Effects Of FKBP12.6 Gene On Myocardial Structure And Function In Mouse H9c2(2-1) Cells Transfected By Ultrasound-mediated Destruction Of Microbubbles

Background and purpose:Myocardial cells exhibit characteristic disorders of excitation-contraction coupling under multiple pathological conditions. FK506 binding proteins 12.6 (FKBP12.6) plays an important role in regulation of intracellular Ca2+ release and calcineurin activity. Decreased expression of FKBP12.6 and its impaired interaction with the ryanodine receptor 2 (RyR2) causes the disorder of excitation-contraction coupling, therefore, FKBP12.6 might be a therapeutic target for myocardial dysfunction. With the development of ultrasound imaging technique, more and more studies use the microbubble acoustic contrast to take therapeutic genes into cells or tissues. The site-specific release of target genes by disrupted the contrast microbubbles with ultrasound is a new targeted gene transfection technique. The microbubbles used in the technique are less than 8μm in diameter. When the microbubbles are delivered to the target tissues through pulmonary circulation, diagnostic ultrasound can increase microvascular permeability and thus make the targeted gene easier to go into the parenchymal cells. Previous studies show that ultrasound disrupted microbubbles increase the efficiency of gene transfection and gene expression.To investigate the effects of FKBP12.6 on the growth, structure and function of myocardial cells, we studied the cardiac cells transfected with FKBP12.6 by ultrasound-disrupted microbubbles.Methods:1.pcDNA3.1-FKBP12.6 gene expression vector: The synthesized whole objective gene was cloned by PCR, inserted into the digestive point, connected to pGEM-T easy vector, and identified by digestion and sequencing. The recombinant plasmids were inserted into the expression vector pcDNA3.1(-). The pcDNA3.1- FKBP12.6 gene expression vector was amplified after identification.2.Experimental study: The pcDNA3.1-FKBP12.6 plasmid, mingled with albumin-coated microbubbles agents, was transfected into H9c2(2-1) cells by ultrasound-mediated destruction of microbubbles. The H9c2(2-1) cell growth state and cell ultrastructural change were checked by inverted microscope and electron microscope; the intracellular calcium (Ca2+) concentration was determined by laser scanning confocal microscope, the FKBP12.6 mRNA and protein expressions were checked by RT-PCR, western blotting and immunohistochemistry.Results:1.To construct and amplify pcDNA3.1-FKBP12.6 gene expression vector: The pcDNA3.1- FKBP12.6 gene expression vector was successfully constructed and amplified.2.Experimental study:(1) H9c2(2-1) cells, transfected with FKBP12.6 gene, grew well, and the reticulum was larger as compared with control cells, indicating that there is an active synthesis.(2) As compared with control cells, there was higher gene expression for FKBP12.6 gene in H9c2 (2-1) cells transfected by ultrasound disruption of microbubbles.(3) The intracellular Ca2+ concentration was higher in FKBP12.6 transfected cells than that in control cells, indicating an augmented myocardial cell contraction.Conclusions:1.There is higher FKBP12.6 gene expression in H9c2 (2-1) cells by ultrasound disruption of microbubbles.2. FKBP12.6 gene transfection by ultrasound disruption of microbubbles significantly improved the structure and function of myocardial cells, which may provide a new approach for treatments of myocardial dysfunction.