Korean J Physiol Pharmacol.  2020 Nov;24(6):529-543. 10.4196/kjpp.2020.24.6.529.

Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane

Affiliations
  • 1Department of Physiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon 16419, Korea
  • 2Department of Anesthesiology and Pain Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea

Abstract

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+ channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

Keyword

Atrium; Rat; Scanning ion conductance microscopy; Transverse tubule

Figure

  • Fig. 1 Scanning ion conductance microscopy (SICM) imaging of rat ventricular myocyte. (A) Normal control ventricular myocyte. (B) Formamide-induced detubulated ventricular myocyte. Transmission: Optical transmission images of the cells with SICM scanning areas (red boxes, scale bars = 20 µm). 3D SICM: 3D SICM surface images are presented with an indication of Z-grooves (dotted yellow lines), crests (dotted blue lines), and surface T-tubule openings (red arrows). 2D SICM: In 2D SICM images, several lines were drawn on the X- or Y-axis to get topographic line profiles of the surface images. Arrowheads indicate T-tubule openings. Line profiles: X-Z axis line profiles of the lines (i) and (ii) show a regular appearance of Z-grooves and T-tubule openings along the longitudinal X-axis. The line profile of the line (iii) indicates Z-grooves without openings. Two arrowheads (1 and 2) present the traceable depth of the T-tubules. Line profiles (line iv and v) obtained from detubulated ventricle show a relatively flat surface and closed openings. FFT: Fast Fourier transformation (FFT) analysis of the control cell (line i) gives a peak of 1.88 µm of T-tubule intervals (n = 48). FFT of the line (iii) on a Z-groove has no peak. FFT of the detubulated cell surface (lines iv and v) has no regularity (n = 36).

  • Fig. 2 Confocal imaging of the intracellular T-tubules of the ventricular myocyte. (A) Normal control myocyte. (B) Detubulated ventricular myocyte. A plane of di-8-ANEPPS confocal image (green) is overlaid with an optical transmission image of the cell (scale bar, 10 µm). Representative X-Y, X-Z, and Y-Z confocal plane images are presented. Line profiles: The di-8-ANEPPS fluorescence intensity (arbitrary unit, AU) profile on the longitudinal direction (solid red line in the X-Y plane) shows regular striations of intracellular T-tubules. Fast Fourier transformation (FFT) of the fluorescence profile gives a peak of 1.92 µm intervals (n = 16). (B) Confocal images of a detubulated ventricular myocyte. The di-8-ANEPPS image overlaid with transmission image, representative X-Y, X-Z, and Y-Z confocal images, fluorescence intensity line profile, and FFT analysis are presented for detubulated myocyte. FFT of detubulated cell has no regular peaks (n = 11).

  • Fig. 3 Classification of rat atrial cell surface structure by scanning ion conductance microscopy (SICM) imaging. The 3D SICM and 2D SICM images, topographic line profiles, and Fast Fourier transformation (FFT) analyses are presented for each subtype (A: smooth-type, B: longitudinal fissures (LFs)-type, C: ventricular-type, and D: mixed-type). Optical transmission images (scale bar, 20 µm) are presented with the SICM scanning area (red boxes). The lines presented on each 2D SICM images are used for the construction of topographic line profiles and FFT analysis. FFT was not performed for mixed-type. (A) SICM images and topography of the smooth-type have no particular pattern or regularity. (B) SICM images of the LFs-type show two parallel running LFs (arrows). The arrowheads indicate multiple T-tubule openings inside LFs. FFT analysis (lines i and ii) gives no regularity. (C) SICM images of the ventricular-type show the Z-grooves and the T-tubule openings (arrowheads). FFT shows the T-tubules are regularly opened at 1.84 µm intervals. (D) SICM images of the mixed-type show multiple LFs and T-tubule openings (arrowheads in line i and ii). The average intervals between T-tubule openings (i.e., intervals between arrowheads in longitudinal line i) were ~1.8 µm, though FFT has no regularity.

  • Fig. 4 Comparison of T-tubule openings between the ventricular myocyte and the ventricular-type of the atrial myocyte. (A) Ventricular myocyte. (B) Ventricular-type of atrial myocytes. The comparison of the Z-groove ratio (C) and T-tubule opening size (D) between the two cells. The Z-groove ratio was calculated from the ratio of the actual Z-groove (dotted lines) to the total extrapolated Z-grooves (sum of dotted and solid lines). The opening area (µm2) was calculated from the measured diameter of the oval T-tubules orifice. **p < 0.01, ***p < 0.005.

  • Fig. 5 Relationship between the atrial surface structure and the intracellular T-tubule networks. The 3D scanning ion conductance microscopy (SICM), 2D SICM, and confocal plane images obtained from each subtype are presented (A: smooth-type, B: longitudinal fissures (LFs)-type, C: ventricular-type, and D: mixed-type). Confocal images of intracellular T-tubules and the surface SICM images were recorded from the same cell. A plane of di-8-ANEPPS confocal image (green) is overlaid with an optical transmission image, with an indication box of the scanning area (scale bar, 10 µm). The topographic line profiles are not presented in the figure. Arrowheads indicate the opening sites of T-tubules or LFs. Representative X–Y, X–Z, and Y–Z confocal plane images are presented for each subtype.

  • Fig. 6 Effect of formamide-induced detubulation on the structure of longitudinal fissures (LFs) and T-tubule openings. After formamide treatment of the cells, SICM images were obtained from the LFs-type (A) or the ventricular-type cells (B). The LFs remain intact after detubulation (A), but the T-tubule orifices are closed by detubulation in the ventricular-type (B). Arrowheads in each group indicate the LF openings and the Z-groove remnants, respectively. The line profile and Fast Fourier transformation (FFT) of the detubulated ventricular-type surface show regular positioning of Z-groove remnants at ~1.8 µm intervals (n = 6).

  • Fig. 7 Validation of formamide-induced detubulation in the atrial myocytes. (A) Atrial cells were stained with di-8-ANEPPS after formamide-induced detubulation procedure. Confocal and surface scanning ion conductance microscopy (SICM) images were taken from the same scanning areas of the cell. SICM images of the detubulated cell surface show the existence of longitudinal fissures (LFs). A plane of the di-8-ANEPPS confocal image is overlaid with an optical transmission image, with an indication box of the scanning area. Arrowheads in the confocal image indicate presumable LF membranes. Representative X–Y, X–Z, and Y–Z confocal plane images are presented. (B) Detubulation reduced the cell membrane capacitance (*p < 0.05). (C) Proportions of each subtype, classified by SICM surface structure, are compared between control and detubulation cells.

  • Fig. 8 The change of atrial action potential and L-type Ca 2+ channel (LTCC) current by detubulation. (A) The representative trace shows the shortening of atrial action potential (AP) by detubulation. (B) AP amplitudes are not changed by detubulation. (C) Change of action potential duration (APD)20, APD50, and APD90 by detubulation (*p < 0.05). Representative LTCC traces (D) and I-V curves (E) show the reduction of LTCC by detubulation.

  • Fig. 9 The change of atrial myocyte contraction by detubulation. (A) Representative contraction traces that evoked by varying electric field stimulation (EFS) frequencies. Sarcomere length (SL) shortening traces recorded at 1 Hz are overlaid to compare the change of contraction kinetics. (B) The decrease of SL shortening by detubulation. (C) Systolic time was not changed by detubulation. (D) At every frequency of EFS, half-relaxation time was significantly prolonged by detubulation. Control (n = 47). Detubulation (n = 48). *p < 0.05.


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