Identification And Functional Studies Of Biomarkers In Plasma Involved In Transposition Of Great Arteries Children Undergoing Left Ventricular Training Surgery The Expression Of Ca²⁺-regulatory Proteins In Hypertrophic Right Ventricle In Childr

Background:The left ventricle of complete congenital transposition of great arteries (TGA) children was connected with pulmonary artery. With the decline in pulmonary vascular resistance after birth, the left ventricular pressure load decreased gradually. The long-term low left ventricular pressure load will lead the functional degradation of left ventricle. Increased left ventricular pressure load induced by left ventricle training with pulmonary artery banding surgery would stimulate left ventricular development and cardiomyocyte hypertrophy, which provides an unique and valuable human disease model to study pressure overload hypertrophy and development of immature myocardium. Identification of the plasma biomarkers involved in transposition of great arteries children undergoing left ventricular training surgery will provide new clues for studying the molecular mechanisms of hypertrophy and development of immature myocardium. Additionally, using these biomarkers as a new potential molecular target can bring hope for developing new myocardial hypertrophy intervention drugs.Methods:Differential gel electrophoresis (DIGE) proteomics was used to screen and identify the differential proteins from the plasma of the anterior and posterior left ventricular training surgery in TGA children. Then, enzyme linked immunosorbent assay (ELISA) was used to detect the expression level of the differential proteins including ceruloplasmin (CP), alpha-1-antitrypsin (SERPINA1), transferrin (TF), and parvalbumin (PVALB) in the preoperative and postoperative plasma of TGA children undergoing left ventricular training surgery, the preoperative and postoperative plasma of children undergoing off-pump cardiac surgery, and the preoperative plasma of children undergoing non-congenital heart disease surgery. Furthermore, the in vitro cultured neonatal rat cardiomyocytes was treated with TF or Endothelin-1(ET-1) to observe changes in the size of cells, in the mRNA expression of myocardial hypertrophy biomarkers including natriuretic peptide precursor A (Nppa) and natriuretic peptide precursor B (Nppb), as well as in the PVALB expression.Results:By using DIGE proteomics,25differential proteins were successfully identified in postoperative plasma of TGA children undergoing left ventricular training surgery.19of them were involved in heart development, iron ion homeostasis, protein biosynthesis and protein folding, metabolism, signal transduction and others. ELISA analysis showed that compared with the levels in their preoperative plasma,3differential proteins including CP, TF and PVALB levels rose significantly in postoperative plasma of TGA children undergoing left ventricular training surgery, while there was no significant change in children undergoing off-pump cardiac surgery. The in vitro study showed that after48hours'incubation with TF or ET-1, the size of cardiomyocytes increased significantly, the expression of hypertrophic biomarkers including Nppa and Nppb was significantly enhanced, as well as the PVALB mRNA expression level and PVALB positive protein percentage rose significantly. Both the apotransferrin (apo-TF) and holotransferrin (holo-TF) could induce cardiomyocyte hypertrophy and enhanced expression of Nppa and Nppb mRNA.Conclusions:Iron homeostasis related proteins CP and TF and heart development-associated protein PVALB were plasma biomarkers of posterior left ventricular training surgery in TGA children, so were the biomarkers associated with hypertrophy and development of human immature myocardium. The in vitro study confirmed that TF was a factor which can induce immature cardiomyocyte hypertrophy effectively, and the same dose of apo-TF or holo-TF has similar effect on cardiomyocyte hypertrophy. The in vitro study also confirmed that PVALB was a biomarker of the immature cardiomyocyte hypertrophy. Background:Congenital heart disease (CHD) is a major birth defect around the world. Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Ca2+-regulatory proteins regulate intracellular free Ca2+concentrations and maintain intracellular Ca2+homeostasis so they are very important for excitation-contraction coupling and for myocyte contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Research about the alteration of Ca2+-regulatory proteins in right ventricular hypertrophy (RVH) with or without hypoxia will help to understand the cellular and molecular bases of RVH and hypoxia in the populations of children with CHD.Methods:Right ventricle tissues were collected from children with pulmonary stenosis [n=25, hypertrophy group (H group)], tetralogy of Fallot [n=25, hypoxia and hypertrophy group (HH group)], or small isolated ventricular septal defect [n=25, control group (C group)] during open-heart surgery. Paraffin sections of tissues were stained with3,3'-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), ryanodine receptor2(RyR2), sodiumcalcium exchanger (NCX), sarcolipin (SLN) and phospholamban (PLN)] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase1(PP1) were evaluated using western blot.Results:Mild cardiomyocyte hypertrophy of the right ventricle in H (+31%, P<0.001vs C group) and HH groups (+22%, P<0.001vs C group) was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P<0.01) was observed in the HH group compared with the C group, and a similar trend of decrease was detected in the H group, but there was not a statistically significant difference. No change in the mRNA or protein expression of RyR2, NCX, SLN and PLN in H group and HH group when compared with the C group. The level of Ser16-phosphorylated PLN was down-regulated (P<0.01) and PP1was increased (P<0.01) in the HH group compared to that in the C group.Conclusions:The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser'6dephosphorylation, and early surgical repair might accelerate the recovery of the phosphorylated state of PLN and thereby contribute to improved cardiac contractility in cyanotic CHD with right ventricular hypertrophy. Increased PP1could result in the decreased PLN-Ser16and inhibition of PP1is a potential therapeutic target for heart dysfunction in pediatrics.