Description
Caveolae are highly specialised lipid rafts whose high content in caveolins confers them a typical flask-like shape, 50-100nm in diameter and are found on the plasma membrane of most cell types. They are involved in cell signalling and also act as a membrane storage to allow membrane expansion in response to hypoosmotic events [1]. A better understanding of caveolae structure could shed new insights into their function. Here we have investigated how two three-dimensional (3-D) electron microscopy techniques, electron tomography and serial block face scanning electron microscopy (SBF-SEM), can be applied to study the structure and the distribution of caveolae in the healthy myocardium and in a pig model of myocardial infarction (MI). A porcine model of MI was employed for this study as described previously [2]. All animal work was approved by the University of Manchester local ethics committee and was covered by the necessary UK Home Office project and personal licences. Left ventricular heart tissue (~0.5 mm3) was collected from the border zones of the MI pigs and corresponding regions from control animals and processed for electron microscopy [3]. To obtain high resolution structural information we used electron tomography. Briefly, plastic sections were cut at ~250 nm and tilt series images were collected at a resolution of ~ 1nm between +65° and -65° at every degree, for each section investigated, using a Tecnai G2 Polara. Tilt series were aligned and reconstructed in IMOD [4] producing computational stacks containing the 3-D information enclosed inside a single section. Caveolae distribution information was obtained using an FEI Quanta 250 FEG SEM equipped with a Gatan 3View system which takes an image of the block face after a slice has been cut away at a specific thickness (SBF-SEM). Serial images were collected at different magnifications ranging from 6 to 15 nm per pixel in the X-Y plane, while the cutting depth along the Z-axis was fixed at 50 nm for all the datasets. Images were segmented and rendered in IMOD [4] or Fiji [5]. 3-D reconstructions obtained with electron tomography reveal that both control and MI pigs are characterised by open caveolae, while closed caveolae are uncommon and are likely the result of the cutting plane not passing through the neck of the caveolae. Furthermore, analysis of the 3-D data revealed how cross sections of the network sarcoplasmic reticulum could be misinterpreted as small closed caveolae. Interestingly caveolae tend to fuse together creating structures reminiscent of the cubic membranes investigated by Landh [6]. The SBF-SEM data revealed that the sarcolemma undergoes heavy remodelling in disease with prominent clusters of caveolae on the sarcolemma comparable to those seen in endothelial cells. Our electron microscopy data reveal gross remodelling of the sarcolemma within the peri-infarct cardiomyocytes in the MI pigs with caveolar clustering. Given that this region is involved in arrythmogenesis this remodelling may represent a pro-survival response, with the caveolae acting to concentrate ion channels in order to re-equilibrate, at least locally, the ionic imbalance of the cell. Our future studies will characterise the morphological changes and distribution of the caveolae within the peri-infarct region employing 3-D electron microscopy techniques and immunogold labelling to examine if patches of caveolae are characterised by specific ion channels linked to arrhythmias, complemented with biochemical and functional studies| Period | 29 Jul 2016 → 31 Jul 2016 |
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| Event title | Physiology 2016: A Joint APS-TPS Meeting |
| Event type | Conference |
| Location | Dublin, IrelandShow on map |
| Degree of Recognition | International |