomethacin was dissolved in dimethyl sulphoxide (10 µmol l–1), which had no effect on cardiac function at its final concentration of 0.1%.
Statistics
Data are expressed as means ±S.E.M. Starling curves were analysed by a two-way ANOVA for repeated measures. Other data were analysed by Student's unpaired t test or one-way ANOVA with Bonferroni post hoc testing, as appropriate. P < 0.05 was considered significant.
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Results |
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Values for Vp, measured by the saline dilution method, in the three study groups were: normal mice, 28.9 ± 5.4; banded hearts, 26.1 ± 2.7; and sham banding, 27.6 ± 2.9; in all cases r2= 0.95. Values for were: normal mice, 0.78 ± 0.06; banded hearts, 0.71 ± 0.04; and sham banding, 0.73 ± 0.03. There were no significant differences in either Vp or values between groups.
Baseline haemodynamic parameters
Baseline haemodynamic data from isolated ejecting hearts are shown in Table 1. Values for the maximal rate of LV pressure rise, LV dP/dtmax, were in the region of 6000 mmHg s–1, which were at least as high as those reported previously in ex vivo preparations (Bittner et al. 1996; Gauthier et al. 1998; De Windt et al. 1999). These data are also comparable with recent in vivo reports from well-established groups (Esposito et al. 2000; Reyes et al. 2003), although they are significantly lower than some data reported in vivo (Georgakopoulos et al. 1998; Yang et al. 1999); the latter may be related to differences in the level of sympathetic activation between preparations and the denervation of the isolated heart with a consequent absence of the ß-adrenergic stimulation. Furthermore, values for cardiac output were comparable to those found in mice in vivo (Barbee et al. 1992; Hartley et al. 1995), and aortic flow accounted for almost 70% of the total cardiac output, which is comparable to or significantly greater than previous ex vivo studies (Bittner et al. 1996; Gauthier et al. 1998; De Windt et al. 1999). Since this is the first study to measure pressure–volume relations ex vivo, there are no directly comparable volume data available. However, values for volume-related indices, such as LV end-systolic volume (ESV), end-diastolic volume (EDV), stroke volume (SV) and ejection fraction, were similar to those previously reported in vivo (Georgakopoulos et al. 1998; Reyes et al. 2003). The slope of the ESPVR under basal conditions was typically found to be about 50% of the in vivo values reported previously (Georgakopoulos et al. 1998; Yang et al. 1999; Feldman et al. 2000). However, this decrease was in proportion to the level of LV dP/dtmax as compared to in vivo values and is likely to be a consequence of differences in ß-adrenergic stimulation between preparations (see Discussion).
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In a series of 10 individual hearts, aortic occlusions were repeated after an interval of at least 10 min. The values for the slope of the ESPVR were almost identical between occlusions (4.80 ± 0.41 versus 4.78 ± 0.43; not significant, n.s.).
Effect of ß-adrenergic stimulation and blockade on steady-state pressure–volume relations
Haemodynamic parameters after incubation with isoproterenol and propranolol are presented in Table 1. ß-Stimulation caused a marked potentiation of LV contractile function, reflected by a significant increase in maximum LV systolic pressure (LV Pmax), LV end-systolic pressure (ESP) and LV dP/dtmax. These changes were accompanied by a significant decrease in ESV, although EDV and SV were not significantly altered. There was a concomitant acceleration of relaxation, in terms of an increase in the maximal rate of LV pressure fall dP/dtmin and a decrease in the time constant of isovolumic LV pressure fall, 
. These changes were associated with an increase in coronary flow, which just failed to reach statistical significance. Conversely, ß-blockade caused a decrease in LV dP/dtmax and LV Pmax, although these changes were not statistically significant. This attenuation of LV contractility was, however, accompanied by a significant increase in ESV, although EDV and SV were not significantly altered. Neither isoproterenol nor propranolol had any significant effect on LV end-diastolic pressure (EDP).
