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Figure 1. TEM analysis of the morphology of CTs in the Xenopus tropicalis heart. A, Schematic of the upper region, middle region and base of the X tropicalis heart for TEM analysis. B, Representative CTs with a hallmark ultrastructural morphology: a thin perinuclear rim of cytoplasm with few organelles and thin cytoplasmic veils containing mitochondria. Long telopodes (up to 100 μm), which represent cellular prolongations of the telocytes with moniliform (segments approximately 100 nm thick, named podoms) processes. C, A representative CT cell body (arrowhead: microvesicle). D, A representative telopode with podom (white arrow: podom; arrowhead: microvesicle). E, A representative telopode with many microvesicles (arrowhead) and secreted microvesicle (white arrowhead). F, A representative podom containing mitochondria. G, CTs in mitosis. CT: Cardiac telocyte; M: Cardiomyocyte; Mt: Mitochondria; N: Nucleus; Scale bar: Size as shown in the figures; Tp: Telopode
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Figure 2. TEM identified CTs in myocardium is câ€Kit–positive but vWFâ€negative. Double immuofluorescent staining for anti–câ€Kit (red) and vWF (green) demonstrated câ€Kit+ and vWF†cells with very small cell bodies (open arrow), a nucleus (approximately 1:1 ratio of the cytoplasm to the nucleus; white asterisk) and extremely thin prolongation (telopode) around trabeculae in the Xenopus tropicalis myocardium (white arrow). Showing that TEM identified CTs express câ€Kit, a generally accepted marker of CTs, but not vWF, a unique marker of endothelial cell. A, Anti–câ€Kit (red); B, Antiâ€vWF (green); C, DAPI; D, merged of A, B and C. A2, C2 and D2 are higher magnification of A1, C1 and D1, respectively, which are showing the cell body and part of telopode of CTs. Scale bar: 20 μm. Tr: Trabecula of the myocardium
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Figure 3. Distribution of CTs in the upper region, middle region and base of the Xenopus tropicalis myocardium. CTs mainly concentrated on the outer surface of trabeculae containing cardiomyocytes in the upper region (A), middle region (B) and base (C) of the X tropicalis myocardium. Most of the trabeculae are twined around one, two or several CTs and their telopodes or with telopodes alone. A′, B′ and C′: CTs of A, B and C that are not included in the trabecular structure; CT: Cardiac telocyte; Scale bar: Size as shown in the figures; Tr: Trabecula of the myocardium
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Figure 4. Vesicles and caveolae of CTs. Many vesicles (arrowhead) or coated vesicles (white arrowhead) are present in the telopodes. In addition, many caveolae are present in the membrane of the cell body and telopode (small triangle). The opening site of caveolae faces the extracellular space, and some vesicles or coated vesicles are located near the opening site of caveolae (Aâ€C). C: Collagen and extracellular matrix; CT: Cardiac telocyte; M: Cardiomyocyte; Mt: Mitochondria; N: Nucleus. Scale bar: Size as shown in the figures; Tp: Telopode
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Figure 5. Telopodeâ€telopode contact. Most of the CTs link with other CTs via connections between the far ends of their telopodes (Aâ€C). Two types of telopodeâ€telopode contacts are observed: (1) a gapâ€junction–like structure coming into nanometerâ€range contact, in which some areas make nanometerâ€range contact and other areas have a structure resembling one to two gap junctions (white arrow); (2) a nanometerâ€range contact that does not have a wellâ€established junction at the far end of the telopodes in which two telopodes closely connect at a distance of approximately 1â€2 nm (white small triangle). Scale bar: Size as shown in the figures. C: Collagen and extracellular matrix; M: Cardiomyocyte; Tp: Telopode
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Figure 6. TEM analysis of the Xenopus tropicalis myocardium 2 d after injury. A, Schematics of the region of TEM analysis. Two days after amputation, red blood cells (B) and inflammatory cells (C) accumulate in the wound. Myofibres are disorganized in some cardiomyocytes on the border of the wound (white triangle), and disorganized telopodes of some CTs (white asterisk) are found. There are some clot structures in the extracellular space (open triangle) (D). In addition, some network structures consisting of disorganized telopodes and extracellular matrix tissue but lacking cardiomyocytes are present in the wound area (E). Scale bar: Size as shown in the figures. Asterisk: Disorganized telopode; C: Collagen and extracellular matrix; CT: Cardiac telocyte; M: Cardiomyocyte; Open triangle: Clot structure; R: Red blood cell; Tp: Telopode; W: Inflammatory cell; White Triangle: Disorganized myofibre
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Figure 7. TEM analysis of the Xenopus tropicalis myocardium 8 d after injury. Eight days after injury, some of the injured muscle fibres regenerated via a novel muscle fibre characterized by an irregular muscle fibril arrangement and irregular sarcomere (RM) as well as regenerated sarcolemma (open arrow). An accumulation of mitochondria is seen in the regenerated muscle fibres (A, B), and a karyokinesisâ€like nucleus was found in the border cardiomyocytes of regenerated myofibres (C). In addition, CTs with normal morphology are found on the outer surface of regenerated myofibres (A, B). B: Higherâ€power view of the dotted line rectangle in A. Scale bar: Size as shown in the figures. Asterisk: Cardiomyocyte; CT: Cardiac telocyte; Mt: Mitochondria; N: Nucleus; RM: Regenerated myofibres; Tp: Telopode; W: Inflammatory cell
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Fig. S1: Three-dimensional reconstruction of Figure 2D. A: Three-dimensional reconstruction of Figure 2 in Z-axis. B: 45 degree rotation of A in y-axis. C: 135 degree rotation of A in y-axis. D: 180 degree rotation of A in y-axis. Showing CTs (c-Kit positive) is around the trabeculae of X. tropicalis myocardium (white arrow) and the representative cell body (open arrow) and part of telopode of CTs (white arrow) in higher magnification (A2-D2) of selected area (white dot line rectangle; A1-D1) respectively. Red: anti-c-Kit; Green: anti-vWF, Blue: DAPI. Tr: Trabecula of the myocardium.
