| Objective:(1)To observe the ultrastructure of cardiac telocytes in old rat heart using the transmission electron microscope(TEM);(2)To investigate the distribution of CTs in aged heart using immunofluorescent staining,and observe the age-related change of CTs;(3)To analyze the mi RNA profile of cardiac telocyte exosomes under hypoxia and their possible biofunctions,signal pathways,regulatory networks using small RNA sequencing and Ingenuity Pathway Analysis software.Methods:(1)The female SD old rat hearts was imbedded for TEM and was imaged for the representation sections from the base,the medium and the atrium-atria part.The Image J software was used to measure the cell body area?nucleus area?telopodes length and podoms diameter of cardiac Telocytes;(2)The female SD old rat hearts was used for paraffin imbedded,and the representative sections from the base,the medium,and the atrium-atria of the heart were selected for immunofluorescence staining with c-kit antibody(marker for CTs).The c-kit positive with unique morphology of CTs were applied as standard to identify the distribution of CTs in the old rat heart and compared with the young rat studied in our previous research to investigate the age-related change of CTs;(3)The CTs isolated by anti-c-kit magnetic bead were cultured under hypoxia condition(5%O2,5%CO2,90%N2).The supernatant was collected and secreted exosomes are extracted.The small RNA sequencing was applied to analyze the mi RNA profile of CT-exosomes under hypoxia.Ingenuity Pathway Analysis software was applied to analyze the biofunctions,signal pathways and regulatory networks of mi RNAs included in CT-exosomes.Results:(1)TEM results showed that: a lot of cells which had unique morphology of CTs were found in the intercellular space of cardiac myoctyes and around microvessels.The morphology and the unique parameters of CTs were similar in the three representative dimensions of heart.The difference of the morphological parameters is not statistic significance;(2)The immunofluorescence staining for c-Kit revealed that: in aged heart,the CT density in the atrium-atria part(47.65±4.01 cells/mm2)was significantly higher than that of the base part(33.68±2.53 cells/mm2)and the medium part(26.49±2.11 cells/mm2).The differences among them are statistic significance(p<0.05).In addition,compared with our previous reported data [53],it was found that the CT density in aged heart is significantly higher than that of the parallel positions of young heart and the differences are statistic significance(p<0.05);(3)The results of small RNA sequencing for CT-exosomes under hypoxia shown that: there are a total number of 12,690,873 raw reads,and 10,878,946 clean reads were obtained after wiping out the low-quality sequences,the match rate to rat genome 5 is 98.6%.There are 159 known mature mi RNAs were identified in CT-exosomes after comparing with the sequences from mi RNA databases(mi RBase 20.0).There were 36 mi RNAs with expression levels above 100,and mi R-21-5p was listed as the highest expression level among them.There are 53 mi RNAs with the expression levels between 10 and 100,while the expression levels of the left were lower than 10.Acording to the IPA core analysis,there are 20 mi RNAs were connected with cardiotoxicity.To investigate the probable cardiac functions of the 20 indentified cardiactoxicity mi RNAs,we used mi Randa,mi RDB,Targent Scan,mi RWalk and IPA to predict the possible target genes of the 20 mi RNAs,and a total number of 210 target genes were predicted.IPA core analysis for these 210 target genes revealed that these mi RNAs target genes were involved in Nitric Oxide signaling ? ERK/MAPK Signaling ? CXCR4 Signaling ? TGF-? Signaling and Cholecystokinin/ Gastrin-mediated Signaling to regulate cardiac physiopathology.Conclusion:(1)The morphology and the unique parameters of CTs were similar in the three representative parts of heart;(2)The CT density of the atrium-atria part was significantly higher than that of the base part and the medium part,and CT density of the base part was higher than the medium part.The CT density in aged heart was significantly larger than that of parallel parts of young heart;(3)The 159 matured mi RNAs were identified in CT-exosomes under hypoxia.Among them,there are 20 mi RNAs were connected with cardiotoxicity.There were 210 target genes were predicted mediated by these 20 mi RNAs which might regulate the cardiac physiopathology: Nitric Oxide signaling in the Cardiovascular System?ERK/MAPK Signalin?CXCR4 Signaling?TGF-? Signaling and Cholecystokinin/ Gastrin-mediated Signaling. |
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