Animals

Homeodomain-only protein transgenic mice ( HopX ^Tg ) develop cardiac hypertrophy with cardiomyocyte enlargement as the result of HDAC recruitment, as previously described. We studied 10 HopX ^Tg and 10 age-matched wild-type (WT) mice weighing 25 to 35 g and aged 14 to 18 weeks. All protocols used conformed to the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care and were approved by the University of Pennsy-lvania Animal Care and Use Committees. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996).

Transthoracic Echocardiographic Imaging

A comprehensive transthoracic two-dimensional and Doppler echocardiographic study was conducted using a high-resolution (30 MHz) Vevo 770 imaging system (VisualSonics Inc., Toronto, Canada) as previously described. Mice were lightly anesthetized with a mixture of 1% to 2% isoflurane gas and 100% oxygen while supine on a heated platform. Several studies have compared different anesthetic regiments on murine cardiovascular function, and inhaled anesthetics seem to be the most advantageous because they have the least cardiodepressive effects with a relatively short half-life. Furthermore, we compared 1% isoflurane versus 2% isoflurane on the echocardiographic and invasive parameters we measured in a subset of HopX ^Tg and WT mice, and found no significant differences in these measurements at either dose, suggesting the cardiovascular effects of isoflurane are close to maximal at a concentration of 1% in these mice ( Appendix 1 ). The mouse’s core temperature was monitored using a rectal temperature probe, and an infrared heating lamp was used to maintain body temperature at 37°C ± 0.5°C throughout the procedure. An electrocardiogram signal was also monitored through electrode pads on the heated platform. Chest hairs were removed using a chemical depilator (Nair, Church & Dwhite Co. Inc., Princeton, NJ) to minimize ultrasound attenuation. The ultrasound probe (RMV-707B, VisualSonics Inc., Toronto, Ontario, Canada) was placed on the mouse’s chest using warm ultrasound gel as a coupling medium.

Two-Dimensional Echocardiography

Two-dimensional images were recorded in the parasternal long- and short-axis views to guide M-mode recordings obtained at the mid-ventricular level. Wall thicknesses of the interventricular septum and LV posterior wall, as well as the LV diastolic and systolic internal dimensions, were measured, and the average values from both views are reported. LV systolic function was computed from the M-mode measurements according to the recommendations of the American Society of Echocardiography Committee. Relative wall thickness was calculated using the formula: (interventricular septum dimension + LV posterior wall dimension)/LV diastolic internal dimension, and left atrial (LA) area was measured in the 4-chamber view during LV systole using the polygon region of interest area measurement tool in the Visualsonics software suite (Visualsonics Standard Measurements, version 23, VisualSonics Inc.) with the program’s B-mode generic measurement feature.

Transmitral Doppler Echocardiography

Pulsed Doppler studies of LV diastolic function were performed in the apical 4-chamber view with the Doppler cursor oriented parallel to the long-axis plane of the left ventricle. The sample volume was placed just below the level of the mitral annulus and adjusted to render the highest early diastolic flow velocity peak of the transmitral Doppler flow signal. There was often a need for angle correction, which was always less than 20 degrees. The early and late diastolic peak velocity (E, A) and their ratio (E/A) were derived from the transmitral Doppler waveform. Left ventricular systolic intervals of the isovolumic contraction time, the ventricular ejection time, diastolic intervals of the isovolumic relaxation time (IVRT), and the acceleration and deceleration times of the E-wave (E _AT and E _DT , respectively) were also derived from the transmitral Doppler waveform.

Pulmonary Venous Doppler Echocardiography

The pulmonary vein (PV) was visualized by orientating the transducer in the parasternal long-axis view with the left atrium, left ventricle, and aorta in the field ( Figure 1 A, B). The transducer was then moved laterally and rotated clockwise by 10 to 20 degrees, and the beam was then oriented slightly inferiorly and anteriorly to visualize the PV ( Figure 1 C, D). The single PV connects to the LA close to the LV atrioventricular groove ( Figure 1 D). The PV Doppler waveform was obtained by placing the Doppler sample volume approximately 1 mm away from the LA-PV junction with an angle correction of less than 15 degrees. To locate the PV, we would first identify the aorta in the parasternal long-axis view and then tilt the transducer posteriorly until the aorta disappeared and the PV came into the field. In mice, the coronary sinus is in close anatomic proximity to the PV, and so the coronary sinus could be mistaken for the PV while trying to obtain Doppler recordings. This type of confusion may be avoided by analyzing the Doppler spectra, where blood flow in the coronary sinus is affected to a much larger degree by respirations than in the PV. Pulmonary venous flow velocity and the velocity-time integral (VTI) of all waves were then measured. All Doppler parameters are reported as the average value obtained from three consecutive cardiac cycles.

