The Molecular And Functional Specialization Of The Cardiac Telocytes And Their Implications In Heart Biology

Recent investigations identified a novel population of interstitial cells, which projected long (tens to hundreds of um) and thin (0.1-0.5um in diameter) filaments. They were thus named as telocytes (TCs) and their filaments as telopodes (TPs). To date, colonies of TCs have been identified from multiple mammalian tissues, including the heart. Through their TPs, TCs interacts with other cardiac cells, capillaries and nerve endings; collectively, they cultivated an integrated interstitial system that was essential for cardiac renewal, regeneration and repair. Multiple aspects of such functional coordination are being explored, such as regulation on cardiomyocyte (CM) progenitors in the epicardial stem cell niches, and angiogenesis in the experimental myocardial infarction. However, a systematic examination of these mechanisms requires purified CTCs from the heart.In addition to their signature contour, a panel of unique molecular markers has been identified in CTCs. Accordingly, we established a procedure to successfully isolate CTCs by targeting cell surface proteins CD34and CD117with flow cytometry. Subsequently, we conducted an array of electrophysiological, biochemical and proteomic analyses to define the fundamental attributes of these cells. Results also demonstrated that CTCs exhibited a pro-mitogenic effect on CMECs via paracrine and autocrine pathways. These discoveries afforded new prospective to comprehend the dynamics of angiogenesis in cardiac physiology and protection.Part OneIsolation, Cultivation, Purification and Identification of Cardiac Telocytes and Medium Optimization for Cardiac TelocytesObjective Purification of cardiac telocytes and optimal medium for cardiac telocytesMethod Cardiac cells in situ of6-week male C57BL/6J mice were digested and purified by flow cytometry, targeting both CD34and CD117positive, following gradient centrifugal, adhere to growth method and primary cell culture. And then, the expression of Vimentin and the characteristic morphology of telocytes were observed by laser scanning confocal microscope and common microscope, respectively. Four media were evaluated for the efficacy to culture CTCs by CCK-8:(1)low glucose-DMEM with10%FBS;(2) low glucose-DMEM with20%FBS;(3) high glucose-DMEM with10%FBS;(4) high glucose-DMEM with20%FBS.Result Isolated CTCs were cultured to evaluate the efficiency of our purification. The third passage of CTCs was inspected via fluorescence-activated cell sorting, targeting FITC-anti CD34and PE-anti CD117antibodies, with CD34-/CD117"24.0%, CD34+/CD117-7.4%, CD34-/CD117+4.6%, CD34+/CD117+64%. And the morphological structure of CD34+/CD117+cells were observed by common microscope, which was consistent with the signature filamentous TPs of CTCs, and they were also positive for vimentin. The highest proliferation potency was recorded with high glucose and10%FBS,which was adapted to support all the following studies.Conclusion CD34+/CD117+cells were accordance with the characteristic structure of CTCs and Vimentin expression, following primary cell culture.And the optimal medium for telocytes was DMEM with high glucose and10%FBS. Part TwoThe Proliferation of Cardiac Telocytes, the Development of Telopodes and analysis of telomerase activity of cardiac telocytesObjective The time-dependent observation of the proliferation of telocytes and growth process of telopodes.Method Heart tissues were excised from the6-week old male C57BL/6J mice. CD34/CD117positive telocytes were selected out from the primary cardiac cells by flow cytometry, and transferred into a plastic culture dish. And then the proliferation of telocytes and growth process of telopodes were monitored by using Cell Imaging System. Images were captured every20min, last for96h. The video signal was digitally processed and the video was produced at1392×1040pixels in size. Telomerase activity among CTCs, BMSCs, CFBs, and CMs were detected by Real-Time quantitative telomeric-repeat amplification protocol assay, respectively.Result The developmental process of telocytes division and the growth process of telopodes were monitored by cell imagining system. CTCs entered interphase within12hours in culture, and then committed to mitosis at48hours, with binuclear and invagination of the body. TPs gradually emerged as the daughter CTCs migrated away from each other at56h. At the period of telocytes attachment from16h to24h, the characteristic TPs could detach from cell body and grow alternatively, leaving behind scattered podomers and podoms. The telomerase concentrations were measured at0.0668,0.1637,0.0974, and0.0006amoles/uL, respectively.Conclusion Telocytes with longitudinal splitting and a relatively low telomerase activity grow slow, and telopodes detach from cell body and grow alternatively during cell growth process, leaving behind scattered podomers and podoms. Part ThreeElectrophysiological Properties of Cardiac Telocytes and Significance of Whole-cell Proteomics of Cardiac TelocytesObjective Investigation of the electrophysiological properties of cardiac telocytes and function trend of whole-well comparative proteomics of cardiac telocytesMethod Heart tissues were excised from the6-week old male C57BL/6J mice. CD34/CD117positive telocytes were selected out from the primary cardiac cells by flow cytometry, and transferred into Glass Bottom Dish, and Na+, K+, Ca2+channel expression were observed by laser scanning confocal microscope; CTCs were incubated on small round glass covers. The recording chamber was perfused with an oxygenated solution (140mmol/L NaCl,5mmol/L KC1,2mmol/L CaCl2,2mmol/L MgCl2,10mmol/L HEPES,12mmol/L glucose,2mmol/L CoCl2,2mmol/L4-AP,2mmol/L BaCl2, pH7.4) at a rate of1.5mL/min. The electric resistance of CTCs was measured with a borosilicate glass pipette filled with a measurement solution (130mmol/L K-aspartate,6mmol/L NaCl,10mmol/L HEPES,2mmol/L MgCl2,14mmol/L