Cardiac telocytes in normal and diseased hearts.

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Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

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Review

Cardiac telocytes in normal and diseased hearts Sawa Kostin Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Ludwigstrasse 43, Germany

a r t i c l e

i n f o

Article history: Received 8 February 2016 Accepted 16 February 2016 Available online xxx Keywords: Telocytes Telopodes Heart failure Heart regeneration

a b s t r a c t Our previous studies suggested that an important variable of the progression of contractile dysfunction to terminal heart failure is the imbalance between myocyte cell death and myocyte renewal. For this reason, preventing myocyte cell death and an increasing generation of new myocytes may represent attractive targets in the treatment of human heart failure. Prospective clues to enhance myocardial regeneration are the newly discovered cells termed telocytes, formerly called interstitial Cajal-like cells, which are believed to nurse or guide the endogenous and exogenous stem cells for activation and commitment, but they also act as supporting cells for progenitor cells migration toward injured myocardium. We have recently found that telocytes are reduced in the diseased and failing myocardium. Importantly, the imbalance between telocyte proliferation and telocyte death is responsible for the telocytes depletion in cardiac diseases leading to heart failure. We have also demonstrated that telocytes are influenced by the extracellular matrix protein composition such that the telocytes are almost absent in areas of severe fibrosis. It is plausible that the reduction in telocytes in diseased human hearts could participate in the abnormal threedimensional spatial organization and disturbed intercellular signalling of the myocardium. Decreased telocytes in diseased hearts would also be predicted to alter the property of telocytes to maintain cardiac stem cell niche by decreasing the pool of cardiac stem cells and thereby impairing cardiac regeneration. © 2016 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4. 5. 6.

From the interstitial Cajal-like cells to telocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Identification of telocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. The ultrastructure and cellular compartments of telocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Phenotype(s) of telocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Differences between telocytes and other interstitial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Distribution and quantity of telocytes in the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Telocytes in cardiac stem cell niche and heart regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Telocytes in heart diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. From the interstitial Cajal-like cells to telocytes Originally, interstitial Cajal-like cells were named because of their apparent and similar morphological features with the gastrointestinal interstitial cells of Cajal, the gut pacemaker cells. The major reason was the identification of interstitial Cajal-like cells in

Abbreviation: TEM, transmission electron microscopy. E-mail address: [email protected]

a plenty extra-gastrointestinal organs [1–10], including the heart [11–17]. Numerous studies using electron microscopic examination revealed that the interstitial Cajal-like cells possess unique morphological characteristics and are totally different from other stromal cells. In addition, the electrophysiological properties of the interstitial Cajal-like cells differed substantially from the interstitial Cajal cells in the gut [18–24]. To avoid further confusion and to give a precise identity to interstitial Cajal-like cells (e.g., fibroblasts, fibrocytes, fibroblast-like cells, pericytes, mesenchymal cells and interstitial Cajal cells), Popescu LM and Faussone-Pellegrini MS

http://dx.doi.org/10.1016/j.semcdb.2016.02.023 1084-9521/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: S. Kostin, Cardiac telocytes in normal and diseased hearts, Semin Cell Dev Biol (2016), http://dx.doi.org/10.1016/j.semcdb.2016.02.023

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ARTICLE IN PRESS S. Kostin / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

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Fig. 1. TEM images of rat left ventricular myocardium. (A) Digitally coloured electron micrograph showing a typical cell body (telocyte, blue) in between two cardiomyocytes (CM). Note the sharp transition from cell body containing rough endoplasmic reticulum (rER) to long tortuous process (telopode). EC-endothelial cell, m—mitochondria. The circles indicate dilations of the telopode (podoms), which contains the so-called “Ca++ handling unit” composed of: one to two mitochondria, one to two elements of rER and one or a few caveolae. The black arrow indicates the thin segments of the telopodes called podomers. (B) A typical example of telopodes (arrows) forming a labyrinthine system and located in vicinity of endothelial cells (EC) and cardiomyocytes. The presence of caveolae (arrowheads) is a typical feature of telopodes and ECs. Note the cross-section of intermediate filaments and microtubules (mT, arrow) inside the telopodes. (C) Telopodes (arrows) located in close vicinity of EC. Cell-to-cell contacts of telopodes are indicated with circles. Note the presence of the basal lamina (BL, arrowhead), which can occasionally be observed in telopodes. (D) Higher magnification of the right part of (C). Reproduced with permission from Ref. [15].

