In Vivo Echocardiographic Detection of Enhanced Left Ventricular Function in Gene-Targeted Mice With Phospholamban Deficiency
Abstract We evaluated the ability of M-mode and Doppler echocardiography to assess left ventricular (LV) function reliably and repeatedly in mice and tested whether these techniques could detect physiological alterations in phospholamban (PLB)–deficient mice. Anesthetized wild-type mice (n=7) and mice deficient in PLB (n=8) were studied with two-dimensional guided M-mode and Doppler echocardiography using a 9-MHz imaging and 5- to 7.5-MHz Doppler transducer. Data were acquired in the baseline state and after intraperitoneal isoproterenol administration (2.0 μg/g IP). Interobserver and intraobserver variability and reproducibility were excellent. PLB-deficient mice were associated with significant (P<.05) increases in several physiological parameters (mean±SD) compared with wild-type control mice: normalized mean velocity of circumferential shortening (7.7±2.1 versus 5.5±1.0 circ/sec), peak aortic velocity (105±13 versus 75±9.2 cm/s), mean aortic acceleration (57±16 versus 31±4 m/s2), and peak early-diastolic transmitral velocity (80.0±7.2 versus 66.9±7.7 cm/s). LV dimensions, shortening fractions, heart rates, late diastolic transmitral (A) velocities, and early to late (E/A) diastolic velocity ratios were similar in both groups. Isoproterenol administration resulted in significant increases in Doppler indices of ventricular function in control but not PLB-deficient mice. These findings indicate that assessment of LV function can be performed noninvasively in mice under varying physiological conditions and that PLB regulates basal LV function in vivo.
Manipulation of the mammalian genome with transgenic techniques is emerging as a powerful method for definitively identifying the molecular mechanisms underlying cardiac development and function. Although a variety of animal species have been used in transgenic experiments, the mouse has been studied most extensively. The predominant use of mice in transgenic investigations reflects their relatively low cost, their well-characterized genome, the availability of numerous in-bred lines, and the ability to perform rapid linkage analysis.1 Despite these considerable advantages, the functional analysis of the transgenic phenotype in the intact animal has been limited by the small size of the mouse.
Recently, investigators have attempted to image the murine heart in vivo. Right ventricular function was assessed by quantitative angiography in a murine model of right ventricular pressure-overload hypertrophy (Rockman et al2 ). However, ventriculography is invasive, requires microsurgical techniques and contrast injections, and has limited temporal resolution. Moreover, in the study of Rockman et al, 40% of densitometric studies were unsuitable for analysis. Although radiolabeled microsphere and indicator dilution techniques have been used for cardiac output and stroke volume determinations in both conscious and anesthetized mice,3 these methods are technically demanding and do not directly assess ventricular function.
M-mode echocardiography is an attractive alternative because of its ability to noninvasively and repeatedly assess ventricular dimensions and shortening with both high temporal and (with appropriate transducer frequency) spatial resolutions. M-mode echocardiography has been shown by Manning et al4 to provide accurate estimates of murine LV mass; however, in that study, ventricular function was not assessed, and the ability to quantify physiological changes by using in vivo Doppler echocardiographic techniques was not determined. More recently, Gardin et al5 presented data to support the feasibility of M-mode echocardiographic evaluation of LV mass and systolic function in mice.
LV function can also be quantified by Doppler echocardiographic interrogation of transvalvular flows. Aortic Doppler waveform analysis can be used to estimate LV stroke volume and assess LV systolic performance.6 7 Peak aortic velocity and mean acceleration were highly correlated with maximal dP/dt, peak flow, and maximum flow rate in a highly instrumented canine model; moreover, correlations were not appreciably influenced by adjustments for heart rate and preload.6 Transmitral Doppler waveform analysis provides insight into the temporal distribution of LV filling; diastolic filling patterns (eg, E/A ratios) characteristic of impaired LV relaxation and reduced ventricular compliance are described.8
Accordingly, the overall goal of the present investigation was to develop reliable methods to noninvasively assess LV function by using M-mode and pulsed-wave Doppler echocardiography in the mouse. An important related objective was to evaluate the ability of these techniques to characterize a well-defined genetic model with altered cardiac biochemical and physiological parameters. Recent studies using the isolated working heart indicate that the phosphoprotein PLB plays critical roles in SR function, basal myocardial contractility, and the contractile responses to β-adrenergic agonists ex vivo.9 Therefore, we performed echocardiographic studies before and after isoproterenol administration in transgenic mice with PLB deficiency and in wild-type control mice to test the hypothesis that PLB regulates basal LV contractile function and modulates the sensitivity to β-adrenergic agonists in vivo.