Starling curves showing changes in steady-state LV pressure–volume relations in response to ß-stimulation and blockade are shown in Fig. 2. ß-Adrenoceptor stimulation with isoproterenol caused a significant increase in LV dP/dtmax, whereas inhibition with propranolol caused a significant decrease in LV dP/dtmax compared to baseline (Fig. 2A). Consistent with their positive and negative inotropic actions on LV contractility, isoproterenol and propranolol caused a significant increase and decrease in ejection fraction, respectively (Fig. 2B). Stroke work, which is calculated as the area of the pressure–volume loop, was significantly increased by isoproterenol and decreased by propranolol (Fig. 2C). This is better demonstrated in Fig. 3A, in which a single steady-state loop is shown after each intervention. The area of the loop (i.e. stroke work) is influenced by changes in both LV pressure and stroke volume. In previous pressure-only studies, stroke work was estimated rather crudely using pressure and flow data (Bittner et al. 1996; Larsen et al. 1999); the microconductance catheter allows a more accurate measurement of stroke work.
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Beat-to-beat analysis of the effect of ß-stimulation and blockade on the LV pressure–volume relation is shown in Fig. 3B and C. Figure 3B shows a representative series of pressure–volume loops during transient occlusion of the aortic outflow tract before and after each intervention. Consistent with its demonstrated effects on LV contractile function in the steady state, isoproterenol caused a significant increase in the slope of the ESPVR. Likewise, propranolol caused a significant blunting of the ESPVR. Figure 3C shows a comparison of mean ESPVR data from 10 experiments. In all cases r2 values for the fit of the ESPVR were 
0.9.
Consistent with their lack of effect on LV EDP and EDV, isoproterenol and propranolol were found to have no significant effect on the slope of the EDPVR (see Table 1). In all cases r2 values for the fit of the EDPVR were 0.75.
Steady-state pressure–volume relations during early decompensated cardiac hypertrophy
Table 2 shows morphometric and haemodynamic parameters of isolated hearts taken from banded and sham-operated animals. There was an approximate 30% increase in LV mass in the banded compared with sham-operated animals. Banding resulted in a significant decrease in LV dP/dtmax, LV Pmax and LV ESP; this was accompanied by a significant increase in LV ESV, although LV EDV and SV remained unaltered. Banding also resulted in a significant decrease in LV dP/dtmin and increased compared to sham-operated animals. LV EDP was also elevated in banded animals, although this increase failed to reach statistical significance. Importantly, coronary flow was not significantly different between groups.
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Beat-to-beat analysis of the effect of pressure overload on the LV pressure–volume relation is shown in Fig. 5B and C. Consistent with its negative inotropic effects on LV contractile function, Fig. 5B demonstrates that pressure overload caused a reduction in the slope of the ESPVR. Figure 5C shows a comparison of mean ESPVR data between banded and sham-operated animals. In all cases r2 values for the fit of the ESPVR were 0.9.
In pressure-only studies, diastolic function can be interpreted only from changes in LV EDP or minimum diastolic pressure. The additional measurement of volume allows analysis of the EDPVR during variably loaded beats. Since the relation is exponential in shape, the slope increases as LV EDP increases (Little & Downes, 1990). In the present study, the slope of the EDPVR was calculated from the following elastic model equation: LV EDP =P0+(eßV– 1), where P0 is the LV pressure offset, i.e. Y intercept, V is LV EDV and ß is the slope of the EDPVR (Kawaguchi et al. 2003). A representative example of the calculation of the EDPVR is shown in Fig. 6A. Figure 6B shows a comparison of mean EDPVR data between banded and sham-operated animals. Consistent with the reported increase in LV EDP in response to pressure overload, the slope of the EDPVR was significantly increased in banded compared to sham-operated animals, confirming the presence of diastolic dysfunction. In all cases r2 values for the fit of the EDPVR in were 0.75.
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Discussion |
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In contrast, the effects of residual anaesthetic agents used at the time of euthanasia are minimal in the isolated ejecting heart preparation. The isolated heart is denervated so that values of LV dP/dtmax are typically less than 50% of those found in anaesthetized preparations in vivo (Georgakopoulos et al. 1998; De Windt et al. 1999; Yang et al. 1999; Grieve et al. 2002), but are comparable to in vivo preparations in which denervation has been employed (Esposito et al. 2000). The ex vivo perfusion system also allows heart rate to be easily controlled. In the present study, hearts were atrially paced at 450–500 beats min–1, which was found to be optimal for cardiac performance in our preparation. This is higher than pacing rates previously published for isolated murine heart models (Bittner et al. 1996; Gauthier et al. 1998; De Windt et al. 1999), but lower than the typical heart rates reported for conscious mice under physiological conditions (Georgakopoulos et al. 1998; Yang et al. 1999; Reyes et al. 2003). A controlled heart rate that is not excessively high may allow the optimal study of ß-adrenergic responses independent of the confounding effects of ‘basal’ sympathetic influences in vivo. Indeed, it has previously been shown that ß-adrenergic stimulation only slightly enhances murine cardiac function in vivo, but causes a marked increase in myocardial contractility in the ex vivo preparation (Huke et al. 2002). Furthermore, the isolated ejecting heart system is well suited for the study of the effects of specific pharmacological agents at precise concentrations, independent of the effects of additional neurohumoral influences in vivo.
