👤 W Scott Watkins

Most sarcomere gene mutations that cause hypertrophic cardiomyopathy are missense alleles that encode dominant negative proteins. The potential exceptions are mutations in the MYBPC3 gene (encoding cardiac myosin-binding protein-C [MyBP-C]), which frequently encode truncated proteins. We sought to determine whether there was evidence of haploinsufficiency in hypertrophic cardiomyopathy caused by MYBPC3 mutations by comparing left ventricular muscle from patients undergoing surgical myectomy with samples from donor hearts. MyBP-C protein and mRNA levels were quantitated using immunoblotting and RT-PCR. Nine of 37 myectomy samples had mutations in MYBPC3: 2 missense alleles (Glu258Lys, Arg502Trp) and 7 premature terminations. No specific truncated MyBP-C peptides were detected in whole muscle homogenates of hypertrophic cardiomyopathy tissue. However, the overall level of MyBP-C in myofibrils was significantly reduced (P<0.0005) in tissue containing either a truncation or missense MYBPC3 mutation: 0.76+/-0.03 compared with 1.00+/-0.05 in donor and 1.01+/-0.06 in non-MYBPC3 mutant myectomies. The absence of any detectable truncated MyBP-C argues against its incorporation in the myofiber and any dominant negative effect. In contrast, the lowered relative level of full length protein in both truncation and missense MYBPC3 mutations argues strongly that haploinsufficiency is sufficient to cause the disease. Show less

Fibroblast Growth Factors (FGFs) represent a large family of secreted proteins that are required for proper development and physiological processes. Mutations in mouse and zebrafish FGFs result in abnormal embryogenesis and lethality. A key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis. Expression of Dual Specificity Phosphatase 6 (dusp6, also known as Mkp3) is controlled by FGF signalling throughout development. The Dusp6 promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein (d2EGFP) in transgenic embryos (Tg(Dusp6:d2EGFP)). Expression of d2EGFP is initiated as early as 4 hours post-fertilization (hpf) within the future dorsal region of the embryo, where fgf3 and fgf8 are initially expressed. At later stages, d2EGFP is detected within structures that correlate with the expression of Fgf ligands and their receptors. This includes the mid-hindbrain boundary (MHB), pharyngeal endoderm, otic vesicle, hindbrain, and Kupffer's vesicle. The expression of d2EGFP is under the control of FGF signalling as treatment with FGF Receptor (FGFR) inhibitors results in the suppression of d2EGFP expression. In a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific FGF inhibitors within the class of indolinones. These compounds were counter screened with the transgenic line Tg(Fli1:EGFP)y1, that serves as an indirect read-out for Vascular Endothelial Growth Factor (VEGF) signalling in order to determine the specificity between related receptor tyrosine kinases (RTKs). From these assays it is possible to determine the specificity of these indolinones towards specific RTK signalling pathways. This has enabled the identification of compounds that can block specifically the VEGFR or the FGFR signalling pathway. The generation of transgenic reporter zebrafish lines has allowed direct visualization of FGF signalling within the developing embryo. These FGF reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the RTK family. Show less