Twenty-seven adult male New Zealand White rabbits were studied, 7 were fed normal rabbit chow (group 1) and the remaining 20 were fed a high cholesterol diet as previous described [¹⁴,¹⁵] for 6 months. Briefly, rabbits were fed a high cholesterol diet (1%, Harlan-Sprague Dawley, Inc, Indianapolis, IN) alternating with normal chow on a monthly basis, 10 were treated with ezetimibe (1 mg/kg/day) (group 2) throughout the 6 months and the remaining were not (group 3). All studies were performed with the approval of Michigan State University Animal Care and Use Committee on Animal Investigations following NIH guidelines.

After 6 months echocardiographic images were obtained after inducing general anesthesia with ketamine (15 - 25 mg/kg) and xylazine (3 - 5 mg/kg). Food was withheld overnight and the rabbits were placed in prone position without restraint.

All images were obtained with a 7 MHz probe (Vivid 7/Vingmed General Electric, Milwaukee, WI). Parasternal and apical views were obtained with the probe positioned at a cranial angle over a shaved area on the lower portion of the thoracic wall as previously reported [¹⁶,¹⁷]. The exact position of the transducer was adjusted as necessary to acquire standard images. Echocardiographic measurements by both 2D and M-mode were obtained in the parasternal long axis view. These included septal and posterior left ventricular (LV) wall thickness, LV cavity size (end-diastolic (LVEDD) and end-systolic (LVESD) dimensions), aortic root, and left atrial anteroposterior diameter. Fractional shortening was computed as follows: [(LVEDD-LVESD)/LVEDD] ? 100 and as global systolic function was balanced the ejection fraction was calculated with the Teicholz formula [¹⁸].

Doppler imaging of the mitral valve was obtained from the apical four-chamber view. The following measurements were obtained at the mitral inflow velocity image: peak E and peak A wave velocities, E/A ratio, E wave acceleration and deceleration times, and isovolumetric relaxation time.

TDI was performed from an apical four-chamber view with very low contrast and a 5 mm tissue sampling volume at the mitral annulus from both septal and lateral walls. From the acquired images, the following diastolic function parameters were measured: E', A', E'/A' and E/E' wave ratio; the systolic parameter measured was S'.

All the measurements were obtained at the time of examination by the echosonographer and subsequently read by two separate physicians who were blinded to the groups, the average of the three measurements was used for subsequent analysis.

GraphPad InStat 3.06 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. One way ANOVA was used to compare the E/A, E'/A', E/E', S', myocardial and serum cholesterol levels for all three groups. Pearson Correlation analysis was done between echocardiographic, TDI variables; and serum and myocardial cholesterol levels. A two tailed P value of < 0.05 was noted to have statistically significant value.

A full echocardiographic study was obtained in 24/27 rabbits (89%); TDI were not obtainable in three due to imaging difficulties involving adequate image resolution at the mitral annulus. The 2-D and M-mode measurements were similar and consistent without significant differences among all three groups and with the published data [¹⁷] (Table 1), and there were no significant differences noted between the independent measurements obtained by the echosonographer and the blinded readers.

Several TDI derived parameters were significantly different among the groups (Figures 1, 2). The E'/A' ratio measured in the septal wall was higher in group 1 (1.93 ? 0.42) and group 2 (1.82 ? 0.22) compared to group 3 (1.26 ? 0.62) (p = 0.03). Similar values and statistical differences were noted for the lateral wall for group 1 (2.16 ? 0.5) and group 2 (2.25 ? 0.8) compared to group 3 (1.38 ? 0.66; p = 0.02) (Figure 3). However, the mitral Doppler inflow velocity showed no difference with regards to E wave acceleration or deceleration. Also, there was a borderline significant decrease in the E/A ratio in group 3 when compared to groups 2 and 1 respectively (1.55 ? 0.49 vs. 1.92 ? 0.37 and 1.80 ? 0.51; p = 0.07 and p > 0.1; student t-test).

Group 1 and group 2 had higher S' measurements compared to group 3 respectively (6.42 ? 2.5 and 6.55 ? 1.66 vs. 4.6 ? 0.69; p = 0.03). Also, Group 3 was noted to have an elevated E/E' ratio (11.5 ? 4.9) in comparison to both group 2 (8.72 ? 2.0) and group 1 (8.91 ? 1.54) with a trend towards statistical significance (respectively p = 0.07; p = 0.08; student t-test).

Peak serum cholesterol levels were 495 ? 305 mg/dl vs. 114 ? 95 mg/dl and 87 ? 37 mg/dl; p < 0.01. Myocardial cholesterol content was also significantly greater in group 3 vs. group 2 (0.105 ? 0.04 mg/g vs. 0.068 ? 0.02; p = 0.05; one tailed student t-test).

There was a significant negative correlation between serum cholesterol levels and the TDI derived measurements including E' (r² = 0.28, p = 0.009), S' (r² = 0.16, p = 0.04), E'/A' (r² = 0.37, p = 0.001) with a positive correlation with E/E' (r² = 0.24, p = -0.01). Also, there was a significant negative correlation between myocardial cholesterol content and E' (r² = 0.16 p = 0.04) and S' (r² = 0.15, p = 0.05) with a positive correlation with E/E' (r² = 0.41, p < 0.01). However, there was no correlation between serum or myocardial cholesterol levels and the E wave peak velocity, the peak A wave velocity, E/A ratio or ejection fraction (Table 2).

The mechanism by which hypercholesterolemia alters myocardial function is not clear, and it may overlap with its established effects on the coronary macro and microvasculature (A). Other studies have demonstrated the deleterious effect of high glucose levels on myocardial function with microvascular dysfunction and progressive LV remodeling being implicated for such changes [³⁰]. It has been suggested that the deposition of cholesterol in the myocardium may cause a cholesterol myopathy similar to other infiltrating diseases [³¹]. Alternatively, myocardial metabolism may shift ATP production from a glucose based to a free fatty acid state [³²] and flood the Krebs cycle to create more free radicals that injure the myocardium [³³,³⁴].

This study does not establish causality between higher cholesterol and myocardial function. We used an aggressive dietary regimen to attempt to produce effects that may otherwise take years to develop in humans. Extrapolating these findings to the clinical arena may be relevant based on a lone study that found similarities between cholesterol accumulation in lipotoxic rat hearts to human transplanted hearts [³⁵].

E wave = Peak E velocity, E dec = E wave decelation time IVSd = Intraventricular septum diastole, IVSs = Intraventricular septum systole, LVIDd = Left ventricular internal dimension diastole, LVIDs = Left ventricular internal dimension systole, LVFWd = Left ventricular free wall diastole, LVFWs = Left ventricular free wall systole, FS = Fractional shortening, EF = Ejection fraction, Ao = Aortic root diameter, LA = Left atrial diameter.

From: Fontes-Sousa, et al. M-mode and Doppler echocardiographic reference values for male New Zealand white rabbits. AJVR 2006;67:1725-1729 (17).

* =p < 0.05 for group 1 vs. group 3

? =p < 0.05 for group 1 and group 2 vs. group 3

*=p < 0.05