Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium

Notch proteins function as receptors for membrane-bound ligands (Jagged and Delta-like) to regulate cell-fate determination. We have investigated the role of Notch signaling in embryonic endothelium of the mouse by expressing an activated form of the Notch4 protein in vasculature under the regulation of the Flk1 (VEGFR) locus. Expression of activated Notch4 results in a growth and developmental delay and embryonic lethality at about 10 days postcoitum. The extent of the developing vasculature in mutant embryos was restricted, fewer small vessels were seen, and vascular networks were disorganized. The brain periphery of mutant embryos contained large dilated vessels with evidence of compromised vessel-wall integrity and large areas of necrosis; yolk-sac vasculature was abnormal. Expression of an activated form of Notch4 in embryonic vasculature leads to abnormal vessel structure and patterning, implicating the Notch pathway in phases of vascular development associated with vessel patterning and remodeling.

Cardiac βARK1 inhibition prolongs survival and augments β blocker therapy in a mouse model of severe heart failure

Chronic human heart failure is characterized by abnormalities in β-adrenergic receptor (βAR) signaling, including increased levels of βAR kinase 1 (βARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of βARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of βARK1 (βARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca2+-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 ± 1 weeks). In contrast, CSQ/βARKct mice exhibited a significant increase in mean survival age (15 ± 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/βARKct, left ventricular end diastolic dimension 5.60 ± 0.17 mm vs. 4.19 ± 0.09 mm, P < 0.005; % fractional shortening, 15 ± 2 vs. 36 ± 2, P < 0.005). The enhancement of the survival rate in CSQ/βARKct mice was substantially potentiated by chronic treatment with the βAR antagonist metoprolol (CSQ/βARKct nontreated vs. CSQ/βARKct metoprolol treated, 15 ± 1 weeks vs. 25 ± 2 weeks, P < 0.0001). Thus, overexpression of the βARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with β-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of βARK1 inhibition.

A Tropomyosin-2 Mutation Suppresses a Troponin I Myopathy in Drosophila

A suppressor mutation, D53, of the held-up2 allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up2, including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human α-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).

A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

A locus on chromosome 7 determines myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

Dystrophic cardiac calcinosis, an age-related cardiomyopathy that occurs among certain inbred strains of mice, involves myocardial injury, necrosis, and calcification. Using a complete linkage map approach and quantitative trait locus analysis, we sought to identify genetic loci determining dystrophic cardiac calcinosis in an F2 intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains. We identified a single major locus, designated Dyscalc, located on proximal chromosome 7 in a region syntenic with human chromosomes 19q13 and 11p15. The statistical significance of Dyscalc (logarithm of odds score 14.6) was tested by analysis of permuted trait data. Analysis of BxH recombinant inbred strains confirmed the mapping position. The inheritance pattern indicated that this locus influences susceptibility of cells both to enter necrosis and to subsequently undergo calcification.

Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development.

Mapping a gene causing cerebral cavernous malformation to 7q11.2-q21.

Cerebral cavernous malformation is a common disease of the brain vasculature of unknown cause characterized by dilated thin-walled sinusoidal vessels (caverns); these lesions cause varying clinical presentations which include headache, seizure, and hemorrhagic stroke. This disorder is frequently familial, with autosomal dominant inheritance. Using a general linkage approach in two extended cavernous malformation kindreds, we have identified linkage of this trait to chromosome 7q11.2-q21. Multipoint linkage analysis yields a peak logarithm of odds (lod) score of 6.88 with zero recombination with locus D7S669 and localizes the gene to a 7-cM region in the interval between loci ELN and D7S802.