Roles And Mechanisms Of TG2-Mediated SERCA2 Serotonylation On Hypoxic Pulmonary Vein Remodeling

PartⅠ.Effects of Hypoxia on TG2 Activity and ExpressionObject:To investigate effects of chronic hypoxia on TG2 activity and expression.Methods:Human venous smooth muscle cells（hPVSMC）were isolated and cultured from the intrapulmonary veins(4^th level)by tissue explants adherence method.hPVSMCs were identified by positive immunostaining with an anti-α-SMA monoclonal antibody.qPCR,Western blotting（WB）and（biotinamido）pentylamine（BP）were used to detect expression and activity of transglutaminase 2（TG2）under chronic hypoxia（1%O2）and normoxia.Immunohistochemistry was used to detect location of TG2 protein on pulmonary vessels.qPCR and Western blotting（WB）were used to detect expression of TG2 on pulmonary vessels.Results:3th-4th generation hPVSMCs were used for cell experiments.It was identified that 3-4th human pulmonary vein smooth muscle cells were successfully isolated.Cells were high purity and in good condition,and cells can be used in experiments.Chronic hypoxia enhanced TG2 expression and activity in hPVSMC,and was time-dependent.Chronic hypoxia enhanced TG2 expression in the pulmonary veins of mice.TG2localized on pulmonary smooth muscle layer.Conclusion:Hypoxia enhanced TG2 expression and activity,and TG2 localized on pulmonary smooth muscle layer.Part Ⅱ.To investigate the mechanisms of TG2-mediated SERCA2 Serotonination and disruption Ca²⁺ balanceObject: To investigate mechanism of TG2-mediated SERCA2 Serotonination and hypoxia promoted calcium influx through TRPC6-mediated SOCE.Methods: siRNA and adenovirus were used to silence and overexpress the interest gene.co-Immunoprecipitation（co-IP）and micro-Ca²⁺ ATP enzyme kit were used to detect sSERCA2 expression and SERCA2 activity when the TG2 gene silenced oroverexpressed under normoxia and hypoxia.Intracellular calcium ion was measured by using Fluo-4AM probe under normoxia and hypoxia.q PCR and WB were used to detect TRPC1 and TRPC6 expression on hPVSMCs under normoxia and hypoxia.Fluo-4AM probe was used to detect basal [Ca²⁺]i and SOCE when the TRPC1 and TRPC6 gene silenced and overexpressed under hypoxia.Results: Cells produced more inorganic phosphorus when the TG2 gene was silenced,whereas cells produced less organic phosphorus when the TG2 gene overexpressed under normoxia.Hypoxia significantly prevented cells from producing inorganic phosphorus,which was regulated by TG2.When compared to the normoxia group,silencing of the TG2 gene did not increase the production of inorganic phosphorus under hypoxia.Expression of s-SERCA2 protein was significantly increased when the TG2 gene was overexpressed,whereas the expression of s-SERCA2 protein decreased when the TG2 gene was silenced under normoxia.When compared to the normoxia group,the expression of s-SERCA2 protein significantly increased under hypoxia,however the expression did not significantly increase when the TG2 gene was silenced under hypoxia.Hypoxia（1% O2）induced a marked increase in basal [Ca²⁺]i from 6.81±1.14 to 13.88±0.26.In the normoxia group,the cell basal [Ca²⁺]i decreased from 6.81±1.14 to 1.40±0.04 when the TG2 gene was silenced,and increased from 6.81±1.14 to 8.30±0.03 when the TG2 gene was overexpressed.In the hypoxia group,the cell basal [Ca²⁺]i decreased from 13.88±0.26 to 6.95±0.16 when the TG2 gene was silenced,and increased to 20.48±0.25 when the TG2 gene was overexpressed.Hypoxia induced a marked increase in the peak of time-Δfluorescence curve from 164.33±7.64 to 228.00±14.42.In the normoxia group,the peak Δ[Ca²⁺]i decreased from 164.33±7.64 to 130.33±4.51 when the TG2 gene was silenced,and increased to 207.00±6.25 when the TG2 gene was overexpressed.In addition,in the hypoxia group,the peak Δ[Ca²⁺]i decreased from 228.00±14.42 to 165.33±11.01 when the TG2 gene was silenced,and increased to 383.67±13.50 when the TG2 gene was overexpressed.Hypoxia increased expression of TRPC6,not TRPC1,on hPVSMCs.The basal [Ca²⁺]i increased from 2.81±0.29 to 5.04±0.05 after TRPC6 gene overexpression,but decreased to 1.64±0.06 after TRPC6 gene silencing.The basal [Ca²⁺]i of silencing and