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Fig. S2: TEM identified CTs in myocardium is c-Kit positive but CD31 negative. Double immunofluorescent staining for anti-c-Kit (red) and CD31 (green) demonstrated c-Kit+ and CD31- cells with very small cell bodies (open arrow), a nucleus (approximately 1:1 ratio of the cytoplasm to the nucleus; white asterisk) and extremely then prolongation (telopode) around trabeculae in the X. tropicalis myocardium (white arrow). Showing that TEM identified CTs express c-Kit, a generally accepted marker of CTs, but not CD31, a unique marker of endothelial cell. A: Anti-c-Kit (red); B: Anti-CD31 (green); C: DAPI; D: merged of A,B and C. Scale bar: 20 micro meters. Tr: Trabecula of the myocardium.
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Fig. S3: Three-dimensional reconstruction of Figure S2D. A: Three-dimensional reconstruction of Figure S2 in Z-axis. B: 45 degree rotation of A in y-axis. C: 135 degree rotation of A in y-axis. D: 180 degree rotation of A in y-axis. Showing CTs (c-Kit positive) is around the trabeculae of X. tropicalis myocardium (white arrow) and the representative cell body (open arrow) and part of telopode of CTs (white arrow) in higher magnification (A2-D2) of selected area (white dot line rectangle; A1-D1) respectively. Red: anti-c-Kit; Green: anti-vWF; Blue: DAPI. Tr: Trabecula of the myocardium.
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Fig. S4: Distribution of CTs among trabeculae in the X. tropicalis myocardium. Among the trabeculae, CTs twined around the outer surface are able to connect using their telopodes in the upper region (A), middle region (B) and base (C). Scale bar: Size as shown in the figures. CT: Cardiac telocyte. Tr: Trabecula of myocardium.
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Fig. S5: Contact between CTs and cardiomyocytes. The CT cell body does not contact or form a junction with cardiomyocytes. Many microfilaments that are arranged as a vertical and horizontal network with collagen fill the gap to link the CT cell body with cardiomyocytes (A-F). In addition, the cell body of one CT is able to closely connect with the cell body of another CT (D), and the cell body of one CT is able to form a nanometer-range connection with the telopode of another CT (E,F; circle). Distinct from contacts between the far ends of telopodes from different CTs, two telopodes from different CTs are able to form a nanometer-range connection via a gap-juction-like structure (E, F; dotted line circle). Scale bar: Size as shown in the figures. CT: Cardiac telocyte. C: Collagen and extracellular matrix. Mt: Mitochondria. M: Cardiomyocyte. Tp: Telopode.
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Fig. S6: Contact between CT telopodes and cardiomyocytes. The telopodes of CTs do not directly contact or form a junction with cardiomyocytes (A-C). Many microfilaments that are arranged as a vertical and horizontal network with collagen fill the gap to link the telopodes with cardiomyocytes (A-C). Many vesicles (arrow head) are present in the telopodes. Scale bar: Size as shown in the figures. C: Collagen and extracellular matrix. Mt: Mitochondria. M: Cardiomyocyte. Tp: Telopode.
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Fig. S7: Vesicles and caveolae of CTs and cardiomyocytes. Some vesicles are present under membrane of the cardiomyocyte (A; arrow head). Many single vesicles are concentrated in the podoms of the telopode (B). In addition, many caveolae are present in the membrane of the cell body and the telopodes (A-C; small triangle). The opening site of caveolae faces the extracellular space, and some vesicles or coated vesicles (C; open arrow) are located around the opening site of caveolae. Scale bar: Size as shown in the figures. C: Collagen and extracellular matrix. M: Cardiomyocyte. Tp: Telopode.
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