have coined the term TELOCYTE for these cells and TELOPODES for their prolongations [25]. The name of telocytes (from the Greek prefix “telos,” meaning goal, end, or fulfillment) has been given owing to their major features characterized by their extremely long prolongations, called telopodes. Now this terminology is highly accepted by many investigators working in different research areas [26–42] and therefore, for the sake of simplicity and standardization, this nomenclature will be used in this review. 2. Identification of telocytes 2.1. The ultrastructure and cellular compartments of telocytes Transmission electron microscopy (TEM) examination is fundamental in identifying the telocytes. The ultrastructural features of telocytes comprise distinct cellular compartments: (1) cell body (the proper telocytes), (2) cellular prolongations (telopodes) and (3) the labyrinthine system made of telopodes. Fig. 1 shows a typical telocyte (cell body) characterized by a thin perinuclear rim of cytoplasm with few cytoplasmic organelles and thin cytoplasmic veils with mitochondria. Telopodes representing cellular prolongations of the telocytes, are unique and probably the longest structures in the body (except some axons). Telopodes are constituted by an alternation of dilated segments (podoms—which contain mainly mitochondria and caveolae) and thin segments (podomers). Fig. 1 shows some of the major distinct ultrastructural features of the telopodes including: (1) characteristic long, moniliform cell processes with a dichotomous branching pattern; (2) caveolae and coated vesicles; mitochondria, relatively well developed smooth and rough endoplasmic reticulum and (3) intermediate and thin filaments, microtubules and undetectable thick filaments. Another

distinctive ultrastructural feature of telopodes is the formation of labyrinthine apparatuses by three-dimensional convolution and cytoplasmic overlapping. Fig. 1B through Fig. 1D present several examples of labyrinthine systems of telopodes which are interconnected via cell-to-cell contacts, thereby generating a real cellular network in the entire heart. Fig. 2 presents the location of telocyte convoluted processes between cardiac myocytes and blood capillaries in human hearts. Moreover, Fig. 2B clearly shows that the tentacular telocytes and telopodes may surround (completely) other cardiac myocytes, or blood capillaries, or both. Hereby, telocytes and telopodes suggest the existence of a three-dimensional network. This impression is strengthened by comparing the bidimensional view of Figs. 1 and 2 with the scanning electron micrograph presented in Fig. 3. The three-dimensional image shows, unequivocally, the silhouette of a very long, slender bipolar telocytes, which has close contacts with at least two blood capillaries and at least four telocytes. In summary, the ultrastructural features of myocardial telocytes comprise: 1. Characteristic long (up to 100 ␮m), moniliform (with tortuous thin segments—up to 100 nm) cell processes (telopode) with a dichotomous branching pattern. 2. The telopodes comprise thin segments (podomers) alternating with dilated segments (podoms), which contain mitochondria, endoplasmic reticulum and caveolae. 3. Caveolae and coated vesicles. 4. Mitochondria and relatively well developed smooth and rough endoplasmic reticulum.




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Fig. 2. Electron micrographs of human atrial (A) and ventricular myocardium (B) showing the interstitial network of telocytes and their telopodes (digitally colored in blue). Many different types of nonmyocytes are present in atrial interstitium: telocytes fibroblast, blood vessel, Schwann cell and numerous nerve endings (n). Telopodes (Tp) of different telocytes are visible among the interstitial cells. Telopodes Tp1 and Tp2 enfold a group of working cardiomyocytes. Mention also that in comparison with telocytes, the fibroblast shows a large cell body with short (about 15 ␮m long) and thick processes and is reach in mitochondria and cisternae of rough endoplasmic reticulum (rER). Reproduced with permission from Ref. [58]. (B) Typical telopodes (Tp) forming a network in the narrow spaces between cardiomyocytes (CM) and surrounding blood capillaries (A and B). m—mitochondria, Z–Z band, ID—intercalated disc, RBC—red blood cell, E—endothelium. Reproduced with permission from Ref. [15].

5. 6. 7. 8. 9. 10.

