Methodological details of stereological parameters.

<p>Legend. V_V = volume density, N_V = numerical density, L_V = length density, myo = cardiomyocytes, int = interstitium, lv = left ventricle, mit = mitochondria, mf = myofibrils, sp = residual sarcoplasm, nuc = nucleus, ld = lipid droplets, N_N(nuc/myo) = mean number of nuclei per myocyte, nf = nerve fiber, Q_Q(ax/nf) = mean number of axon profiles per nerve fibre profile, FOV = fields of view, h = height, a(p) = area per point.</p

Ultrastructure of cardiomyocytes and nerve fibers.

<p>Electron micrographs of myocardium from control (A, C, E) and tumor-bearing mice (B, D, F). A and B demonstrate the the increased volume of sarcoplasm and the loss of myofibrils in more or less longitudinally sectioned myocytes. Scale bar = 5 µm. C and D clearly show the increase in lipid droplet (ld) size and number in the tumor group in diagonally sectioned myocytes. Scale bar = 1 µm. E and F show nerve fibres containing three axons (asterisk) each. There were no apparent differences in the morphology. The neighbouring myocytes are more or less transversely cut. Abbreviations: cap = capillary, mf myofibrils, mi = mitochondria.</p

Relative expression of various innervation-related targets.

<p>Legend. Data are expressed as mean ± standard deviation.</p><p>*indicates p<0.05,</p><p>**indicates p<0.01,</p><p>***indicates p<0.001.</p><p>TNF-R1 = tumor necrosis factor receptor 1, TNF-R2 = tumor necrosis factor receptor 2, β1-R = β1 adrenergic receptor, NGF = nerve growth factor, NPY = neuropeptide Y, TH = tyrosine hydroxylase.</p

Morphology of myocardium.

<p>Semithin cross-sections (1 µm thickness) stained with toluidine blue did not show any differences in the dimensions of cardiomyocytes between control (A) and tumor-bearing mice (B). However, the subcellular organization of cardiomyocytes appeared less dense in the tumor group. Paraffin longitudinal sections (5–7 µm thickness) stained with Masson-Goldner's trichrome did not provide evidence for an enhanced collagen deposition in the tumor-bearing mice (D) as compared to control mice (C). Scale bar for A and B = 10 µm. Scale bar for C and D = 20 µm.</p

Body and tumor weight.

<p>Legend. Data are expressed as mean ± standard deviation.</p><p>* indicates p<0.05,</p><p>**indicates p<0.01,</p><p>***indicates p<0.001.</p

Demonstration of the point counting method.

<p>A multi-purpose grid with 10 points (crosses or endpoints of test lines) is projected on an electron micrograph. The number of points hitting a structure of interest, e.g. mitochondria (here, 2), is divided by the total number of points hitting cardiomyocytes (here, 9) to provide the volume fraction of mitochondria within cardiomyocytes (here, 2/9 = 22.22%). The volume fraction is further multiplied by the reference volume, viz. the total volume of cardiomyocytes, to provide the total volume of mitochondria in the left ventricle.</p

Primers used for quantitative RT-PCR.

<p>Legend. TNF-R1 = tumor necrosis factor receptor 1, TNF-R2 = tumor necrosis factor receptor 2, β1-R = β1 adrenergic receptor, NGF = nerve growth factor, NPY = neuropeptide Y, TH = tyrosine hydroxylase.</p

Stereological results.

<p>Neither the volume of cardiomyocytes (A, open circles) nor the volume of the interstitial space (A, filled squares) was different between the groups. In addition, the number of cardiomyocytes was similar among the groups (B). The volume of sarcoplasm (C, filled squares)was significantly increased in the tumor group while the volume of myofibrils (C, open circles) tended to be smaller. A greater volume of lipid droplets was found in the cardiomyocytes of tumor-bearing than of control mice (D). Note the logarithmic scale on the y-axis (D). The total length of axons was reduced to approximately one half in the tumor group (E) while the relative frequency of nerve fibers with a certain number of axons did not differ between the groups (F). The number of LDVC per axon profile was reduced in the tumor group (G) while the diameter of the axons was similar in both groups (H). Each circle or square represents the data for an individual animal. The horizontal bars demonstrate the group mean.</p

Immunohistochemistry of nerve fibers.

<p>Nerve fibers were detected by immunostaining for protein gene product 9.5 (PGP9.5). Scale bar = 50 µm. Nerve fibers (arrows) were counted in an unbiased counting frame. The dashed line is the inclusion line, the closed line and its extensions represents the exclusion line (see Methods section).</p

Lipid content, lipid peroxidation and caspase 3/7 activity.

<p>Legend. Data are expressed as mean ± standard deviation. MDA = malondialdehyde, TBARS = thiobarbituric acid reactive substances, LU = luminescence units.</p><p>* = p<0.05.</p