Materials and Methods
Mice of either sex (Charles River Laboratories, Wilmington, Mass), 10 to 12 weeks of age and weighing 23.6 to 40 g (29.1±5.2 g), were used in these studies. Mice were anesthetized with 0.1 mL/g of a mixture of 65% ketamine, 22% acepromazine, and 13% xylazine (at a concentration of 10 mg/mL each and prepared as a 1:10 dilution) and were allowed to breathe spontaneously. Oxygen was delivered at 2 L/min via a nose cone. The chest was shaved, and ECG leads were attached to each limb with needle electrodes (Grass Instruments). The extremities were secured to the examining surface with paper tape. A warming pad (Deltaphase isothermal pad, Braintree Scientific) was used to maintain normothermia.
Studies were performed in mice with PLB deficiency that were obtained by gene-targeting methodology (PLB-KO mice, n=8) and in age-matched CF-1 mice (control mice, n=7). The control group included two wild-type littermates and five CF-1 mice. PLB-targeted mice were generated from CF-1 and C57Bl/6 strains by using methods previously described.9
Cardiac ultrasound studies were performed with an Interspec Apogee X-200 ultrasonograph (Interspec-ATL). A dynamically focused 9-MHz annular array transducer (axial resolution, 0.2 mm) was placed on a layer of acoustic coupling gel that was applied to the left hemithorax; care was taken to maintain adequate contact while avoiding excessive pressure on the chest. Mice were imaged in a shallow left lateral decubitus position; short- and long-axis views of the LV were obtained by slight angulation and rotation of the transducer. Two-dimensional targeted M-mode studies were generally taken from the short axis (at the level of the largest LV diameter), but occasionally, endocardial echoes were best defined from the long-axis view.
Aortic outflow and diastolic transmitral LV inflow velocities were interrogated from angulated parasternal long-axis views by using a pulsed-wave Doppler (5- to 7.5-MHz) transducer with a sample volume length of 3.5 to 7.5 mm. The Doppler instrument has a sampling frequency of 4 to 35.7 kHz and a minimum computation time of 1 millisecond. Echoes from the mitral valve annulus and aortic root were readily defined, and color flow-mapping Doppler assisted the sample volume placement. Attempts were made to align the ultrasound beam as parallel as possible to flow and to record the highest velocities.
Studies were recorded on 1/2-in S-VHS videotape (Sony 9500 VCR, Sony Corp). Freeze frames were printed (either on-line or off-line from digital archival storage) on a Sony color video printer (UP-5200, Sony Corp). The limb-lead ECG was patched into the ultrasonograph for timing purposes.
Two-dimensionally targeted M-mode and color flow mapping–guided pulsed-wave Doppler studies were performed at baseline and after the administration of 2.0 μg/g IP isoproterenol. Heart rate responses to isoproterenol were maximal within 1 to 2 minutes after injection. Imaging sequences were generally completed within 10 to 15 minutes. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of Cincinnati.
M-mode measurements of LV EDD and ESD were made from original tracings (Fig 1⇓) (Sigma Plot, Jandel Scientific) by using the leading-edge convention of the American Society of Echocardiography and by using the steepest continuous endocardial echoes. End diastole was taken at the onset of the QRS complex, and end systole was taken at the peak of posterior wall motion. Three beats were averaged for each measurement.
LV FS was calculated as follows: (EDD−ESD)/EDD.
