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1.
Am J Physiol Renal Physiol ; 295(2): F462-70, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18524855

ABSTRACT

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.


Subject(s)
Cardiomegaly/genetics , Hypertension/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Blood Pressure/drug effects , Blood Pressure/physiology , Cardiomegaly/metabolism , Disease Models, Animal , Endosomal Sorting Complexes Required for Transport , Epithelial Sodium Channels/metabolism , Hypertension/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nedd4 Ubiquitin Protein Ligases , Sodium Chloride, Dietary/pharmacology , Ubiquitin-Protein Ligases/metabolism
2.
Am J Physiol Heart Circ Physiol ; 293(5): H2680-92, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17766470

ABSTRACT

The intercalated disk protein Xin was originally discovered in chicken striated muscle and implicated in cardiac morphogenesis. In the mouse, there are two homologous genes, mXinalpha and mXinbeta. The human homolog of mXinalpha, Cmya1, maps to chromosomal region 3p21.2-21.3, near a dilated cardiomyopathy with conduction defect-2 locus. Here we report that mXinalpha-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinbeta likely provides partial compensation and accounts for the viability of the mXinalpha-null mice. Ultrastructural studies of mXinalpha-null mouse hearts reveal intercalated disk disruption and myofilament disarray. In mXinalpha-null mice, there is a significant decrease in the expression level of p120-catenin, beta-catenin, N-cadherin, and desmoplakin, which could compromise the integrity of the intercalated disks and functionally weaken adhesion, leading to cardiac defects. Additionally, altered localization and decreased expression of connexin 43 are observed in the mXinalpha-null mouse heart, which, together with previously observed abnormal electrophysiological properties of mXinalpha-deficient mouse ventricular myocytes, could potentially lead to conduction defects. Indeed, ECG recordings on isolated, perfused hearts (Langendorff preparations) show a significantly prolonged QT interval in mXinalpha-deficient hearts. Thus mXinalpha functions in regulating the hypertrophic response and maintaining the structural integrity of the intercalated disk in normal mice, likely through its association with adherens junctional components and actin cytoskeleton. The mXinalpha-knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects.


Subject(s)
Arrhythmias, Cardiac/physiopathology , Cardiomegaly/physiopathology , Cardiomyopathies/physiopathology , DNA-Binding Proteins/metabolism , Heart Conduction System/physiopathology , Nuclear Proteins/metabolism , Sarcolemma/metabolism , Animals , Arrhythmias, Cardiac/pathology , Cardiomegaly/pathology , Cardiomyopathies/pathology , Heart Conduction System/pathology , Mice , Mice, Knockout , Sarcolemma/pathology
3.
Am J Physiol Renal Physiol ; 292(5): F1334-44, 2007 May.
Article in English | MEDLINE | ID: mdl-17229678

ABSTRACT

In mammals, the hormonal regulation of water homeostasis is mediated by the aquaporin-2 water channel (Aqp2) of the collecting duct (CD). Vasopressin induces redistribution of Aqp2 from intracellular vesicles to the apical membrane of CD principal cells, accompanied by increased water permeability. Mutations of AQP2 gene in humans cause both recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. In this study, we generated a line of mice with the distal COOH-terminal tail of the Aqp2 deleted (Aqp2(Delta230)), including the protein kinase A phosphorylation site (S256), but still retaining the putative apical localization signal (221-229) at the COOH-terminal. Mice heterozygous for the truncation appear normal. Homozygotes are viable to adulthood, with reduced urine concentrating capacity, increased urine output, decreased urine osmolality, and increased daily water consumption. Desmopressin increased urine osmolality in wild-type mice but had no effect on Aqp2(Delta230/Delta230) mice. Kidneys from affected mice showed CD and pelvis dilatation and papillary atrophy. By immunohistochemical and immunoblot analyses using antibody against the NH(2)-terminal region of the protein Aqp2(Delta230/Delta230) mice had a markedly reduced protein abundance. Expression of the truncated protein in MDCK cells was consistent with a small amount of functional expression but no stimulation. Thus we have generated a mouse model of NDI that may be useful in studying the physiology and potential therapy of this disease.