Intermediate filaments (vimentin) and microtubules. Discontinuous basal lamina. An organized network (labyrinthic system). Cellular contacts with working myocytes. Close vicinity with capillaries and nerve fibres. Stromal synapses with other interstitial cells macrophages, mast cells, lymphocytes).

(e.g.,

Taken together, all these characteristic ultrastructural features of telocytes/telopodes, which are already established as a diagnostic panel, underscore and make these cells completely different

from other types of myocardial interstitial cells, especially from fibroblasts (Table 1). In addition to TEM, the focused ion beam scanning electron microscopy (FIB-SEM) tomography, which is a new and powerful technique, has recently further provided a three-dimensional reconstruction and a spatial view of human cardiac and skin telocytes [43,44]. This technique and three-dimensional imaging of human cardiac telocytes has confirmed a tortuous and convoluted course of cardiac telopodes and that the podoms are bulged from the podomer plane. 2.2. Phenotype(s) of telocytes

Fig. 3. Representative scanning electron micrograph of ventricular myocardium showing a typical telocyte (digitally coloured in blue) located across the cardiomyocytes. Another (possible) telocyte appears located along the cardiomyocytes (upper left). The three-dimensional vision reveals close interconnections of telocytes with cardiomyocytes and capillaries (cap); compare with Fig. 2. Note that telocyte processes (telopodes) begin from the cell body abruptly, as very thin prolongations (arrows); ID—intercalated disc. Arrowheads denote the opening of cardiomyocyte T-tubuli. Reproduced with permission from Ref. [15].

In adult hearts, telocytes were found to be variably positive for ckit/CD117, CD34, vimentin, and PDGFR-␤ [12,15,17,30,34,45–48]. It has recently been demonstrated that cardiac telocytes also express CD34/PDGFR-␣ [49]. Fig. 4A and B shows representative confocal images of c-kit-positive cells in human hearts. Telocytes in engineered heart tissue consistently express c-kit and CD34 [47]. Noteworthy, cardiac telocytes in primary culture positively express embryonic stem cell marker Nanog and myocardial stem cell marker Sca-1, indicative of pluripotent properties of telocytes [50] similar to adult lung telocytes [51]. It should be mentioned that double immunolabeling is recommended to make differential diagnosis of cardiac telocytes from other cells in primary culture or tissue sections [27,33]. For example, it has been shown that double immunofluorescent staining for c-kit/CD34, CD34/vimentin, CD34/PDGFR-␣ and CD34/PDGFR-␤ discriminates cardiac telocytes from fibroblasts, because the latter cells are only positive for vimentin and PDGFR-␤ [48]. It is noteworthy that the immunophenotype of telocytes varies with the organ and/or the animal species examined, possibly because of the existence of subpopulations of telocytes raising the possibility for the existence of organ- or tissue-specific telocytes. As an example, it has been shown that telocytes in human myometrium and fallopian tube express estrogen and progesterone receptors [8,52,53]. On the other hand, we have shown that telocytes in adult lung mouse tissue comprise Oct4-positive




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Table 1 Comparison of the characteristics of telocytes and fibroblasts. Feature

Telocytes

Fibroblasts

Cell body

Small; piriform/spindle/triangular/stellate shaped Small amount One, oval/rod-shaped Heterochromatin dominates Rarely visible

Pleomorphic (phenotypic heterogeneity)

Cytoplasm Nucleus Chromatin Nucleolus Organelles Golgi complex Mitochondria Endoplasmic reticulum (ER)

Membrane Caveolae Junctions Number of prolongations Branching Conformation Emergence from the cell body Length Podomers

Podoms

Gene expression profile (from Ref. [55])b Proteomic analysis (from Ref. [56]b

Telomerase activity (from Ref. [103])

Large amount One, oval Typically euchromatic 1–2 nucleoli

Small amount 2–5% of cell cytoplasm; present in podoms (not in podomers) Smooth or rough ER is located in podoms and occupies ∼2% of cell volume

Prominent ∼5%of cell cytoplasm Smooth ER virtually absent, but rough ER prominent (8–12% of cell volume), located mainly in cell body

