The inter-relationship between the junctional SR and the sarcolemma; a big hug that makes your heart beat.

The ultrastructural morphology of the sarcoplasmic reticulum (SR) is not well understood being represented as a sequence of compartmentalised domains limited by the sarcomere [1] or as a continuous network spread across the cell [2]. Since structure is dictated by function it is of primary importance to investigate it. We used serial block face imaging SEM to produce the first 3D reconstruction of the SR within a cardiomyocyte and identified junctional domains at the sarcolemma. We used sheep heart since its size and rate are similar to those of man. Sheep (n=4) were killed with an overdose of pentobarbitone (200mg/kg iv). All procedures were carried out in accordance to the United Kingdom Animals Act of 1986 and the University of Manchester's ethical review process. Small portions of left ventricle were put in 4% PFA and 2.5% GA in 0.1M sodium cacodylate buffer with 50mM CaCl2 to enhance the staining of the SR [3]. After washings, samples were put in 1% OsO4 and 1.5% K4Fe(CN)6, dehydrated and embedded in Taab LV. Resin blocks were trimmed to ∼ 0.5 mm3, gold coated and positioned in a 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 at a specific thickness, with images taken after each slice to generate 3-D data with a resolution of ∼15nm in the X and Y directions (block surface) and ∼ 50 nm in the Z direction (entering into the block). Images were aligned, analysed, reconstructed and segmented in Fiji [4] or IMOD [5]. The size of the tissue examined means that the SEM images at least 5-8 cells with the removal of each slice, generating volumetric data for each myocyte. Using the staining described the SR is highly contrasted and appears black. 3D reconstruction showed that the SR runs along the mitochondria throughout the cell and is also in close contact to the myofilaments parallel to the mitochondria. It also forms connections perpendicular to the myofilaments mostly located at the Z-line and is a continuous network connecting one end of the cell to the other. We found that junctional portions of the SR (jSR) not only form patches in apposition to the t-tubules but also adopt a similar organisation with the sarcolemma. This novel technique has generated detailed images of entire SR network within a cell at a resolution close to 15 nm. The identification of the jSR patches in close apposition to the sarcolemma is consistent with a functional role for spark generation in myocytes at the surface of the cell. A similar organisation would be relevant to myocytes which do not have a developed t-system like atrial myocytes of small mammals. The SR is also in close contact with the mitochondria which may underlie a critical role in local Ca2+ transfer between the two organelles. [ABSTRACT FROM AUTHOR]

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