Subject(s)
Aquaporin 2/genetics , Diabetes Insipidus, Nephrogenic/genetics , Disease Models, Animal , Mice , Mutation , Animals , Aquaporin 2/metabolism , Atrophy , Cell Line , Deamino Arginine Vasopressin/pharmacology , Diabetes Insipidus, Nephrogenic/pathology , Diabetes Insipidus, Nephrogenic/physiopathology , Diabetes Insipidus, Nephrogenic/urine , Diuresis , Dogs , Drinking , Gene Deletion , Heterozygote , Homozygote , Kidney Concentrating Ability , Kidney Medulla/pathology , Kidney Pelvis/pathology , Kidney Tubules, Collecting/pathology , Osmolar Concentration , Protein Structure, Tertiary
4.
Am J Physiol Renal Physiol ; 291(1): F107-15, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16571596

ABSTRACT

The present studies were designed to determine whether mice heterozygous for deletion of beta-ENaC exhibited defects in Na+/K+ transport and blood pressure regulation. In response to an acute KCl infusion, +/-mice developed higher serum [K+] and excreted only 40% of the K+ excreted by +/+mice. After 6 days on a low (0.01%)-Na+ diet, the cumulative Na+ excretion from days 3-6 was greater for +/-mice. This low-Na+ diet caused higher serum [K+] and lower K+ excretion rates in +/-mice than in +/+mice, but the rectal potential differences were not different. Analyses of mRNA from mice on this diet showed the expected approximately 50% reduction of beta-ENaC in kidney and colon of +/-mice. Unexpectedly, the level of gamma-ENaC mRNA was similarly reduced. NHE3 mRNA was approximately 30% higher in +/-mice whereas mRNA of the Na-K-2Cl cotransporter was not different. Also unexpectedly, the amount of beta-ENaC proteins was similar in both groups of mice but there was a reduction of one form of gamma-ENaC in +/-mice. These experiments demonstrate that mice heterozygous for beta-ENaC have a small but detectable defect in their ability to conserve Na+ and a more readily apparent defect in the ability to secrete K+.


Subject(s)
Blood Pressure/genetics , Gene Deletion , Heterozygote , Potassium/metabolism , Sodium Channels/genetics , Aldosterone/blood , Animals , Blood Pressure/drug effects , Blood Pressure/physiology , Epithelial Sodium Channels , Female , Homeostasis/drug effects , Homeostasis/genetics , Homeostasis/physiology , Kidney/chemistry , Kidney/drug effects , Kidney/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , RNA, Messenger/analysis , RNA, Messenger/genetics , Sodium/metabolism , Sodium Channels/physiology , Sodium, Dietary/pharmacology , Sodium-Potassium-Chloride Symporters/analysis , Sodium-Potassium-Chloride Symporters/physiology , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/drug effects , Sodium-Potassium-Exchanging ATPase/physiology
5.
Physiol Genomics ; 22(2): 244-56, 2005 Jul 14.
Article in English | MEDLINE | ID: mdl-15886330

ABSTRACT

alpha-Sarcoglycan-deficient (Sgca-null) mice provide potential for elucidating the pathogenesis of limb girdle muscular dystrophy type 2D (LGMD 2D) as well as for studying the effectiveness of therapeutic strategies. Skeletal muscles of Sgca-null mice demonstrate an early onset of extensive fiber necrosis, degeneration, and regeneration, but the progression of the pathology and the effects on muscle structure and function throughout the life span are not known. Thus the phenotypic accuracy of the Sgca-null mouse as a model of LGMD 2D has not been fully established. To investigate skeletal muscle structure and function in the absence of alpha-sarcoglycan throughout the life span, we analyzed extensor digitorum longus and soleus muscles of male and female Sgca-null and wild-type mice at 3, 6, 12, and 18 mo of age. Maximum isometric forces and powers were measured in vitro at 25 degrees C. Also determined were individual myofiber cross-sectional areas and numbers, water content, and the proportion of the cross section occupied by connective tissue. Muscle masses were 40-100% larger for Sgca-null compared with age- and gender-matched wild-type mice, with the majority of the increased muscle mass for Sgca-null mice attributable to greater connective tissue and water contents. Although the greater mass of muscles in Sgca-null mice was primarily noncontractile material, absolute forces and powers were maintained near control levels at all ages, indicating a successful adaptation to the deficiency in alpha-sarcoglycan not observed at any age in LGMD 2D patients.


