Electron microscopy

Neonate hearts were dissected in cold Krebs buffer without calcium, and fixed open with 2% glutaraldehyde in cacodylate buffer (Na Cacodylate 150 mmol l⁻¹, CaCl2 2 mmol l⁻¹, pH 7.3). The left ventricular papillary muscles were excised and fixed again in 2% gluteraldehyde in cacodylate buffer, post-fixed in 1% OsO4, contrasted in 1% uranyl acetate, dehydrated and embedded into Durcupan. Ultrathin (58–60 nm) longitudinal sections were cut by Power-Tome MT-XL (RMC/ Sorvall, USA) ultramicrotome, placed on copper slot grids covered with formwar and stained with lead citrate. The sections were examined in a JEM 2000FX (Jeol, Japan) electron microscope and recorded using a Gatan DualVision 300W CCD (charge-coupled device) camera (Gatan Inc., USA).