Abnormal peri-lacunar remodeling in cochlear fibrous dysplasia.

<p>(<b>A</b>) 12-week-old cochlea from male WT and ColI(2.3)⁺/Rs1⁺ cochleae were examined for markers of peri-lacunar remodeling in a specific area of the otic capsule. (<b>B–D</b>) TRAP, a marker for osteoclast activity, was elevated in moderate and severe ColI(2.3)⁺/Rs1⁺ otic capsules compared to the low levels observed in WT controls. (<b>E</b>) The expression of osteocyte secreted remodeling factor, MMP-13 was significantly increased 7-fold in moderate (**, p<0.005) and 100 fold in severe ColI(2.3)⁺/Rs1⁺ cochleae (***, p<1×10⁻⁰⁵) compared to WT cochleae. (n = 6 male WT, n = 5 male ColI(2.3)⁺/Rs1⁺ moderate, and n = 5 male ColI(2.3)⁺/Rs1⁺ severe cochlea). Error bars are mean +/− SEM of triplicate measurements. (<b>F–H</b>) Immunohistochemistry showed that MMP-13 expression was confined to the otic capsule in WT cochleae, but the domain of expression is increased in ColI(2.3)⁺/Rs1⁺ moderate and severe cochlea. (<b>I–K</b>) Thionin staining of the canalicular network was examined in WT and ColI(2.3)⁺/Rs1⁺ moderate and severe cochleae. The canalicular network in WT cochlea shows normal elliptical osteocyte morphology and connectivity. ColI(2.3)⁺/Rs1⁺ moderate and severe cochleae show an abnormal rounded osteocyte morphologies with disrupted and disorganized canalicular networks of varying severity (n = 5 males per genotype).</p

Sensorineural structures of ColI(2.3)⁺/Rs1⁺ mice are normal despite the bony overgrowth affecting the ossicular chain.

<p>(<b>A</b>) Histological analysis of the sensorineural structures of 12-week-old WT and ColI(2.3)⁺/Rs1⁺ revealed no gross abnormalities in the organ of Corti (OC), tunnel of Corti (TC), inner hair cells (IHC), or outer hair cells (OHC). (<b>B</b>) Whole mount immunofluorescence preparations of the outer hair cells revealed no visible differences in hair cell structure or number in ColI(2.3)⁺/Rs1⁺ in comparison to WT structures. (<b>C</b>) Micro computed x-ray tomography of the temporal bones was examined. A region of interest including the middle ear was selected in WT and ColI(2.3)⁺/Rs1⁺ at 12-week-old mice. ColI(2.3)⁺/Rs1⁺ temporal bones showed significant bony overgrowth of the middle ear compared to WT. The ossicles are structurally identifiable and are affected in the ColI(2.3)⁺/Rs1⁺ mice [malleus (M), incus (I), and stapes (S)].</p

Irregular lesions in ColI(2.3)⁺/Rs1⁺ cochlea involve the apex and the labyrinth causing thickening of the otic capsule.

<p>(<b>A–C</b>) Cochlea from 12-week-old WT and ColI(2.3)⁺/Rs1⁺ mice were stained for toluidine blue and examined histologically. Mixed boney fibrous lesions were often observed surrounding ColI(2.3)⁺/Rs1⁺ cochlea compared to WT controls. ColI(2.3)⁺/Rs1⁺ cochlea had multiple fibrous boney overgrowths of the vestibular bone compared to normal WT morphology. Scale bar, 150 µm. (<b>D–F</b>) The walls of ColI(2.3)⁺/Rs1⁺ cochleae showed significant thickening in comparison to WT cochleae, possibly due to the overall thickening of the surrounding otic capsule. The stria vascularis (SV) in the ColI(2.3)⁺/Rs1⁺ mice appear normal. Scale bar, 100 µm.</p

AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy

<div><p>Background</p><p>A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown.</p><p>Methodology/Principal Findings</p><p>To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction.</p><p>Conclusions</p><p>These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy.</p></div

The gene-trap induced truncations of AKAP13 disrupt the expected protein domains.

<p>(A) Expression constructs corresponding to the AKAP13 gene-trap events were generated using V5-tagged AKAP-Lbc truncation mutants. (B-D) These expression constructs were transfected into HEK293 cells and protein complexes were co-immunoprecipitated using anti-V5 antibody. (B) Rho-GEF activity was measured after immunoprecipitation (IP). Both AKAP-Lbc-ΔGEF and -ΔBrx had disrupted Rho-GEF activity, compared to AKAP-Lbc-WT and -ΔPKD. Immunoblotting (IB) for AKAP-Lbc-V5 with anti-V5 antibody confirmed that the AKAP-Lbc truncation mutants were expressed and immunoprecipitated at an equivalent extent. (C) Protein kinase D (PKD) activity was measured following IP. The AKAP-Lbc-ΔPKD, -ΔGEF, and -ΔBrx protein complexes lacked PKD activity compared to AKAP-Lbc-WT. Immunoblotting for GFP-PKD1 with anti-GFP antibody confirmed that only AKAP-Lbc-WT bound PKD1. The bottom gel image confirmed that GFP-PKD1 was expressed at the same level in all conditions. (D) Protein kinase A (PKA) activity was measured after IP. All AKAP-Lbc truncation mutants immunoprecipitated PKA activity and bound PKAc. The means and standard deviations are graphed for three independent experiments. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). *, p<0.05; ***, p<0.001.</p

AKAP13-ΔGEF mutant mice had normal cardiac structure.

