Cardiac telocytes exist in the adult Xenopus tropicalis heart

Abstract Recent research has revealed that cardiac telocytes (CTs) play an important role in cardiac physiopathology and the regeneration of injured myocardium. Recently, we reported that the adult Xenopus tropicalis heart can regenerate perfectly in a nearly scar‐free manner after injury via apical resection. However, whether telocytes exist in the X tropicalis heart and are affected in the regeneration of injured X tropicalis myocardium is still unknown. The present ultrastructural and immunofluorescent double staining results clearly showed that CTs exist in the X tropicalis myocardium. CTs in the X tropicalis myocardium were mainly twined around the surface of cardiomyocyte trabeculae and linked via nanocontacts between the ends of the telopodes, forming a three‐dimensional network. CTs might play a role in the regeneration of injured myocardium.


| BACKG ROU N D
Regeneration of the damaged mammalian myocardium is a major challenge in clinical settings. After cardiac injury, such as myocardial infarction (MI), adult humans and non-human mammals show very limited regenerative ability to replace the lost cardiomyocytes, as adult mammalian cardiomyocytes have very low capacity for cell proliferation and division. Necrotic cardiomyocytes are replaced with scar tissue, impairing the contractility of the remaining myocardium and even resulting in heart failure and death if the damage is severe. 1 Thus, regeneration of the damaged myocardium is pursued as a therapeutic goal.
Recent studies have revealed that stromal cells communicate responsiveness to physiopathological stimuli through continuous bidirectional crosstalk between cardiomyocytes and noncardiomyocytes, such as cardiac fibroblasts, endothelial cells and cardiac telocytes (CTs), which act as 'cardiovascular units' (CVUs) and functional and structural building blocks of the heart to maintain the integrity of myocardial function. 2-7 During development and under physiopathological conditions, cardiac stromal cells and endothelial cells control the proliferation, growth and differentiation of cardiomyocytes in the myocardium. [8][9][10] One important discovery is the identification of a novel type of stromal cell named telocytes, which are found in humans and rodents in the interstitium of the heart, 7,11-17 skeletal muscle, 18 trachea and lung, 19,20 intestine, 21 uterus and fallopian tubes, 22 placenta 23 and mammary glands 24 and in the interstitium of the leech Hirudo medicinalis. 25 CTs were found to be niche supporting cells that nurse cardiac stem cells and other cardiac cells in the myocardium and play an important role in regeneration following MI. 26 Recently, we reported that the death of CTs is an important mechanism that contributes to the structural damage and poor healing and regeneration observed in MI. [27][28][29] This evidence reveals that CTs provide a unique structural and functional microenvironment for maintaining the integrity of the myocardium and the regeneration of damaged myocardium.
Lower vertebrates, such as newts and zebrafish, display an extraordinary ability of cardiac tissue regeneration. [30][31][32] Among anurans (frogs and toads), it is known that frog tadpoles can regenerate their tails, 33 and adult Xenopus has a high capacity for retinal regeneration. 34,35 Recently, we reported for the first time that the adult Xenopus tropicalis heart can regenerate perfectly in a nearly scar-free manner after injury via apical resection. 36 However, whether telocytes exist in the X tropicalis heart and are affected in the regeneration of injured X tropicalis myocardium is still unknown. This study is designed to investigate this intriguing issue.

| Experimental animals
Xenopus tropicalis frogs (Nigerian strain) were purchased from NASCO (USA) and maintained in a freshwater tank at 26°C under a 12-hour/12-hour light/dark cycle. All the experimental protocols related to X tropicalis were approved by the Jinan University Animal Care Committee.

| Collection of Xenopus tropicalis heart
Xenopus tropicalis frogs (4 females; 12 months old) were used in the present study. Representative sections of the upper region, middle region and base of the individual ventricles ( Figure 1A) were collected for transmission electron microscopy (TEM) analysis.

| Apical resection of the Xenopus tropicalis heart
Apical resection of the X tropicalis heart was performed based on our recently established protocol. 36 Briefly, X tropicalis frogs were placed in a tricaine methanesulfonate (MS-222; 1 mg/mL; TCI) bath that was prepared with sterile double-distilled water at room temperature for 4 minutes, incubated on ice for 60 seconds and then positioned ventral side up on an ice pad. The skin of the chest and upper abdomen was sterilized with iodine and 75% alcohol. A small incision was made near the heart using ophthalmic scissors. The pericardial sac was then opened, and the ventricle was exposed.
Approximately 10% (approximately 1 mm in length) of the ventricle tissue from the cardiac apex was resected with Vannas scissors ( Figure 6A). The opened cavity was sutured with 4-0 suture after amputation. The animals were subsequently transferred to and maintained in freshwater at 26°C. The injured hearts were collected at 2 or 8 days after apical resection (daar). A cross-section (approximately 1.5 mm) that included the wound zone was collected at 2 or 8 daar for TEM ( Figure 6A).

| Transmission electron microscopy
The samples from cross-sections of X tropicalis heart were fixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 30 minutes at room temperature. After washing in PBS (pH 7.2) for 5 minutes at room temperature, specimens were immersed overnight in 0.1% osmium tetroxide in PBS at room temperature and then processed for TEM observation.

| Semiquantitative analysis of CTs
ImageJ version 1.48 was used to measure and analyse the CT cell bodies, telopodes, podoms, contacts, vesicles and caveolae. The longest and shortest diameters of vesicles, caveolae, CT cell bodies, CT nuclei and CT podoms; gaps between a CT and a cardiomyocyte; and gaps between a telopode and a cardiomyocyte were measured.
The values are presented as the means ± standard deviation (SD).
The counting numbers for all above-observed parameters are listed in Tables S1 and S2.