Schematic diagram exemplifying the technique used to locate and visualize the murine PV. (A) The transducer was first positioned to obtain a standard parasternal long-axis view. (B) Representative image displaying the LA, LV, and aorta in the field with the transducer positioned as shown in A . (C) To image the PV, the transducer was moved a little laterally and rotated clockwise 10 to 20 degrees; the beam was then slightly oriented anteriorly and inferiorly. (D) Representative image showing the PV that would appear with the LV and LA, just after the aorta would disappear from the field, with the transducer oriented as in C . LV, Left ventricle; LA, left atrium; Ao, aorta; PV, pulmonary vein.

Invasive Hemodynamics

Protocols for invasive hemodynamic recordings have been described. Briefly, anesthesia was achieved using 1% to 2% isoflurane with 100% oxygen by ventilation, and core body temperature was maintained at 36°C to 37°C using a heating pad and rectal temperature probe. Intracardiac pressures were recorded with a 1.4F microtip catheter (SPR-839; Millar Instruments, Houston, TX) and zeroed in a saline bath before obtaining measurements from each animal. The catheter was inserted via the right carotid artery into the left ventricle to record intracardiac pressures. Signals were digitized at 2 kHz using a PowerLab/16 SP A/D converter (ADInstruments Ltd., Mountain View, CA) and stored on the hard drive of a Core Duo-based PC computer for off-line analysis. Data were analyzed using the PVAN3.2 analysis suite (Millar Instruments) and normalized to the mean arterial pressure to account for load-dependency. The LV relaxation time constant (tau) was calculated by two different methods: 1) the Weiss method (tau [w]): regression of log (pressure) versus time and 2) the Glantz method (tau [g]): regression of dp/dt versus pressure.

Intraobserver and Interobserver Variability

To evaluate intraobserver and interobserver variability of Doppler echocardiographic measurements, one same observer and two independent observers remeasured 12 sets of values for E _AT , pulmonary venous first diastolic wave velocity and IVRT in 12 animals using the same cardiac cycles.

Statistical Analysis

All values are reported as the mean ± 1 standard deviation. Differences between groups were analyzed using the 2-tailed Student’s t test. Linear regression analysis was used to determine correlations between echocardiographic and invasive relaxation parameters. Agreement between two measurements was determined according to the method of Bland and Altman. A P value < .05 was considered significant for all analysis (Prism 5, GraphPad Inc., San Diego, CA). Data were not corrected for multiple comparisons to reduce type I error due to the risk of introducing type II error.

Evaluation of Left Ventricular Relaxation in HopX ^Tg Mice Using Invasive Hemodynamics

Although HopX ^Tg mice clearly develop LV hypertrophy, we next sought to determine the extent to which LV diastolic function was affected in these animals. Invasive hemodynamic recordings revealed HopX ^Tg mice had reduced LV relaxation compared with WT control mice, where tau (g) and LVEDP were significantly higher and -dP/dt _min was much lower ( Figure 2 A, B; Table 2 ). These data confirm that HopX ^Tg mice with LV hypertrophy have impairments in LV relaxation. Furthermore, measurements of the LV peak pressure and +dp/dt _max revealed that LV systolic function is preserved in HopX ^Tg mice ( Table 2 ).

Left ventricular relaxation indices in HopX ^Tg and WT mice. Invasive hemodynamic recordings reveal HopX ^Tg mice have reduced LV relaxation compared with WT mice, where -dP/dt _min is significantly decreased (A, B) in HopX ^Tg mice. Similarly, Doppler echocardiography provides evidence for reduced LV relaxation in HopX ^Tg mice, which shows prolongation in the E-wave acceleration time ( D, the interval between the two vertical solid lines ) compared with WT littermates (C) . Note that the E/A ratio appears normal in this HopX ^Tg mouse, suggesting it is pseudo-normalized. Furthermore, the A-wave duration in the transmitral Doppler profile ( the interval between the two dotted lines ) and the a -wave duration in the pulmonary venous flow profile ( the interval between the two dashed lines ) are distinctly different in both WT and HopX ^Tg mice (C–F) . E , Transmitral early diastolic peak flow velocity; A , transmitral late diastolic peak flow velocity; S , pulmonary venous flow during LV systole; D , pulmonary venous flow during LV diastole; a , pulmonary venous flow during LA contraction; WT, wild type.

