A methodological approach for the study of the cytoarchitecture of the sarcotubular membrane system in cardiac health and disease

Heart failure (HF) is responsible for the death of over 3.6 million people in Europe each year. HF has a poor prognosis with approximately 40% of patients dying within the first year of diagnosis (www.heartstats.org/) and has an increased prevalence and mortality in the aged. In Western countries it is estimated that by 2050 approximately a quarter of the population will be older than 60 and in some places the elderly may account for nearly 50% of residents (www.globalaging.org/waa2). Disruption of the transverse tubule (TT) membranes and Ca2+-signalling dysfunction are both associated with the development of HF. Our studies involve the application of advanced 3-D electron microscopy methods to investigate the ultrastructural features of cardiac myocytes in particular the dyadic cleft, formed by the TT and junctional sarcoplasmic reticulum (jSR) membranes and map the changes that occur in HF. We have examined tissue from an ovine tachypacing model of heart failure. Pacemakers were implanted under inhalational anaesthesia (1-3 % isoflurane) and analgesia (0.5 mg/kg meloxicam s/c). Sheep were humanely killed (pentobarbitone (200 mg/kg iv) and left ventricle samples were collected, cut to 2 mm3 size blocks immediately after animal expiration and immersed in primary fixative 2.5% glutaraldehyde 2% paraformaldehyde 2.5mM CaCl2 in 0.1M sodium cacodylate buffer pH 7.2. Blocks were cut to size for mould embedding and left in the primary fixative for 3 hrs followed by 1.5 hrs post fixation in 1% OsO4 and 1.5% K4Fe(CN)6 or 2% OsO4 and 0.75% K4Fe(CN)6. Post fixation was followed by washes with water, dehydration in an ethanol ascending series and embedding in Taab low viscosity resin; 250 nm sections (according to interference colour chart) were cut on a Reichert microtome. To aid alignment and reconstruction 10 nm gold colloid particles (Sigma Aldrich) were added to sections. Single axis tilt series were collected between -70 and +70 degrees in one degree increments using a Polara FEG transmission electron microscope operated at 300 kV. Projections were aligned, reconstructed and segmented with IMOD (1). The nanoscale resolution of the 3-D tomograms permitted delineation of the TTs and SR membranes, including visualization of ryanodine receptor complexes localized to the dyadic cleft. Examination of myocytes from both control sheep and heart failure sheep has allowed morphological features of both health and disease states to be analysed in 3-D. Thus understanding the factors that precipitate HF at the cellular level will be pivotal for the development of new treatment strategies and is clearly a global healthcare priority.