Many Homo- and heterocellular junctions 2–5 telopodes Dichotomic pattern, forming 3D convoluted network(s) Overall moniliform aspect (alternating podoms and podomers) Thin Very long (tens, up to hundreds, of micrometers) Very thin (mostly 4000 downregulated in telocytes compared with mesenchymal stem cells and fibroblasts [55]. A recent comparative proteomic analysis revealed and confirmed that functionally, fibroblasts are mainly involved in the synthesis of collagen and other matrix components, while telocytes are oriented

to intercellular signaling, either by direct contact (junctions) with surrounding elements or at long distance by release of extracellular vesicles and stem cell niche modulation [56]. A particular and difficult issue pertains to the differentiation between telocytes and pericytes, especially with activated and migrating pericytes [57]. It should be noted that telocytes are CD34/c-kit-positive and ␣-SMA weak positive while pericytes are CD34-negative but ␣-SMA-positive. In addition, telocytes are CD34/PDGFR-␤-positive while pericytes were CD34-negative but PDGFR-␤-positive. These data indicate that telocyte are also different from pericytes. 3. Distribution and quantity of telocytes in the heart Growing evidence provide support to the concept that telocytes are not distributed uniformly in the heart. Although there no available quantitative data of telocytes comparing their number in atria with that in ventricles, it seems very likely that atria contain more telocytes than ventricles [11,12,58]. The distribution of telocytes/telopodes within the left ventricle is also distinct in different myocardial layers: subendocardially [59], intramyocardially, as cellular networks [12,15,26,30], and subepicardially, as accumulations of these cells in the loose connective tissue (cardiac stem cell niche) [16,17,45,49,60]. Intramyocardial telocytes represent less than 1% of interstitial cells in the human heart [61]. Despite a relatively low quantity, the intramyocardial telocytes contribute to form a complex three-dimensional network by interacting with cardiomyocytes and surrounding stromal cells, including endothelial cells, fibrob-




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Fig. 4. Deconvoluted confocal images and immunoelectron microscopy of c-kit positive cardiomyocytes in normal human myocardium. (A) Two telocytes located in close vicinity with cardiomyocytes. Arrows denote telopodes (Tps) originating from the cell body. Asterisks indicate labyrinthine systems. (B) Identical image as in panel A viewed in XY, XZ and YZ axes. Arrowhead points the very small rim of the perinuclear cytoplasm. Thin and long telopodes (Tp) are indicated with arrows and the asterisk indicates the labyrinthine system. Cardiomyocytes are stained red with phalloidin and nuclei are stained blue with DAPI. (C) Immunoelectron microscopy showing a c-kit-positive telocyte and numerous c-kit-positive telopodes (Tp, arrows) cut longitudinally or transversely. Arrowhead denotes that the telocyte cell body is positive for c-kit. CM—cardiomyocyte, EC—endothelial cell, Coll—collagen fibers. Panels A and B are reproduced with permission from Ref. [62].

lasts, macrophages, Schwann cells, pericytes, etc. (see Figs. 2–4). Our own quantitative analysis revealed that 1 mm2 of the normal human myocardium contains 14.9 ± 3.4 telocytes and 41.6 ± 5.9 telopodes [62]. The values of c-kit-positive telocytes found by Zhao et al. [63] in the rat heart ranged from 13.92 ± 2.8 telocytes in zones bordering myocardial infarctions to 44.69 ± 1.4 telocytes per 1 mm2 in the base part of control hearts. Moreover, a recent morphometric study of TEM images revealed 22 ± 2 TCs per 1 mm2 myocardial area of the neonatal human heart and 19 ± 3 telocytes per 1 mm2 myocardial area of the adult human heart [61]. Taken together, all these data obtained independently by other groups concur very close and presumably reflect the biologic reality. 4. Telocytes in cardiac stem cell niche and heart regeneration Our previous studies have demonstrated that myocyte cell death, apoptotic, oncotic or autophagic in nature, has to be regarded as a critical variable of the multifactorial events implicated in the alterations of cardiac anatomy and myocardial structure of the diseased human heart [64,65]. It has also been suggested that an

