Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography

Figures

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  Fig. 1. The jSR in rabbit ventricular myocytes is wider in HPF preserved cells, compared to CFD. A: Comparison of jSR (red arrows) in representative TT-perpendicular ET slices extracted from 3D volumes of rabbit ventricular (coronary perfusion-) chemically fixed and dehydrated tissue (tissue CFD), isolated CFD-preserved cells (cell CFD), and high-pressure frozen isolated cells (cell HPF, same batch as cell CFD). B: jSR width at its widest projection (red arrow) in rabbit cardiomyocytes is highest in cells preserved by HPF when compared both to isolated cells or tissue, preserved by CFD; ***p < 0.001; one-way ANOVA with Bonferroni's post-hoc test; n = 20 TT-jSR pairs/ 16–20 cells/ 3 hearts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Partial jSR/TT volume ratio is higher in HPF vs CFD preserved rabbit ventricular cardiomyocytes. TT (green) and jSR (blue) were segmented and quantified in ET data of isolated cells preserved by HPF or CFD, and in (coronary perfusion-)CFD tissue; ***p < 0.001; one-way ANOVA with Bonferroni's post-hoc test; n = 20 TT-jSR pairs/ 16–20 cells/ 3 hearts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3. The jSR in mouse ventricular myocytes is wider and more voluminous upon HPF, compared to CFD. Top: Comparison of jSR (red arrows) in representative TT-perpendicular ET slices extracted from 3D volumes (tissue CFD and cell HPF) or 2D sections (cell CFD). Bottom: representative 3D models (green: TT; blue: jSR), segmented and quantified in ET data; ***p < 0.001; Student's t-test; n = 21–23 TT-jSR pairs/ 14–21 cells/ 3 hearts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 4. Dyadic geometry differences between HPF and CFD samples. Histograms showing the distribution (%) of the surface area associated with different dyadic spacing (minimum distance between TT and jSR) in ventricular tissue or cells from rabbit (left) and mouse (right), preserved by CFD or HPF. Dyad defined as space of jSR-TT apposition of ≤20 nm. Pooled data from n = 20 TT-jSR pairs/ 16–20 cells/ 3 hearts (rabbit); n = 21–23 TT-jSR pairs/ 14–21 cells/ 2–3 hearts (mouse).

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  Fig. 5. Structured electron densities within the lumen of jSR in rabbit isolated ventricular myocytes, preserved by HPF. Structures of interest (indicated with red arrows) were observed in virtually all HPF-preserved dyads; images representative of n = 20 TT-jSR pairs/ 16–20 cells/ 3 hearts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)