The normalized mean Vcf was calculated as follows: FS/ET, where ET was taken from heart rate–matched aortic Doppler spectra.
Spectral Doppler waveforms were analyzed for peak early- and late-diastolic transmitral velocities, the peak and integral velocities of aortic flow, and aortic acceleration and ETs (Fig 2⇓) from videotape playback using a commercially available image analysis system (Freeland Medical). Mean acceleration was calculated as follows: peak aortic velocity/acceleration time. Three to five beats were averaged for each measurement.
A total of 108 beats were selected at random from nine animals and were analyzed for LV dimensions and wall thickness (n=36) and transmitral (n=36) and aortic Doppler (n=36) velocities by two observers (B.D.H. and S.F.K.). A single observer (S.F.K.) repeated M-mode echocardiographic measurements from 108 beats several days later. To determine reproducibility, studies were repeated on separate days in five mice and analyzed in a blinded fashion.
Interobserver and intraobserver differences were calculated as the difference between two observations divided by the mean of the observations and were expressed as percentages. Interobserver and intraobserver variability was also quantified by the limits of agreement, defined as the mean difference between observations (±2 SD).10 Reproducibility was calculated as the difference between two determinations divided by the mean of the two determinations and was expressed as a percentage. In view of the small number of animals studied, variability was also quantified as the mean difference of two determinations and the range of those differences.
All data are presented as mean±SD. M-mode echocardiographic and Doppler parameters were compared at baseline in control and PLB-deficient mice with unpaired t tests. Paired t tests were used to compare baseline and isoproterenol-stimulated states in each group of animals. A value of P<.05 was considered significant.
Interpretative Variability and Reproducibility
Technically adequate M-mode and Doppler studies were obtained in all mice. Interobserver and intraobserver variability for M-mode dimension and wall thickness and Doppler measurements is summarized in Table 1⇓. The interobserver and intraobserver variabilities for LV cavity dimensions and FS and Doppler-determined variables were excellent; LV wall thickness determinations exhibited greater variability.
Reproducibility data for M-mode and Doppler studies are summarized in Table 2⇓. LV dimension and FS measurements made in the same animals on separate days were highly reproducible; wall thickness measurements were less reproducible. Doppler determinations exhibited considerably less reproducibility; these indices were influenced greatly by the disparate heart rates on the two separate days.
Baseline Echocardiographic Measurements
M-mode and Doppler echocardiographic measurements in PLB-deficient and wild-type control mice are compared in Table 3⇓. EDD, ESD, and the FS were similar in both groups. In contrast, Vcf was significantly greater in PLB-deficient than wild-type control mice.
Compared with control mice, PLB-deficient mice had higher peak aortic velocity, shorter acceleration time, and consequently, greater mean aortic acceleration. The aortic velocity time integral, however, was similar in both groups.
Peak early-diastolic transmitral velocity was greater, and there was a trend (P=.09) toward greater late-diastolic velocity in PLB-deficient mice than in control mice; the E/A ratio did not differ in the two groups.
Heart rates were slightly but not significantly greater in PLB-deficient mice than in control mice. To determine whether differences in M-mode and Doppler-derived indices resulted partly from heart rate effects, data were analyzed after excluding two (PLB-deficient) outliers with rapid basal heart rates (604 and 504 bpm). Despite nearly identical heart rates, significant differences in peak aortic and early-diastolic velocities, aortic acceleration time, and mean acceleration persisted; differences in Vcf became of borderline significance (P=.07).
Effects of β-Adrenergic Stimulation
The effects of the intraperitoneal administration of the β-adrenergic agonist isoproterenol on M-mode and Doppler measurements in control and PLB-deficient mice are illustrated in Fig 3⇓ and are summarized in Table 3⇑. In control mice, isoproterenol caused significant increases in heart rate, LV FS, Vcf, and Doppler-derived aortic and early- and late-diastolic velocities and significant decreases in aortic acceleration time and the E/A ratio. In PLB-deficient mice, isoproterenol caused significant increases in heart rate, LV FS, and Vcf; however, peak aortic and early- and late-diastolic transmitral velocities and aortic acceleration did not change significantly.
