Effect Of Carbon Nanohorns/collagen Hybrids Substrates On Expression And Maturation Of Intercalated Disc Related Proteins Of Neonatal Rat Ventricular Myocytes

The existing methods for roaring heart diseases,especially for end-stage heart disease,are turning to increasing limitations.For end-stage heart disease the main strategy is medicine treatment,intervention treatment and heart transplantation.However,all of them cannot perform perfectly.For example,medicine treatment and intervention treatment only can relieve patients’ syndrome but not to cure or reverse heart function.Heart transplantation is a way to cure but it cannot be a prevalent treatment owing to immunological rejection and lack of donor organ et al.Thus,recently with the development of technology,a new promising treatment-cardiac tissue engineering(CTE)has developed rapidly and it may be an effective treatment in the future.Objective: To manufacture carbon nanohorns/collagen(CNH/Col)hybrids substrates material via ultrasonic method in ice and select the optimal concentration of CNH/Col substrates for growth and differentiation of neonatal rat ventricular myocytes(NRVMs).Then study the effect of CNH/Col substrates on adhesion,proliferation,differentiation and functional maturation of NRVMs and proliferation of cardiac fibroblasts(CFs)and evaluate the application of CNH for CTE.Method: To manufacture CNH/Col hybrids substrates material via ultrasonic method in ice and characterize CNH/Col hybrids with Transmission electron microscopy(TEM),atomic force microscope(AFM)and electrochemical workstation.NRVMs were obtained via tyrisin digestion.The biocompatibility of CNH/Col hybrids was evaluated through Live/dead staining and CCK-8 assay and selected out the optimal concentration for NRVMs cultivation.The expression and distribution of connexin-43(Cx-43)and N-cadherin(NC)of NRVMs cultured on different substrates weremeasured with immunofluorescence staining and western blotting on day 3and 7.To evaluate the maturation of function of NRVMs,intracellular calcium transient measurements were carried out on day 5 and 7.CFs were obtained via differential velocity adherent,and the proliferation of CF on different substrates was measured through BrdU staining and CCK-8 assay.Results: TEM showed that after sonication under our experimental conditions,CNH aggregates were homogeneously dispersed in the collagen matrix.Small agglomerates containing only a few CNH aggregates were observed by TEM,and the CNH aggregates are coated with collagen molecules,which reveal the good affinity between the CNHs and collagen.AFM showed that the surface roughness of the CNH-Col substrate was higher compared with the collagen control.There were significant differences in conductivity among the collagen and the 0.05 and 0.1 mg/ml CNH-Col groups(P<0.05).We got NRVMs via tyrisin digestion,and Live/dead staining and CCK – 8 assay showed that CNH-Col substrates at concentrations of 0.1 and0.05 mg/ml owned optimal biocompatibility with the lowest cytotoxicity.Immunofluorescent staining showed that Cx-43 and NC turned out to be wide distribution in CNH-Col groups(0.05 and 0.1 mg/ml)compared with that in the collagen group on day 3 and 7.With the cultivation of NRVMs,Cx-43 and NC showed a trend of increased accumulation in the intercellular region of cells,especially for Cx-43 gathered in a linear distribution in CNH-Col groups(0.05 and 0.1 mg/ml).Western blotting also showed that the NC quantification normalized by GAPDH in the 0.1 mg/ml CNH-Col group was significantly higher than that in the 0.05 mg/ml CNH-Col and collagen groups(***P<0.001)on day 3.However,the Cx-43 expression quantities in the three groups showed no significant difference.At day 7,western blotting also showed that the Cx-43 quantification normalized by GAPDH in the collagen group was significantly lower than in the 0.1 mg/ml and 0.05 mg/ml CNH-Col groups(**P<0.01).At the same stage,the NC quantification in the 0.1 mg/ml and 0.05 mg/ml CNH-Col groups was significantly higher than that in the collagen group(***P<0.001,*P<0.05).On day 5,the spontaneousintracellular calcium transients showed the NRVMs cultured on the CNH-Col substrates exhibited higher Ca2+ frequency and amplitudes compared with those cultured on the collagen substrates.On day 7,NRVMs cultured on the collagen substrates performed tiny Ca2+ current,however,those cultured on the CNH-Col substrates exhibited more uniform,stable rhythmic Ca2+fluctuations and higher Ca2+ amplitudes.BrdU staining showed on day 1,there was no significant difference between the collagen group and the CHN-Col groups with respect to the BrdU positive ratio.On day 3,the BrdU positive cells accounted for less than 10 % in the 0.1 mg/ml CNHs-Col group,significantly lower than in the collagen group(***P<0.001).On day 7,in two CNH-Col groups,the BrdU positive cells accounted for less than 5%,which was significantly lower than that in the collagen group(****P<0.0001).However,the CCK-8 assay showed that on day 1 the absorbance in the 0.1mg/ml CNH-Col group was significantly higher than that in the collagen group(*P<0.05).On day 3,The CCK-8 assay showed a significantly decreased absorbance in the 0.1 mg/ml group compared with the other two groups(****P<0.0001).On day 7,in two CNH-Col groups,the absorbance was significantly lower than that in the collagen group(****P<0.0001).Conclusion: In this study,we found both 0.05 mg/ml and 0.1 mg/ml CNH-Col can up-regulate Cx-43 and NC expression and intracellular distribution and promote functional maturation of NRVMs compared with collagen.At the same time,CNHs can inhibit cardiac fibroblast proliferation.Thus,the study showed that CNH may be a promising scaffold material to meet the requirements of CTE.