Electron microscopy imaging of cardiomyocyte ultrastuctures.

<p>Two mice from trastuzumab-treated and two mice from vehicle-treated groups were evaluated by EM. Animals were either treated daily with trastuzumab (10 mg/kg) or with vehicle control. On day 7, mice were euthanized and the hearts were harvested. See materials and methods for details of the fixation, embedding, and staining procedures. (<b>a</b>) A representative section of the left ventricle from a control mouse (<b>a1</b>) showing the typical mitochondrial density and intimate contacts (green arrows), connected myofibers (red arrows) and normal thickness of myofibers (pairs of blue arrows), compared to a section from trastuzumab-treated mice (<b>a2</b>) showing sporadic mitochondrial (green arrows), damaged disconnected myofibers (red arrows) and thinner myofibers (pairs of blue arrows). (<b>b</b>) Bar graph, quantification of damaged myofibers in trastuzumab-treated animals compared to control mice. Data are presented as a percentage of damaged and discontinued myofibers out of the total numbers of myofibers in the segment. (<b>c</b>) Bar graph showing a quantification of the distance between mitochondrial in trastuzumab-treated animals compared to control animals. Data presented in µm representing an average distance between mitochondria in the image. Magnification, 1000X (<b>d</b>) Bar graph showing a measurement of myofibers thickness in trastuzumab-treated animals compared to control mice. Data presented in µm representing the average thickness of myofibers. Magnification, 1000X. (<b>e</b>) Bar graph showing quantification of the number of mitochondria in sections from trastuzumab-treated animals compared to control mice. Data presented as average number of mitochondrial per segment. (f) Bar graph showing the percentage of damaged mitochondria (membrane disintegration, thinning of cristae and significant cavelae formation). In this figure, similar segments from the two animals in each group were used for this quantification. Student’s t-test was used to compare the two groups and significance is determined as *P < 0.05 or ** P < 0.01 vs. the vehicle treated animals.</p

Trastuzumab impairs systolic function in mice.

<p>Mice received either trastuzumab (10mg/kg/day) (N = 10) or vehicle injections (N = 6). VEF (a),FS (b), heart rate (d), LVPW thickness (c), left ventricular systolic (f) and diastolic (e) diameter were evaluated at base line (day 0), and days 3 and 7 post injection. Data are presented as percentage of control animals (mean ± SEM). *P < 0.05 vs. the vehicle treated animals.</p

Trastuzumab altered the expression of genes that are essential for cardiac functions.

<p>(a) Heatmap representation of differentially expressed genes in the trastuzumab-treated animals compared to the control vehicle injected animals (blue = downregulation and red = upregulation); n=4 for each group. (b) Bar graph showing microarray results as Log₂ of fold change. (c) Bar graph showing the qPCR validation of randomly selected genes from the microarray data. Results are shown as log₂ of fold change. Analysis of variance was used to determine those probe sets significantly different between the two groups. The gene list was filtered with a fold-change cutoff of 2.</p

Effect of trastuzumab treatment on the levels of cTn-I and cMLC1 in mice sera.

<p>(<b>a</b>) The results of sandwich ELISA detecting the levels of cMLC1 in the serum of trastuzumab-treated mice compared to control mice injected with vehicle only. (<b>b</b>) The results of sandwich ELISA detecting the levels of cTn-I in the serum of trastuzumab-treated mice compared to control mice injected with vehicle only. Standard curve is constructed by plotting optical density (OD) values obtained from each reference point against its concentration in mg/mL. Absorbance values of vehicle and trastuzumab-treated mice samples are determined by corresponding concentrations from standard curve using linear regression analysis. Student’s t-test was used to compare the two groups. Significance is determined as *P < 0.05 vs. the vehicle treated animals. Six animal were used for each group.</p

Trastuzumab induces oxidative stress and increases Caspase 3/7 activity in cardiomyocytes.

<p>(<b>a</b>) Quantification of ELISA measurement of NT in the hearts of animals treated with trastuzumab compared to the control vehicle injected animals. (<b>b</b>) Quantification of ELISA measurement of 4-HNE adducts in the hearts of animals treated with trastuzumab compared to the control vehicle injected animals. (<b>c</b>) Quantification of ELISA measurement of caspase 3 and 7 in the trastuzumab-treated animals compared to the control vehicle injected animals. For all the graphs in Figure 4, the results are expressed as fold changes relative to that of control vehicle-treated animals. Student’s t-test was used to compare the two groups and significance is determined as *P < 0.05 or **P< 0.01 vs. the vehicle treated animals. At least six animal were used for each group (n=6). </p