| Immunohistochemistry for CTs
The cryo-section of the collected X tropicalis hearts (8 μm) was kept at room temperature for 30 minutes, washed for three times with PBS (pH = 7.4; each for 3 minutes) and then post-fixed with 4% Paraformaldehyde for 30 minutes. After three wash with PBS, the sections were permeabilized and blocked with PBS containing 0.5% Triton X-100 and 1% bovine serum albumin (BSA) at room temperature for 60 minutes. The sections were then successively incubated overnight at 4°C with a combination of the following antibodies: goat anti-c-Kit F I G U R E 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).  This confirms that the identified CTs by TEM were not endothelial cells.

| Distribution and spatial organization of CTs in the Xenopus tropicalis myocardium
CTs are mainly located on the outer surface of the trabeculae in the X tropicalis myocardium. With their cell body and telopodes, CTs closely connect with cardiomyocytes included in the trabeculae.
Most of the trabeculae in the myocardium are twined around one to several CTs and their telopodes or around telopodes alone ( Figure 3; Figure S4). In a given trabecula and among all trabeculae, CTs twined around the outer surface are able to connect using their telopodes ( Figure 3C; Figure S4). These unique characteristics of distribution are similar among the upper region, middle region and base of the X tropicalis myocardium. Taking together the above evidence of the characteristic distribution of CTs in the three-dimensional view indicates that CTs appear to be twined around the surface of the trabeculae and to be linked together as a three-dimensional network in the X tropicalis myocardium (Figure 3; Figure S4).

| Contacts between CT cell bodies and cardiomyocytes
The cell bodies of CTs did not contact or form a junction with cardiomyocytes. The average longest gap between the CT cell bodies and cardiomyocytes was 1.05 ± 0.78 μm, while the average smallest gap was 0.21 ± 0.20 μm (Table S2). Many microfilaments, arranged in a vertical and horizontal network with collagen, fill the gaps to link the CT cell bodies and cardiomyocytes ( Figure S5A-D).

| Contacts between CT telopodes and cardiomyocytes
Similar to the CT cell body, the telopodes of the CTs did not directly contact or form a junction with cardiomyocytes ( Figure S6). The mean longest gap between CT telopodes and cardiomyocytes was 1.59 ± 1.40 μm, while the mean smallest gap was 0.16 ± 0.26 μm (Table S2). Similarly, many microfilaments, arranged in a vertical and horizontal network with collagen, fill the gaps to link the CT telopodes and cardiomyocytes ( Figure S6). In addition, there are many vesicles or coated vesicles in the telopodes and the gaps between long CT telopodes and cardiomyocytes (Figure 4).

| Contacts among CT telopodes
Most CTs linked with other CTs via the far ends of their telopodes.
Two types of telopode-telopode contacts were observed: (a) a gapjunction-like structure coming into nanometer-range contact, in which some areas are in nanometer-range contact and other areas have a structure resembling one or two gap junctions ( Figure 5A);

| Contact between the CT cell body and the telopodes of other CTs
The CT cell body was able to make nanometer-range contact with the telopodes of other CTs ( Figure S5E,F). Distinct from the contacts between the far ends of telopodes from different CTs, telopodes from different CTs are able to form a nanometer-range connection via a gap-junction-like structure ( Figure S5E,F).

| Vesicles and caveolae of CTs
In the scarce cytoplasm of the CTs, some vesicles are present ( Figure 1C; Figure 4A).  (Table S2). The single vesicles are distributed along the telopode (Figure 4; Figure S6A,B), while many single vesicles and most of the coated vesicles are concentrated in the podoms of the telopode (Figures 1D and 4B,C). In addition, many caveolae are present in the membrane of the cell body and telopodes.  (Figure 4; Figure S7). In addition, the average diameter of the caveolae was 57.14 ± 18.75 nm (Table S2).

| CTs recover more quickly than cardiomyocytes in the injured myocardium
To investigate whether CTs are affected in the injured X tropicalis myocardium, approximately 10% of the apex was amputated, and the wound site was observed using TEM at 2 and 8 days. At 2 days, red blood cells ( Figure 6B) and inflammatory cells ( Figure 6C) accumulated in the wound. Myofibre disorganization was found in the border area of the wound ( Figure 6D,E), and disorganized telopodes were found in some CTs ( Figure 6D). Some clot structures were found in the extracellular space between the cardiomyocytes and CTs ( Figure 6D). In addition, the wound area contained some network structures that consisted of disorganized telopodes and extracellular matrix tissue but lacked cardiomyocytes ( Figure 6E). At 8 days, some injured muscle fibres regenerated via a novel muscle fibre characterized by an irregular muscle fibril arrangement and irregular sarcomeres as well as regenerated sarcolemma ( Figure 7A,B).
An accumulation of mitochondria was found in the regenerated muscle fibres, and a karyokinesis-like nucleus was found in the border cardiomyocytes of regenerated myofibres (Figure 7). In contrast, CTs with normal morphology were found on the outer surface of regenerated myofibres ( Figure 7A,B). All these findings suggested that 8 days after the cardiac resection, destructed CTs in the damaged myocardium were recovered, while the cardiomyocyte regeneration was not yet complete, the reconstruction of the CT network in the wound area might be an important step for initiating and maintaining the regeneration of injured myocardium and that mitochondria accumulation is needed in the regeneration of injured myocardium.

| D ISCUSS I ON
In the present study, we first showed that cardiac telocytes exist in the adult X tropicalis heart using TEM according to the presence

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
LL, ZL, JL, HC, HG and JY performed most of the experiments and analysed data; RH, QP, HZ, ZY, SF and XQ contributed to discussion and manuscript writing; DC conceived and designed this work and wrote the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.