Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs

Figures

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  Figure 1. Illustration showing the domains associated with the intercalated disc. (A) The intercalated disc (ID) between cells predominantly accommodates three types of intercellular junctions: the adherens junction, the desmosome, and the gap junction (GJ). Thin filaments from the last sarcomere of the myofibrils insert in the adherens junction passing through a transitional junction (TrJ), which is rich in Z disc proteins (hence, an immature Z disc). At the edge of the GJ, there is an extracellular nanodomain, i.e., the perinexus (see (B) for details). See [6]. (B) Illustration showing the GJ and adjacent perinexus. The GJ comprises a cluster of Cx43 channels, docking within the narrow 2-nm gap. While the α-domain of Nav1.5 has a relatively scarce presence in the GJ, the sodium channels are enriched in the perinexus. In this case, the non-pore-forming β-subunit of Nav1.5 cross-links the two sides of the perinexus. Undocked Cx43 hemichannels are also expressed in the perinexus. See [8].

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  Figure 2. Precision analysis for the longitudinal position of the intercalated disc in the left ventricle of the mouse heart at rest in vivo. (A) Confocal image of connecting myocytes. The intercalated disc (ID) is shown by an arrow with a higher fluorescence signal than in T-tubules (cf. [24,25]). The membrane systems in the surface of the left ventricle were stained by CellMask, according to our previously reported procedure (see Materials and Methods). The longitudinal fluctuation of an ID section (width, 5 pixels (=1.05 μm)) in the yellow rectangle was analyzed under the arrested condition via deep anesthesia with >~5% isoflurane. Bar on the top right indicates 10 μm. (B) Time course of fluctuation of the longitudinal position of the ID section in (A). The SD of fluctuation was 83 nm during the course of observation (0.32 s). The heart in an open-chest mouse was illuminated using a laser at 532 nm. Imaging was performed at 34 frames/s.

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  Figure 3. Imaging of the intercalated disc in the CellMask-treated left ventricle of the living mouse heart in vivo. (A) Confocal images of the intercalated disc (ID) at varying time points. Cell 1 and Cell 2 are located on the left and right side of the ID, respectively. Number on the top left in each figure indicates the time point at which the image was obtained. The ID was traced by a yellow line. Vertical green line, cell width, horizontal blue line, deviation of the ID at the center from the mid-point of vertical green line (see Materials and Methods). The heart in an open-chest mouse was excited using a laser at 532 nm, and imaging was performed at 34 frames/s for 0.58 s. Bar on top right, 10 μm. (B) Time course of changes in the deviation distance for the ID (shown in blue). Deviation distance was defined as the length of the horizontal blue line in (A). The value became zero at 0.18 s. (C) Time course of changes in the ID length and cell width. Black line, ID length (total length of the yellow line in (A)); green line, cell width. Cell width represents the width of Cell 2 and Cell 1, respectively, before and after 0.18 s (at which deviation distance became zero, see (B)).

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  Figure 4. Precision analysis for the longitudinal position of the transitional junction in the resting mouse left ventricle. (A) In vivo confocal image of neighboring myocytes revealed transitional junctions (TrJ), which appeared as a singlet band with a high fluorescence signal (arrow, see also description in text). α-Actinin-AcGFP was expressed in myocytes in the surface of the left ventricle (LV) based on our previously reported procedure (see Materials and Methods). The longitudinal fluctuation of an TrJ section (width, 5 pixels (=1.05 μm)) in the yellow rectangle was analyzed under the arrested condition via deep anesthesia with >~5% isoflurane. Bar on the top right indicates 10 μm. (B) Time course of fluctuation of the longitudinal position of the TrJ section in (A). The SD of fluctuation was 64.5 nm during the course of observation (0.13 s). The heart in an open-chest mouse was excited using a laser at 488 nm. Imaging was performed at 99 frames/s.

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  Figure 5. Imaging of the transitional junction in the α-actinin-AcGFP-expressing left ventricle of the living mouse heart in vivo. (A) Confocal images of the transitional junction (TrJ) at varying time points. Cell 1 and Cell 2 are located on the left and right side of the TrJ, respectively. Number on bottom right in each figure indicates the time point at which the image was obtained. The TrJ in Cell 1 is shown by a yellow trace. Green line, vertical dimension of the TrJ. Horizontal blue line, deviation of the central part of the TrJ from the mid-point of vertical green line (see Materials and Methods). The heart in an open-chest mouse was illuminated using a laser at 488 nm, and imaging was performed at 99 frames/s. Bar on top right, 2 μm. See Movies S5 and S6. (B) Time course of changes in the deviation distance for the TrJ (shown in blue). Deviation distance was defined as the length of the horizontal blue line in (A). Changes in the deviation distance was very small (~0.8 μm) as compared to Figure 2B (i.e., CellMask treatment). (C) Time course of changes in the TrJ length and vertical dimension. Black line, TrJ length (total length of yellow line in (A)); green line, vertical…