Evaluation of Left Ventricular Relaxation in HopX ^Tg Mice by Doppler Echocardiography

Transmitral Doppler echocardiography provided evidence for reduced LV relaxation in HopX ^Tg mice, as revealed by a decrease in the E-wave flow velocity and E/A ratio, and an increase in the acceleration time of the transmitral early diastolic peak flow velocity (E _AT ) ( Figure 2 C, D; Table 2 ). On the other hand, the deceleration time of the transmitral early diastolic peak flow velocity (E _DT ), which is a function of LV compliance and typically affected in later stages of diastole dysfunction, was not different between WT and HopX ^Tg mice ( Table 2 ).

Doppler interrogation of the PV revealed the waveform was composed of a small S-wave, two forward D-waves (D1 and D2), and a small, reversed a -wave in all WT mice examined. Although we could obtain pulmonary venous flows from every mouse we studied, approximately 50% of the HopX ^Tg mice examined did not have a D2 wave. Lack of a D2 wave in half of the HopX ^Tg mice probably accounts for the slight, yet significant, decrease in the pulmonary VTI during diastole (PV VTId) in HopX ^Tg mice compared with control littermates ( Figure 2 E, F; Table 2 ).

Comparison of Left Ventricular Relaxation by Echocardiography versus Invasive Hemodynamics in HopX ^Tg Mice

To validate the Doppler echocardiographic indices for assessing diastolic function in mice, we compared several noninvasive measurements of LV filling and relaxation with invasively measured parameters of LV relaxation. In this model, we found that the E _AT by Doppler echocardiography and relative wall thickness and LA area by non-Doppler echocardiography correlated well with -dp/dt _min , tau (g), and LVEDP ( Figure 3 ; Table 3 ). In contrast, the IVRT did not correlate well with -dp/dt _min , and the E/A ratio did not show good correlation with any of the invasive parameters ( Table 3 ).

Linear regression analysis of LV relaxation indices assessed by echocardiography versus invasive hemodynamics in HopX ^Tg and WT mice. Both –dp/dt _min and tau (g) appear to correlate strongly with E _AT (A, B) , but less well with the E/A ratio (C, D) , IVRT (E, F), and PV VTID (G, H) . Note there are overlapped solid dots in panels A, B, D, F, and G that make it appear as if only 19 data points are shown: blue circles = WT mice; red circles = HopX ^Tg mice. E _AT , Transmitral Doppler E-wave velocity acceleration time; E/A ratio, transmitral early-to-late diastolic peak flow velocity ratio; IVRT, isovolumic relaxation time; PV VTID, pulmonary venous velocity-time integral during diastole; LVSP, left ventricular systolic pressure.

Table 3

Correlation of Echocardiographic and Hemodynamic Indices

	LVEDP (mm Hg)		(-dP/dt min )/LVSP (s-1)		Tau (g) (ms)
Index	r	P Value	r	P Value	r	P Value
E/A ratio	−0.05	.822	0.05	.819	−0.28	.239
E AT (ms)	0.60	.005	0.60	.005	0.74	<.001
E AT /R-R interval	0.56	.010	0.55	.012	0.78	<.0001
E flow velocity (cm/s)	−0.25	.295	−0.23	.335	−0.50	.026
IVRT (ms)	0.37	.106	0.25	.297	0.28	.240
IVRT/R-R interval	0.47	.035	0.25	.295	0.61	.004
PV D1-wave velocity (cm/s)	−0.11	.642	0.03	.899	−0.07	.767
PV VTID (cm)	−0.14	.557	−0.28	.237	−0.20	.403
IVST (mm)	0.66	.002	0.78	<.0001	0.79	<.0001
LVPWT (mm)	0.62	.004	0.66	.001	0.59	.045
RWT	0.68	.001	0.78	<.0001	0.82	<.0001
LA	0.31	.186	0.53	.016	0.52	.018
LVEDV (μL)	−0.55	.012	−0.67	.001	−0.79	<.0001
LVESV (μL)	−0.56	.009	−0.68	<.001	−0.79	<.0001

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