Cardiovascular magnetic resonance validation of fractional changes in annulo-apical angles and tricuspid annular plane systolic excursion for rapid assessment of right ventricular systolic function.

PURPOSE: To evaluate the use of right ventricular (RV) annulo-apical angle (AA) changes acquired by magnetic resonance imaging (MRI), alongside tricuspid annular plane systolic excursion (TAPSE), for its association with RV systolic function. MATERIALS AND METHODS: Thirty patients with varying RV dysfunction and 10 normal controls were enrolled and scanned after obtaining written consent. Systolic change in AAs, alpha (α), beta (ß), and theta (θ), subtended by a triangle connecting the septal and lateral extent of the tricuspid valve annulus and RV apex, and TAPSE were measured. Spearman rank correlations of parameters with RV ejection fraction (RVEF) were performed. Receiver operating characteristic (ROC) analysis was used to determine accuracy of these surrogate markers for detecting abnormal RVEF. RESULTS: Correlations with RVEF included: TAPSE (r = 0.74 P < 0.001), fractional systolic changes in α angle (0.64, P < 0.001), ß angle (-0.39, P < 0.05), and θ angle (-0.77, P < 0.001). The best markers for RVEF <50% were fractional θ angle change ≥ -25.5% (eg, -24%) (sensitivity of 97% and specificity of 91%), and TAPSE ≤1.87 cm (sensitivity of 100% and specificity of 82%). Intra- and inter-observer agreement was excellent, with intraclass correlation coefficients for fractional θ angle change (intra = 0.96, inter = 0.94) and TAPSE (intra = 0.98, inter = 0.87). CONCLUSION: Fractional θ angle change is a useful semiquantitative parameter associated with the presence and severity of RV systolic dysfunction, with high observer agreement.

Optimisation and validation of a high throughput screening compatible assay to identify inhibitors of the plasma membrane calcium ATPase pump--a novel therapeutic target for contraception and malaria.

PURPOSE: ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF® Transcreener® ADP (TR-FRET) assay to screen a drug library. METHODS AND RESULTS: PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/µl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1µM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. CONCLUSIONS: We have optimised the HTRF® Transcreener® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.