Another advantage of studying cardiac pressure–volume relations in an ex vivo model is that loading conditions can be carefully controlled, whereas control of preload in particular is difficult in the mouse in vivo. Thus, detailed analysis of the steady-state Frank–Starling relation is straightforward in the isolated preparation, as illustrated in Figs 2 and 4. Furthermore, both steady-state Starling curves and the effects of transient aortic occlusions were found to be highly reproducible in this preparation.
Measurement of pressure–volume relations is the ‘gold standard’ for the assessment of cardiac function in the whole heart (Kass & Maughan, 1988). Construction of pressure–volume loops over consecutive variably loaded beats allows analysis of the ESPVR, which is generally acknowledged to be the best load-independent measure of ‘contractility’ (Kass et al. 1986); the slope of the ESPVR characterizes the intrinsic inotropic state of the myocardium (Baan et al. 1992). The slope of the ESPVR depends critically on the method used to alter ventricular loading conditions; for example, it has been shown to be more sensitive to pressure intervention (e.g. aortic occlusion) than it is to volume loading via the vena cava (Baan & van der Velde, 1988). The slope of the ESPVR is also dependent on resistance (Jacob et al. 1992), which may vary in vivo, primarily due to changes in heart rate and adrenergic stimulation. Values obtained for the slope of the ESPVR (and for LV dP/dtmax) in the present study were approximately 50% of those previously reported in vivo (Georgakopoulos et al. 1998; Yang et al. 1999; Feldman et al. 2000). We think this is most likely due to the fact that in the in vivo reports the substantially higher ESPVR values may be caused by very high (even maximal) sympathetic tone in the anaesthetized mice; this is supported by the fact that additional effects of exogenous catecholamines are often not demonstrable in these preparations. Alternatively, in our preparation, which is obviously denervated, the ESPVR is lower but both positive and negative effects of ß-adrenergic stimulation can be demonstrated, reaching levels of LV dP/dtmax and ESPVR in the ex vivo heart similar to those previously demonstrated in vivo (Georgakopoulos et al. 1998; Kohout et al. 2001). Isoproterenol caused a significant increase in the slope of the ESPVR, whereas the ß-blocker propranolol resulted in a blunting of the ESPVR, responses that have been previously well documented in vivo (Georgakopoulos et al. 1998). The sensitivity of the ESPVR as a measure of myocardial contractility was further demonstrated in hearts from banded mice, which exhibited a significant decrease in the slope of the ESPVR, consistent with the previously reported systolic dysfunction at this stage in this model (Grieve et al. 2002).
Diastolic properties and the EDPVR are also easily assessed in the isolated ejecting heart preparation. The slope of the EDPVR relates to chamber stiffness (Mirsky, 1984) and in the present study, EDPVR was found to be significantly increased in hypertrophied hearts from banded mice compared with sham-operated control animals at a stage of early decompensation (as would be expected). However, EDPVR was not significantly altered by isoproterenol or propranolol in normal hearts, in contrast to the effects of these agents on LV relaxation as assessed by or LV dP/dtmin.
In summary, this study illustrates some of the advantages of applying the microconductance technique to the ex vivo isolated ejecting murine heart preparation. It is evident that the measurement of LV volume in addition to pressure, combined with the ability to carefully control loading conditions and heart rate, provides an extremely versatile system for detailed analysis of murine cardiac function. While the ex vivo isolated ejecting heart preparation is not without its own problems (e.g. buffer perfusion, potential ischaemic damage during isolation and set-up), the results of the present study indicate that its use in conjunction with pressure–volume analyses may provide a valuable complementary method for the careful assessment of cardiac function in gene-modified mice.
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) and after incubation with isoproterenol (
) or propranolol (
). Each data point represents the mean ±S.E.M. from 10 animals; Significant difference between groups: *P < 0.05, two-way ANOVA for repeated measures.