overexpressing of theTRPC1 gene were 2.82±0.25 and 2.73±0.29.The peak fluorescence increased from 193.00±4.00 to 314.67±2.89 after TRPC6 gene overexpression,and decreased to 114.33±2.31 after TRPC6 gene silencing.The peak fluorescence of silencing and overexpressing TRPC1 gene were 200.67 ±10.60 and 206.00±5.00.Conclusion: Hypoxia activated TRPC6-mediated SOCE to promote the extracellular calcium influx.Hypoxia promoted TG2-mediated s-SERCA2,and s-SERCA2 inhibited activity of SERCA2 and promoted calcium influx through TRPC6-mediated SOCE.Part Ⅲ.Effect of SERCA2 activity on cell biological behavior and calcium signalingObject: To investigate the effects of SERCA2 activity on proliferation,apoptosis,migration and intracellular calcium in hPVSMCsMethods: Cells were treated with CPA（SERCA2 inhibitor）and PST-2744（SERCA2 agonist）for 12 and 24 hours,respectively.CCK-8,flow cytometry,wound scratch,q PCR,WB and Fluo-4AM probe were used to detect hPVSMCs apoptosis,proliferation,cell migration,cell phenotypic protein expression and [Ca²⁺]i,respectively.Results: CPA inhibited cell apoptosis,and promoted cell proliferation and migration,whereas PST-2744 promoted cell apoptosis,and inhibited cell proliferation and migration in a time-dependent manner.CPA promoted transformation from a differentiated phenotype to a dedifferentiated phenotype,and PST-2744 promoted transformation from a dedifferentiated phenotype to a differentiated phenotype.Normal [Ca²⁺]i was 6.97±1.16,the [Ca²⁺]i of cells treated with CPA for 12 hours and 24 hours was 8.69±0.43 and 14.82±0.88.In addition,the [Ca²⁺]i of cells treated with PST-2744 for 12 hours and 24 hours was 5.36±0.46 and 4.03±0.19.The normal peak fluorescence was 171.33±6.11.The peak fluorescence of cells treated with CPA for 12 hours and 24 hours were 221.67±8.62 and 265.00±6.00,and the peak fluorescence the of cells treated with PST-2744 for 12 hours and 24 hours was 143.67±5.69 and 116.67±6.51.Conclusion: SERCA2 activity played an important role in regulating hPVSMCs proliferation,apoptosis,migration,cell phenotype and calcium ion influx.Part Ⅳ.Role of SERCA2 serotonination on hypoxic pulmonary vein remodeling in vivoObject: Establishing PH model to investigate role of SERCA2 serotonination on hypoxic pulmonary vein remodelingMethods: Wild-type（WT）mice（n=6）and TG2 knockout（Tgm2-/-）mice（n=6）were exposed to hypoxia（10% O2）in a normobaric chamber for 6 weeks.Another set of identical WT mice（n=6）and Tgm2-/-mice（n=6）were kept under normal conditions for 6 weeks,and represented the normoxia groups.co-IP and micro-Ca²⁺ ATP enzyme kit were used to detect s-SERCA2 and SERCA2 activity of isolated pulmonary veins.Closed-chest insertion into the right ventricle（RV）was used to detect RVSP.To evaluate the extent of RV hypertrophy,the weights of the RV and the left ventricle plus interventricular septum（LV+S）were measured separately.Hematoxylin-eosin（HE）staining and Image J Plus 6.0 software were used to detect percentage medial layer thickness（MT%=100 × [medial layer thickness]/[vessel semidiameter]）and area（MA%=100×[cross‐sectional vessel area]）of peripheral pulmonary vessels.Results: The RVSP of Tgm2-/-mice and WT mice was 18.15±0.45 mm Hg and 18.35+0.76 mm Hg under normoxia.The RVSP of WT mice increased from 18.15±0.45 mm Hg to 34.05±0.99 mm Hg after hypoxia exposure for 6 weeks.The RVSP of Tgm2-/-mice under hypoxia 22.79±6.79 mm Hg was slightly higher when compared to that of WT mice under normoxia.The RVHI of Tgm2-/-mice and WT mice were 0.20±0.010 and 0.21+0.009 under normoxia,respectively.Moreover,the RVHI of Tgm2-/-mice and WT mice were 0.25±0.063 and 0.34+0.055 under hypoxia.The MT% of Tgm2-/-mice and WT mice were 9.90±0.30 and 10.37±0.67,and MA% of Tgm2-/-mice and WT mice were19.13±1.00 and 18.40±0.62 under normoxia,and respectively.The MT% of Tgm2-/-mice and WT mice were 9.63±0.70 and 43.30±1.67,and MA% of Tgm2-/-mice and WT mice were8.97±0.47 and 70.67±0.50 under hypoxia,and respectively.Conclusion:TG2 was a key enzyme that regulateed SERCA2 serotonination in vivo,and knocking out the TG2 gene reversed pulmonary artery pressure and pulmonary vein remodeling under hypoxia.