EGTA,2mmol/L CaCI2,5mmol/L Na2ATP, pH7.4). With the holding potential of-40mV, whole-cell currents were measured using voltage pulses in the range of-110to+10mV (in steps of20-mV). The extracted proteins from CTCs, CSCs, CMECs, and CNCCs were pooled respectively. Peptides were detected by isobaric tags for relative and absolute quantification and proteins were indentified. The function trend of whole-well cardiac telocytes was conjectured by comparative proteomics among CTCs, CSCs, CMECs, and CNCCs.Result Na+, K+, Ca2+channel positive expression were observed by laser scanning confocal microscope, and CTCs with the infusion of Fura-2were observed under a fluorescence microscope, with high expression on the cell body and low expression on podomers and podoms. The average electrical capacitance was22.53pF and the average electrical resistance was15.65GΩ, however, no current was recorded. Compared to CTCs,16,32and90proteins were found to be differentially expressed in CMECs, CSCs and CNCCs, respectively. Proteins of CTCs were up-regulated:cellular component morphogenesis (K2C7, CTRO),intracellular protein transport (SNAG, FLOT2, GBLP), and metabolic process (GLO2, VPS4B); nuclear mRNA splicing (SFRS5), metabolic process (NOL5A, IDH3A), immune system process (SAA3) and transport (FABP4); cell motion (MYH9, ODFP2), cellular component morphogenesis (MYH9, LMNA, SPTA2, K1C13), intracellular protein transport (RAB31, CHMP3, ARF3, NIPS1), cellular amino acid biosynthetic and metabolic process (AL4A1, SCLY, AATM, MMSA, C1TC, GLSL, PROD2), immune system process (SODM, CATA, SBP1, MOSC2, GSTM1, PRDX6, THTR, DNJA3, BPHL, PPIA, EST31, GST01), protein assembly metabolic process (TCPQ, CH10, TCPB, UK114, CALR, TCPZ, TCPG), respiratory electron transport chain and tricarboxylic acid cycle (QCR1, ETFD, MDHC, ODO1, SUCB2, CP2CT).Conclusion Na+, K+, Ca2+channel expression of cardiac telocytes were positive, however, no current was recorded, which suggested that electric signal transduction did not exist between cardiac telocytes and other cells. Whole-well comparative proteomics indicated that cellular biology process including component morphogenesis and so on were high expression of cardiac telocytes. Part FourThe Spatial Distribution of Cardiac Telocytes and the Proliferative Effect of Cardiac Telocytes on Microvascular Endothelial Cells Objective Observation of spatial relationship between cardiac telocytes and cardiac microvascular endothelial cells, and study the proliferation influence of cardiac telocytes on cardiac microvascular endothelial cellsMethod The hearts from male C57BL/6J mouse (6weeks) were harvested, cut into small pieces about1mm3, and then the tissue mass were fixed in4%glutaraldehyde (pH7.3) at4℃for4h, followed by washed, dehydrated, impregnated and embedded. Ultrathin section70nm was cut by a Leica LKB-Ⅱ. Sections were collected on Formvar-coated copper grids, stained with uranyl acetate and lead citrate, and imaged under transmission electron microscope. Cardiac telocytes and cardiac microvascular endothelial cells were isolated, respectively, and then co-cultured by transwell technology. Cell cycles of co-culture group and control group were investigated with flow cytometry after staining with propidium iodide.Result under transmission electron microscope, the presence of a CTC with characteristic a TP was in the stromal space of heart. And among local cellular population, the presence of a TP of a CTC was in close ties with the location of CMECs. A CTC enveloped the wall of capillary and run between muscle bundle and capillary.15.3%+11.7%of cardiac microvascular endothelial cells in co-culture group were at S+G2/M phases, whereas,11.8%+5.56%in control group.Conclusion The spatial relationship between cardiac telocytes and cardiac microvascular endothelial cells was very important, and paracrine of cardiac telocytes could improve the proliferation of cardiac microvascular endothelial cells significantly. Part FiveFunctional Analysis of Paracrine Factors of Cardiac Telocytes and the Improvement of Autocrine of Microvascular Endothelial CellsObjective Quantitative detection of cytokine secretion of cardiac telocytes, analysis of its biology functions and study VEGF autocrine of cardiac microvascular endothelial cells co-existed with cardiac telocytesMethod Heart tissues were excised from the6-week old male C57BL/6J mice. CD34/CD117positive telocytes were selected out from the primary cardiac cells by flow cytometry, and transferred into a6-hole plate. Culture supernatant (the serum free and antibiotic free DMEM) of CTCs was collected at different time (0,6,12,24,48h). Mouse Cytokine Antibody Array2000was applied to quantitatively detect the concentration of120Mouse cytokines. Cytokines with total concentration exceeded100pg/ml at48h were selected and corresponding biological functions were investigated. VEGF Mouse Elisa Kit was used for quantitatively detecting VEGF secreted by CMECs at different time points (2,6,12,24,48,72h) by enzyme-linked immunosorbent assay, following cell culture in the serum free and antibiotic free DMEM for72h.Result36cytokines with total concentration exceeded100pg/ml at48hr were selected and corresponding biological functions:immune response, inflammatory response, signal transduction, cell-cell signaling, chemotaxis, cell adhesion, poptosis, cell proliferation, development, and cell surface receptor linked signal transduction, were analyzed on the basis of each selected cytokine, with1/3of cytokines related to cell development. The concentration of VEGF secreted by CMECs co-existed with CTCs group was higher than that secreted by CMECs group at all observation time points, and there were obvious difference between two groups at48h and72h.Conclusion Cytokines secreted by cardiac telocytes participated in biological functions, with cell development dominating primary element. And paracrine of cardiac telocytes could improve the VEGF autocrine of cardiac microvascular endothelial cells directly.