Effects Of FKBP12.6 Gene Transfection On Cardiac Function And Myocardium Pathomorphology In Canine With Heart Failure

Background and objective: It has been demonstrated that myocardial cells exhibit characteristic disorders of excitation-contraction coupling under multiple pathological conditions. RyR2 (ryanodine receptor type 2) is the calcium release channel of SR (sarcoplasmic reticulum), and the defects of RyR2 can result in myocardial systolic, diastolic disfunction and arrhythmia. Downregulation of FKBP12.6 (FK506 binding protein 12.6) protein and decline of affinity between FKBP12.6 and RyR2 are the main reasons that cause the defects of RyR2. In vitro researches, it has been indicated that overexpression of FKBP12.6 can correct the RyR2 channel defects, reduce leak of Ca2+ from SR, increase Ca2+ transient, elevate the cytoblastema Ca2+ concentration and increase cardiac muscle contraction, thus overexpression of FKBP12.6 can be used as a therapeutic target for myocardial dysfunction. With the development of ultrasound imaging technique and the preparation of the microbubble acoustic contrast that may carry genes, ultrasound has become a tool both diagnostic and therapeutic. The site-specific release of target genes by disrupting the contrast microbubbles with ultrasound is a new target gene transfection technique. The microbubbles used in the technique are less than 8μm in diameter, which carry gene expression vectors. When the microbubbles are delivered to the target tissues through pulmonary circulation, diagnostic ultrasound is applied to the specific tissues to increase microvascular permeability and thus to make the target gene enter the parenchymal cells through both the microvessels and the inter-endothelial cells space. It has been shown that ultrasound disrupting microbubbles can increase the efficiency of gene transfection and gene expression.In the present study, we got the model of canine heart failure induced by rapid ventricular pacing, and transfected pcDNA3.1- FKBP12.6 into myocardium which was carried by ultrasound microbubble contrast and disrruptted by ultrasound-triggered microbubble destruction. The aims were to investigate the effects of FKBP12.6 gene transfection on structural shape of the canine heart, the cardiac function and the myocardium pathological change of canine with heart failure, and open up a new avenue for the clinical treatment of myocardial dysfunction.Methods: Three weeks after onset of rapid ventricular pacing (250bpm), 28 canines were divided into 4 groups.Either pcDNA3.1-FKBP12.6 plasmid encoding human FKBP12.6 gene (transfection groupⅠ,Ⅱ, observed at the 4th or 14th days respectively after trasnfection) or empty vector(controlⅠ,Ⅱ) was transfected into myocardium with the destruction of microbubbles caused by the ultrasound. After the transfection, maintenance pacing at a reduced rate (190bpm) was continued until the end-point of the experiment.Echocadiographic, hemodynamic and plasmic ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) data were collected three times (before pacing, before transfection and endpoint). The pathological change of myocardium is assessed by HE staining and electron microscope. Semiquantitative RT-PCR (reverse transcriptase-polymerase chain reaction) identified FKBP12.6 expression in myocardium.Results:1. In the transfection groups, EF (ejection fraction), FS (fractional shortening) were significantly improved in transfection groupⅠand this effect was stable for 2 weeks at least but EF and FS were still lower than pre-pacing although they increased in transfection groups. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-diastolic volume (LVEDV) decreased on the 14th day, meanwhile LVEDD remained unchanged on the 4th day, compared with pre-transfection.2. It was found that the level of RAP, mRVP, mPAP and PAWP in canine with heart failure increased comparing with baseline, but CO decreased, and the deference was not observed among the groups. At the endpoint of observation, significant improvement of RAP, mRVP, mPAP and PAWP were observed in transfection groups meanwhile CO (cardiac output) could not recover to the normal level.All parameters maintained stable in two transfection groups or two control groups, though CO reduced in control groupⅡ, no statistical significance was found when compared with control groupⅠ(P=0.279)3. When heart failure was developed, ANP (atrial-natriuretic polypeptide) and BNP (brain-natriuretic peptide) increased drastically. In transfection groups, accompanying with the improvement of cardiac functions, ANP and BNP declined drastically and closed normal level.And these data indicated a significant difference when compared with control group or pre-transfection.4. Gene transfection of FKBP12.6 elevated expression of FKBP12.6 mRNA 3.4 folds on the 4th day,and 1.7 folds on the 14th day respectively, compared with control group. Expression of FKBP12.6 mRNA descended lightly in control groupⅡ, but the deference was not significant (0.75±0.17 vs 0.83±0.19).5. The pathology indicated relatively severe changes were observed in control groups and it further aggravated in group controlⅡ.Conclusions: FKBP12.6 gene transfection mediated by ultrasound disruption of microbubbles significantly elevates expression of FKBP12.6 mRNA, improves cardiac function in the failing heart, reverses myocyte remodeling and promotes recovery from pathological changes.Thereby it is a novel gene therapy for human heart failure.