The major findings of the present study are as follows: (1) M-mode and Doppler echocardiography provide precise repeatable measurements of in vivo LV performance and transvalvular velocities in the mouse. (2) These in vivo noninvasive techniques can detect changes in LV function in mice with altered PLB expression.
The development of gene-targeting technology in mice has provided the opportunity to determine, unambiguously, the functions of a specific gene product in the intact mammal.1 11 However, efforts to quantify basal myocardial function and the changes in function produced by an experimental intervention in the transgenic mouse have been limited. Recently, pressure-derived indices in the murine isolated working heart and myocyte preparations were used to quantify myocardial performance.11 12 13 Despite the ability to determine physiological end points with high resolution and under controlled loading conditions, approaches at the isolated heart and myocyte levels have certain limitations. First, in vitro techniques require killing the animal, precluding serial studies of ventricular function. Second, the isolated heart and myocyte preparations are devoid of autonomic reflexes and ventriculovascular interaction; thus, significant differences in ventricular performance may exist in vivo compared with isolated preparations.
By contrast, echocardiography is a relatively simple technique that allows repetitive noninvasive assessment of LV function. The small size of the murine heart and the rapid heart rates encountered (≈600 bpm in the unanesthetized mouse; Dr I.L. Grupp, oral personal communication, 1994) have, in the past, limited echocardiographic assessment of the murine heart. However, in the present study, the use of a dynamically focused annular array transducer operating at a higher (9-MHz) frequency resulted in highly reliable and reproducible image quality. The annular array focuses the ultrasound beam in axial, lateral, and elevational planes, thereby increasing the signal-to-noise ratio.14 Because of its high sampling rate (1000/s), M-mode echocardiography provides excellent temporal resolution; unfortunately, this occurs at the expense of spatial resolution (ie, a unidimensional “ice pick” view of the heart is produced). Therefore, this technique is well suited only for ventricles with uniform geometry and wall motion. Although two-dimensional echocardiography potentially overcomes problems owing to regional differences, the relatively slow frame rates (30 Hz) and the limited acoustic windows and imaging planes available in the murine model remain significant limitations. Fortunately, Doppler interrogation of transvalvular flows permits assessment of global LV systolic and diastolic function without regard for ventricular geometry.
PLB is an inhibitor of the Ca2+-ATPase in cardiac SR.15 Ablation of PLB leads to enhanced rates of SR Ca2+ uptake (resulting in increased rates of myocardial relaxation) and consequently larger amounts of SR Ca2+ available for release (resulting in increased rates of contraction).16 In isolated work-performing hearts from PLB-deficient mice, the times to peak pressure and half-relaxation were significantly shorter, and peak positive and negative dP/dt values were significantly greater in PLB-deficient mice than in their wild-type littermates.9 In the present study, Vcf, mean aortic acceleration, and peak aortic velocity were significantly greater in PLB-deficient mice than in control mice. However, LV FS was similar in PLB-deficient mice and their wild-type controls. The discrepancy between our in vivo FS results and measurements in isolated work-performing hearts may be explained by different experimental preparations and methods used to characterize ventricular function and/or in vivo compensatory mechanisms that serve to maintain basal LV performance. In the isolated work-performing heart, isovolumic indices were measured, and comparisons between groups were made with similar heart rates, venous return, afterload, and coronary perfusion. In contrast, LV performance in the intact heart is modified by interaction with cardiovascular reflexes, uncontrolled loading conditions, and coupling to the arterial circulation. Moreover, our findings are influenced by the cardiodepressant effects of anesthesia. The ejection phase indices used in the present study are load and heart rate dependent; although preload (as determined from EDD) was similar between the two groups, heart rates tended to be greater in PLB-deficient than control mice. In this regard, it is important to note that when data were analyzed after excluding two PLB-deficient mice with excessively rapid basal heart rates, the differences (except for heart rate and Vcf) between the PLB-deficient and control groups persisted. The trend toward higher heart rates in PLB-deficient mice than in wild-type mice is unlikely to be due to an influence of PLB on the sinus node, since their heart rates are similar in the unanesthetized state9 ; although unlikely, a differential effect of PLB on anesthetic-induced cardiac slowing cannot be excluded.