important variable of the progression of contractile dysfunction to terminal heart failure is the imbalance between myocyte cell death and myocyte renewal [66]. Adult differentiated cardiomyocytes have a limited ability to proliferate. Prospective clues to enhance myocardial regeneration are the telocytes owing to their properties to interact with cardiac stem cells [60,67]. Growing evidence indicate that telocytes “nurse” and guide stem cells in the subepicardially located stem cell niches [27,45,47,60]. Fig. 5A shows an example of transversally and obliquely oriented fibres displaying clusters or isolated round c-kit positive cells in human left ventricular myocardium as they appear in the cardiac stem cell niches [68,69]. Isolated c-kit-positive cells with long processes were also observed under epicardial mesothelial cells (Fig. 5B) and TEM shows that these cells are identifiable as telocytes (Fig. 5C). Using TEM, Popescu et al. [16] and Gherghiceanu and Popescu [60] have provided compelling evidence for the presence of putative cardiac stem cells and cardiomyocyte progenitors accompanied by telocytes and telopodes in subepicardially located cardiac stem cell niche. Fig. 5C provides electron micrographs and a schematic drawing illustrating the relation of telocytes with


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putative cardiac stem cells and cardiomyocyte progenitors and cardiomyoblasts in an epicardial stem cell niche of adult mouse. These results and the observations that telocytes and telopodes are in close contact with putative cardiac stem cells and immature cardiomyoblasts provide support for the hypothesis that telocytes and their telopodes may ‘guide’ and ‘nurse’ the myocardial precursors, thereby contributing to cardiac renewal and repair. The presence of nanocontacts and the influence via exosomes between telocytes and stem cells have been considered an important participation of telocytes in tissue regeneration [70–72], including the heart [47,60,73–75]. In addition, the in vitro time-lapse video microscopy further provided evidence that cardiac telocytes might guide and control the aggregation of immature cardiomyocytes [76]. Strong arguments in favor of such functions of telocytes are provided by comparative biology of the mammalian heart with fishes and amphibians. It is known that these species can fully regenerate their organs, including the heart. In these species, the heart is highly trabeculated. Importantly, trabecula-supporting cells are in fact telocytes, and fulfill all (ultra) structural criteria for telocytes. In these species, after amputation of the apex of the ventricle, the heart regenerates and the telocytes and telopodes are the first cells to be involved in this process, which is characterized by proliferation and reorganization of telocytes and telopodes to form

a three-dimensional network resulting in primitive trabeculae, which further direct and guide myocardial regeneration [26]. A close interaction between telocytes and stem cells exists not only structurally, as has again been elegantly shown in a co-culture system [67], but also functionally. Recently, the effect of cardiac telocyte secretome on cardiac stem cell fate has been assessed in vitro [77]. A higher level of IL-6, vascular endothelial growth factor (VEGF), macrophage inflammatory protein-1a (MIP-1␣), MIP-2, MCP-1, and some chemokines was identified in the supernatant of cardiac telocytes compared to cardiac stem cells. Moreover, co-culture of cardiac telocytes and cardiac stem cells resulted in further increased MIP-1␣ and MIP-2 level, while reduced IL-2 level compared with telocyte or cardiac stem cell mono-culture, indicating a cellular interaction via paracrine effects. It has been supposed that the increased telocyte secretome (IL-6, IL-6-type cytokines, and VEGF) might affect the proliferation and differentiation of cardiac stem cells. Therefore, the cardiac telocytes may synergistically act with cardiac stem cells to promote heart regeneration and repair via either direct contacts or paracrine mechanisms. Another mode of interaction between cardiac telocytes and stem cells has been demonstrated in a recent study using the Cy5-miR-21 oligos and calcein-labelled extracellular vesicles. It was revealed that cardiac telocytes and stem cells could shuttle microRNA via a bidirectional vesicular signalling mechanism [78]. Taken together, these studies strongly indicate that cardiac telocytes, via either direct contacts

Fig. 5. (A) Confocal microscopy of c-kit-positive cells in human left ventricular subepicardium. Three-dimensional shadow projection image of transversally and obliquely oriented fibres image showing niche-like clusters (yellow round marks) or isolated round c-kit-positive cells (arrow) in the subepicardial loose connective tissue. Note small green dots (arrowhead) which represent transversally sectioned telopodes of c-kit-positive cells located in close proximity to cardiomyocytes. F-actin is red stained with phalloidin conjugated with TRITC and nuclei are blue stained with DAPI. Reproduced with permission from Ref. [26]. (B) Confocal microscopy of a c-kit-positive cell (green) in human subepicardial area. Mesothelial cells are indicated with arrows. Cardiomyocytes are stained red with phalloidin conjugated with TRITC and nuclei are stained blue with DAPI. (C) TEM: one telocyte is located near the mesothelial cells of the same human epicardium. Panel A is reproduced with permission from Ref. [15]. Panels B and C are reproduced with permission from Ref. [45]. (D) Schematic drawing and electron micrograph illustrating the relation of several telocytes (blue) with a column of cardiomyocyte progenitors (CMP, brown) in an epicardial stem cell niche of adult mouse. The telopodes run parallel to the main axis of the cardiomyocyte progenitors column and seem to establish the direction of development. SV—shed vesicles, pSC—putative stem cells. Reproduced with permission from Ref. [102].




