Apolipoprotein E knockout (apoE^-/-) mice fed a high-fat, Western-style diet for 6 months are grossly hypercholesterolaemic and develop atherosclerotic lesions in the brachiocephalic artery and aorta [1, 2]. In contrast to their male counterparts, female apoE^-/- mice on high fat diet do not show evidence of occlusive coronary lesions or myocardial infarction. The aim of this work was to investigate whether high-fat diet alters the characteristics of Ca²⁺ transients of myocytes from these female mice during field stimulation at different frequencies. Female apoE^-/- mice at 8 weeks old were fed either a high fat, Western-type diet (21% fat; 0.15% cholesterol) or were maintained on normal rodent diet for approximately 6 months. Cardiomyocytes were isolated as described previously [3]. Cardiomyocytes were also isolated from hearts of female wild type C57Bl/6 mice fed a standard rodent diet for comparison. Ventricular myocytes were superfused with normal Tyrode solution in a chamber on the stage of an inverted microscope. Single myocytes were chosen based on the criteria that they were rod-shaped with clear cross striations and well-defined edges, and were able to contract in response to field stimulation with no spontaneous contractions. Myocytes were loaded with 5 µM of the fluorescent indicator Fura-2. Cardiomyocytes were stimulated for 2 min at 0.2Hz followed by increasing the frequency of stimulation every 30 seconds to 0.5, 1 & 2 Hz and Ca2+ transients (Fura ratio) were measured using photometry. At 0.2Hz, wild type cardiomyocytes had larger transient amplitude, faster time to peak and time to decay compared to both groups of apoE^-/- cardiomyocytes. The Ca²⁺ transient amplitude at 0.2 Hz was significantly smaller in apoE^-/- cardiomyocytes fed high-fat diet compared to those fed normal diet (ratio: 0.41 ±0.03 vs. 0.65 ±0.08, p<0.05, t-test). Data shown are the mean ± S.E. for n=45 cells from 6 mice for apoE^-/- high fat, n=24 cells from 3 mice for apoE^-/- normal diet and n=16 cells from 3 C57Bl/6 wild-type mice. As stimulation frequency increased, the amplitude of the calcium transients decreased, with myocytes from apoE^-/- high fat hearts continuing to show smaller transient amplitudes than myocytes from apoE^-/- hearts fed a standard diet. Times to peak and time to decay of the calcium transients decreased as the stimulation rate increased, and was similar for both groups of cardiomyocytes. This work demonstrates that apoE^-/- cardiomyocytes have altered calcium handling characteristics and that these alterations are accentuated by high fat diet feeding, indicating important differences in myocyte function in the hearts of atherosclerotic female mice in the absence of coronary artery disease.