Noninvasive indices of LV diastolic function derived from transmitral waveform analysis are difficult to interpret because they are influenced by many physiological and pathophysiological factors, such as loading conditions, heart rate, age, and atrial function.17 The peak early-diastolic transmitral velocity, which reflects the early-diastolic transmitral gradient, was significantly greater in PLB-deficient mice than in control mice. However, it is not known whether the difference in early-diastolic velocity that we observed is due to the greater left atrial pressure and/or enhanced LV isovolumic relaxation in PLB-deficient mice than in control mice. Similarly, whether the higher atrial systolic velocities in PLB-deficient mice compared with control mice are the result of increased heart rate or are due to altered left atrial contractility and/or load cannot be determined from our study. As our reproducibility data indicate, Doppler indices of diastolic function should be used cautiously.
Several studies have indicated that phosphorylation of PLB and relief of its inhibitory effects on the SR Ca2+-ATPase is mediated by β-adrenergic activation of adenyl cyclase.16 18 Therefore, we determined the effects of β-adrenergic stimulation on LV function in intact mice. In PLB-deficient mice, although isoproterenol increased LV FS, Vcf, and heart rate, there were no significant effects on peak aortic and diastolic transmitral velocities and mean aortic acceleration. In contrast, isoproterenol caused significant increases in all M-mode and Doppler-derived functional indices in control mice. Thus, these data confirm and extend findings in the isolated work-performing heart9 and support the hypothesis that PLB modulates myocardial contractile sensitivity to β-adrenergic stimulation. Moreover, these data suggest that velocity-dependent indices (eg, mean acceleration) are more likely to be affected by an alteration in SR Ca2+-ATPase activity than are force-dependent indices (eg, FS), which are influenced by the number and strength of actin-myosin cross-bridges. It should be recognized that loading conditions were uncontrolled in the present study; therefore, we cannot exclude the possibility that these indices were influenced differentially by isoproterenol-induced alterations in loading conditions.
In conclusion, noninvasive in vivo assessment of LV performance with M-mode and Doppler echocardiography in the mouse is feasible and reproducible and can assess physiological changes elicited by acute interventions and altered cardiovascular phenotypes. These approaches should prove useful in determining the interplay between altered cardiovascular gene expression and compensatory physiological cardiovascular regulation in the transgenic mouse. Moreover, this in vivo approach permits the assessment of age-related cardiovascular changes serially over time and the effects of pharmacological interventions and/or therapeutic agents in the intact animal. These techniques should complement and extend information derived from isolated myocyte and isolated heart studies.
Selected Abbreviations and Acronyms
|Ao Vel||=||peak aortic velocity|
|Ao VTI||=||time-velocity integral of aortic flow|
|AW Th||=||anterior wall thickness|
|E/A ratio||=||early to late diastolic transmitral velocity ratio|
|LV||=||left ventricular, left ventricle|
|peak A Vel||=||late-diastolic transmitral velocity|
|peak E Vel||=||early-diastolic transmitral velocity|
|PW Th||=||posterior wall thickness|
|Vcf||=||velocity of circumferential shortening|
This study was supported by National Institutes of Health grants HL-33579, HL-26057, and HL-22619 and SCOR grant P50-HL-52318-01. We gratefully acknowledge the expert secretarial assistance of Norma Burns and the technical assistance of Rick Willis and Zouping Zhou. We are grateful to Dr Wusheng Luo for providing the PLB-deficient mice.
Reprint requests to Brian D. Hoit, MD, Division of Cardiology, University of Cincinnati, PO Box 670542, Cincinnati, OH 45267-0542.
- Received February 2, 1995.
- Accepted May 22, 1995.
- © 1995 American Heart Association, Inc.
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