Fig. 6. Representative TEM of the left ventricular myocardium myocardium in a patient with dilated cardiomyopathy showing typical features of a telocyte undergoing apoptosis (boxed region). Note that apoptotic telocyte (arrow) is embedded in densely packed collagen fibres (Coll). Mention also that in comparison with a normal telocyte (Fig. 1A), the fibroblast (Fb) shows a large cell body with short and thick processes and is reach in mitochondria and cisternae of rough endoplasmic reticulum. The coloured box is a confocal image showing a telocyte (red) being TUNEL-positive (arrow). Cardiomyocytes are stained green with phalloidin and nuclei are stained blue with DAPI. Reproduced with permission from Ref. [62].

or paracrine mechanisms, may synergistically interact with cardiac stem cells to promote heart regeneration and repair. 5. Telocytes in heart diseases Heart failure is a common end-stage of many cardiovascular diseases and is associated with the pathological remodeling which is defined as “genome expression resulting in molecular, cellular and interstitial changes” [79]. According to this definition, extracellular matrix remodeling is an important component of myocardial remodeling and comprises changes in the quantity of stromal cells, imbalance between matrix metalloproteinase to tissue inhibitor of metalloproteinase and alterations of the collagen type I and type III metabolism [80,81]. Recently, we have investigated the role of telocytes in explanted human hearts with heart failure because of dilated, ischemic or inflammatory cardiomyopathy [62]. We have demonstrated that in diseased human hearts with heart failure telocytes and telopodes are reduced, and even absent in areas of severe fibrosis as a result of imbalance between telocyte proliferation and telocyte apoptotic death. Reduced numbers of telocytes have been documented in other cardiac diseases leading to heart failure including myocardial infarctions in rats [63], myocardial alterations in systemic sclerosis [82]. It has also been shown that the aging human heart is characterized by a gradual depletion of telocytes [61]. Significant reductions of telocytes have been found not only in diseased hearts, but also in other non-cardiac diseases: liver fibrosis [83], colonic wall in ulcerative colitis [84], the terminal ileum of patients affected by small bowel Crohn’s disease [85], gallstone disease [86], endometriosis [87], skin pathologies [82,88,89], renal ischemia/reperfusion injury [37] and different lung diseases [90]. Using TEM and TUNEL labeling, we have provided evidence for the occurrence of telocyte apoptosis in diseased human hearts (Fig. 6). The exact cause and mechanism of telocyte apoptosis in the heart remains unknown. However several micro-environmental factors may induce telopode retraction and telocyte death. Enciu and Popescu have found that that oxidative stress and aging