<p>(A) Hearts isolated from six wild-type (WT) and six AKAP13^ΔGEF/ΔGEF (ΔGEF) adult male mice at 16–18 weeks of age had normal gross morphology; representative images shown. White scale bar is 1 mm. (B) WT and ΔGEF hearts were the same size as measured by heart weight to tibia length (HW/TL) ratios (in milligrams per millimeter). Means and standard deviations are graphed for six hearts of each genotype. Hearts were sectioned for histology and stained with (C) H&E or (D) Masson’s trichrome. The bottom panels of C&D are higher magnifications of the boxed regions in the top panels. (C) Cardiac structure was normal in ΔGEF hearts (top), and cardiomyocytes had proper organization (bottom). (D) ΔGEF hearts had normal levels of fibrosis as assessed by Masson’s trichrome staining. Black scale bars in C&D are 1 mm (top), 50 µm (C bottom), and 250 µm (D bottom).</p

Full-length AKAP13 mRNA levels are reduced by the gene-trap events.

<p>(A) TaqMan gene expression assays were used to measure the expression of AKAP13 transcripts at the indicated exon-exon junctions (E4-5, Brx-9, & E37-38). (B) Quantitative PCR analysis of wild-type (WT), heterozygote (Het) and homozygote (Hom) neonatal mouse heart and lung RNA for AKAP13 showed that none of the gene-trap mutations affected expression of the E4-5 exon-exon junction. The ΔBrx gene-trap dose dependently decreased expression of the Brx-9 exon-exon junction. Expression of the Brx-9 junction was eliminated in the AKAP13^ΔBrx/ΔBrx mice. All three gene-traps decreased expression of the E37-38 exon-exon junction in a dose-dependent manner. Expression of the E37-38 junction was eliminated in the AKAP13^ΔGEF/ΔGEF mice. The means and standard deviations are graphed for six mice per genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p<0.10; *, p<0.05; **, p<0.01.</p

AKAP13 is expressed in adult heart, kidney, and brain.

<p>Adult AKAP13^+/ΔGEF organs were bisected and stained with X-Gal (in blue) to determine AKAP13-βGeo expression in heart (A), kidney (C) and brain (D). (A) The AKAP13-ΔGEF hearts showed strong staining throughout the entire heart, including the left (la) and right (ra) atria, left (lv) and right (rv) ventricles, pulmonary artery, and aorta. (B) AKAP13-ΔPKD hearts had staining in the atria pulmonary artery, and aorta, as expected, but lacked staining in the ventricles. The blood vessels of the ventricles stained positive. (C) The kidney cortex (c), ureter (u), and arteries (ar) stained positive. (D) The interior of the right hemisphere of the brain showed staining of the olfactory bulb (ob), vasculature (arrow), and part of the cerebellum (cbx). Black scale bars are 1 mm.</p

Six-Lead ECG analysis of AKAP13-ΔGEF mutant mice.

<p>Heart rate is in beats per minute, ms = milliseconds.</p><p>Values are given as the mean ± standard deviation for six mice in each genotype.</p

AKAP13 is broadly expressed during mouse development.

<p>(A–D) Whole-mount AKAP13^+/ΔGEF embryos stained with X-Gal (in blue) to identify AKAP13-βGeo expression at (A&B) E8.5, (C) E9.5, and (D) E10.5. (A&B) E8.5 embryos showed expression in the head folds, notochord, and somites. (C) Right side view of E9.5 embryo showed expression in the heart (ht), brain, eye (arrow), otic pit (arrowhead), gut, and somites. (D) Right side view of E10.5 embryo showed similar expression as in (C) with higher expression in the heart (ht). (E) Frontal view of an E10.5 heart showed high levels of expression in the ventricle (v), bulbous cordis (bc), and outflow tract (oft). (F) Sagittal and (H) transverse sections of E14.5 embryos stained with X-Gal and nucleofast red. E14.5 embryos showed expression in the heart (ht), tongue (t), lung (l), gut (g), kidney (k), skeletal muscle, brain (arrow), and urogenital region (arrowhead). (G&I) Close ups of the hearts boxed in F and H, respectively, showed expression in atrial (at), and ventricular (v) myocardium, endocardium and trabeculae. The right and left atria (ra & la) and ventricles (rv & lv) all showed expression with higher levels in the left ventricle (lv). There was also expression in the aorta (a). (J) X-Gal staining of E9.5 embryos with the yolk sac attached showed expression in the yolk sac (ys). Black scale bars are 0.5 mm.</p