Abstract
Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.
Highlights
The link between electrical excitation and Ca2+ release from the sarcoplasmic reticulum (SR) is a key process in cardiac excitationcontraction coupling (ECC) [1]
To exclude potential artefacts associated with the lack of control over the plane of sectioning, we used electron tomography (ET) to measure the width of junctional SR (jSR) at their widest projection along an axis perpendicular to the associated transverse tubules (TT) (Fig. 1B)
This analysis confirmed quantitatively that the jSR is significantly wider in high-pressure freezing (HPF) rabbit ventricular myocytes than in chemical fixation and subsequent dehydration (CFD) cell samples and in CFD tissue (Fig. 1B)
Summary
The link between electrical excitation and Ca2+ release from the sarcoplasmic reticulum (SR) is a key process in cardiac excitationcontraction coupling (ECC) [1]. In the TT–SR membrane complex, called the dyad, precise control of ECC allows for stable amplification of Ca2+ influx to levels that govern efficient activation of the contractile machinery [2,3]. Conceptual and computational models of cardiac ECC and Ca2+ transport/ storage processes require a detailed understanding of the nanoscopic structure of the dyad and of the distribution of Ca2+ binding proteins [4]. Previous ultra-structural studies of the membrane systems underlying ECC have been based almost entirely on the analysis of twodimensional (2D) transmission electron microscopy (EM) images. Several studies have reported 3D data obtained by serial blockface scanning EM and electron tomography (ET) and reconstruction [5,6,7,8,9,10,11,12,13]
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