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substantially influence the formation of new telocytes and telopodes in culture conditions [91]. Numerous studies have clearly demonstrated that oxidative stress activates processes such as changes in gene expression and cell death that are now accepted components of myocardial remodeling and heart failure (reviewed in [92]). Therefore, it is likely that oxidative stress might also lead to telocyte loss. We have also found that another important factor which affects the number and distribution of cardiac telocytes is the composition of extracellular matrix. For example, we have found that in areas of replacement fibrosis or in zones with tightly packed fibrillar collagens, the telocytes and telopodes are significantly reduced or even absent. Similar observations have been documented in the scar tissue after myocardial infarction in rats [63]. In addition, our findings demonstrating that telocytes are influenced by the extracellular matrix protein composition are in good agreement with a recent in vitro study showing that the dynamics of cardiac telopodes are also influenced by the extracellular matrix proteins: the stronger telopode spreading being produced by fibronectin, while the lowest by laminin [93]. In this study, telocytes seeded on collagen determined the highest dynamics of telopode extensions [93]. Apart from telocyte apoptosis, we found that in heart failure telocytes displayed severe ultrastructural alterations in form of cytoplasmic vacuolization, absence of the labyrinthine components, shrinkage and shortening of the telopodes. Similar degenerative changes of telocytes including destruction and dissolution of telopodes have been documented in endometriosisaffected rat oviduct [87]. It is important to mention that telocyte degeneration and loss in the latter setting were associated with the local inflammation or ischemia micro-environment such as elevated levels of inducible nitric oxide synthase, cyclooxygenase-2 lipid peroxide and estradiol. The pathophysiological consequences of reduced telocytes observed in many heart diseases are largely unknown. It is, however, tempting to speculate that the reduction in telocytes in diseased human hearts could participate in the abnormal three-dimensional spatial organization and disturbed intercellular signalling of the myocardium as telocytes are considered to be as structurally connecting cells with other heart cells and given that telocytes are involved in intercellular signalling via homo- or heterocellular cell-to-cell contacts [58,94], shedding microvesicles, exosomes [95,96] or paracrine secretion including microRNAs [33]. Our preliminary data in diseased human hearts indicate decreased telocytes in the subepicardium suggesting that the property of telocytes to maintain cardiac stem cell niche is impaired thereby decreasing the pool of cardiac stem cells in heart failure [60,70,97]. This notion is supported by the observations that the number of cardiac stem cell is markedly reduced in diseased human hearts [98]. Moreover, the observations that telocyte transplantation after myocardial infarction is beneficial for functional regeneration of the infarcted myocardium, demonstrate the therapeutic utility of telocyte transplantation in diseased hearts [99]. Telocytes have also been proposed to be associated with amyloid deposit formation in patients with long-standing atrial fibrillation. In this process, telopodes play an important role by surrounding the amyloid deposits and showing a strong tendency to limit the amyloid fibrils to spread in the surrounding areas [100]. It is noteworthy to mention other protective functions of telocytes in cardiac diseases. For example, increased cardiac telocytes were detected in the border zone of the infarcted myocardium during the neo-angiogenesis phase after myocardial infarction [101]. It has been proposed that telocytes might contribute to neo-angiogenesis via paracrine secretion of pro-angiogenic factors, including several pro-angiogenic microRNAs and thus regulating the endothelial cells. Our preliminary data indicate increased numbers of telocytes in patients with compensated left ven-




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tricular hypertrophy suggesting the protective function of these cells to maintain the three-dimensional myocardial scaffold of the increased left ventricular wall thickness. 6. Conclusions All the data presented here strongly indicate that telocytes are distinct and unique interstitial cells in the heart. The golden standard technique for the identification of these cells still remains electron microscopy. It is hypothesized that telocytes have an integrative function supporting the three-dimensional organization of the cardiomyocytes, their physiology and their renewal. Telocyte cell tracing and telocyte ablation studies are needed to mechanistically address this hypothesis. A particular aspect of the data presented here is that telocytes are located together with cardiomyocyte progenitors in a particular environment—cardiac stem cell niche. According to the predicted functions of telocytes including progenitor, nurse, intercellular communicating and structurally organizer cells, the possible implications and therapeutic utilities of these cells are their regenerative and anti-remodelling potential in heart diseases. This is crucial given that telocytes are decreased in diseased and aging hearts. Competing interests The author declares that I have no competing interests. Acknowledgments I would like to thank Professor L.M. Popescu and his team for the constant support and fruitful long-term collaboration. I also thank for excellent technical assistance of Brigitte Matzke, Carmen Büttner, Beate Grohmann and Renate Möhren. References [1] L.M. Popescu, S.M. Ciontea, D. Cretoiu, M.E. Hinescu, E. Radu, N. Ionescu, et al., Novel type of interstitial cell (Cajal-like) in human fallopian tube, J. Cell. Mol. Med. 9 (2005) 479–523. [2] L.M. Popescu, M.E. Hinescu, E. Radu, S.M. Ciontea, D. Cretoiu, M. Leabu, CD117/c-kit positive interstitial (Cajal-like) cells in human pancreas, J. Cell. Mol. Med. 9 (2005) 738–739. [3] S.M. Ciontea, E. Radu, T. Regalia, L. Ceafalan, D. Cretoiu, M. Gherghiceanu, et al., C-kit immunopositive interstitial cells (Cajal-type) in human myometrium, J. Cell. Mol. Med. 9 (2005) 407–420. [4] M. Gherghiceanu, L.M. Popescu, Interstitial Cajal-like cells (ICLC) in human resting mammary gland stroma. Transmission electron microscope (TEM) identification, J. Cell. Mol. Med. 9 (2005) 893–910. [5] E. Radu, T. Regalia, L. Ceafalan, F. Andrei, D. Cretoiu, L.M. Popescu, Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture, J. Cell. Mol. Med. 9 (2005) 748–752. [6] D. Cretoiu, S.M. Ciontea, L.M. Popescu, L. Ceafalan, C. Ardeleanu, Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach, J. Cell. Mol. Med. 10 (2006) 789–795. [7] M.E. Hinescu, C. Ardeleanu, M. Gherghiceanu, L.M. Popescu, Interstitial Cajal-like cells in human gallbladder, J. Mol. Histol. 38 (2007) 275–284. [8] L.M. Popescu, S.M. Ciontea, D. Cretoiu, Interstitial Cajal-like cells in human uterus and fallopian tube, Ann. N. Y. Acad. Sci. 1101 (2007) 139–165. [9] L. Suciu, L.M. Popescu, M. Gherghiceanu, Human placenta: de visu demonstration of interstitial Cajal-like cells, J. Cell. Mol. Med. 11 (2007) 590–597. [10] M.E. Hinescu, L.M. Popescu, M. Gherghiceanu, M.S. Faussone-Pellegrini, Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach, J. Cell. Mol. Med. 12 (2008) 260–270. [11] M.E. Hinescu, M. Gherghiceanu, E. Mandache, S.M. Ciontea, L.M. Popescu, Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization, J. Cell. Mol. Med. 10 (2006) 243–257. [12] L.M. Popescu, M. Gherghiceanu, M.E. Hinescu, D. Cretoiu, L. Ceafalan, T. Regalia, Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC), J. Cell. Mol. Med. 10 (2006) 429–458. [13] M. Gherghiceanu, M.E. Hinescu, F. Andrei, E. Mandache, C.E. Macarie, M.S. Faussone-Pellegrini, Interstitial Cajal-like cells (ICLC) in myocardial sleeves of human pulmonary veins, J. Cell. Mol. Med. 12 (2008) 1777–1781. [14] M. Gherghiceanu, M.E. Hinescu, L.M. Popescu, Myocardial interstitial Cajal-like cells (ICLC) in caveolin-1 KO mice, J. Cell. Mol. Med. 13 (2009) 202–206.

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Post-extrasystolic potentiation without a compensatory pause in normal and diseased hearts.

Telocytes in cardiac regeneration and repair.

Vasa vasorum in normal and diseased arteries.

Arylamidases in normal and diseased human muscle.

Isolation, culture, purification and ultrastructural investigation of cardiac telocytes.

Reaching out: junctions between cardiac telocytes and cardiac stem cells in culture.

Electrophysiology of human cardiac atrial and ventricular telocytes.

RPE Cell and Sheet Properties in Normal and Diseased Eyes.

Sigma receptors [σRs]: biology in normal and diseased states.

Carcinoembryonic antigen in normal and diseased liver tissue.

Soluble proteins in normal and diseased human brain.

Expression of VCAM-1 in the normal and diseased kidney.

Human cardiac telocytes: 3D imaging by FIB-SEM tomography.

Intramyocardial transplantation of cardiac telocytes decreases myocardial infarction and improves post-infarcted cardiac function in rats.

Endothelial cell metabolism in normal and diseased vasculature.

Intracellular [Ca2+] in normal and diseased human myocardium.

Mineral density volume gradients in normal and diseased human tissues.

Guanylate cyclase activity in normal and diseased human muscle.

Pattern of electrical activity in normal and diseased muscle.

Anatomic correlates of normal and diseased adenoids in children.

Telocytes - a Hope for Cardiac Repair after Myocardial Infarction.

The noncontact tonometer. Clinical evaluation on normal and diseased eyes.

Radiologic evaluation of the normal and diseased posterior cervical space.

Dipyridamole-induced capillary growth in normal and hypertrophic hearts.