Subject(s)
Muscle, Skeletal/physiology , Sarcoglycans/deficiency , Animals , Body Weight , Connective Tissue , Isometric Contraction/physiology , Mice , Mice, Knockout , Muscle Fibers, Skeletal , Muscle, Skeletal/pathology , Organ Size , Sarcoglycans/metabolism
6.
Science ; 302(5649): 1416-8, 2003 Nov 21.
Article in English | MEDLINE | ID: mdl-14631046

ABSTRACT

Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the alpha1H T-type Ca2+ channel (alpha(1)3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from alpha(1)3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through alpha1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.


Subject(s)
Arteries/physiology , Calcium Channels, T-Type/physiology , Calcium/metabolism , Coronary Vessels/physiology , Vasodilation , Acetylcholine/pharmacology , Animals , Arteries/drug effects , Calcium Channels, T-Type/genetics , Coronary Vessels/drug effects , Coronary Vessels/pathology , Echocardiography , Electrocardiography , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Female , Fibrosis , Ganglia, Spinal/cytology , Gene Targeting , Heart/physiology , Heart Rate , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/physiology , Myocardium/pathology , Neurons/metabolism , Nickel/pharmacology , Nitric Oxide/physiology , Nitric Oxide Donors/pharmacology , Nitroprusside/pharmacology , Patch-Clamp Techniques , Vasoconstriction/drug effects , Vasodilation/drug effects
7.
Obstet Gynecol ; 102(5 Pt 2): 1185-8, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14607051

ABSTRACT

BACKGROUND: Chorioangiomas are benign vascular tumors of the placenta. Complications occur when their size exceeds 5 cm. CASE: We report the use of peak systolic velocity in the middle cerebral artery, fetal hemoglobin levels in maternal circulation, and maternal serum alpha-fetoprotein levels in the diagnosis and management of fetal anemia in a patient with a large placental chorioangioma. CONCLUSION: Fetal hemoglobin levels in maternal circulation and abrupt elevations of middle cerebral artery peak velocity may be useful in detecting fetal anemia.


Subject(s)
Anemia/diagnosis , Fetal Diseases/diagnosis , Hemangioma/complications , Placenta Diseases/complications , Pregnancy Complications, Neoplastic , Prenatal Diagnosis , Adult , Anemia/etiology , Anemia/therapy , Blood Flow Velocity , Female , Fetal Diseases/etiology , Fetal Diseases/therapy , Fetal Hemoglobin/analysis , Hemangioma/diagnosis , Humans , Middle Cerebral Artery/diagnostic imaging , Placenta Diseases/diagnosis , Pregnancy , Pregnancy Complications, Neoplastic/diagnosis , Ultrasonography, Doppler , alpha-Fetoproteins/analysis
8.
Neuron ; 38(5): 747-58, 2003 Jun 05.
Article in English | MEDLINE | ID: mdl-12797959

ABSTRACT

Dystroglycan is a central component of the dystrophin-glycoprotein complex implicated in the pathogenesis of several neuromuscular diseases. Although dystroglycan is expressed by Schwann cells, its normal peripheral nerve functions are unknown. Here we show that selective deletion of Schwann cell dystroglycan results in slowed nerve conduction and nodal changes including reduced sodium channel density and disorganized microvilli. Additional features of mutant mice include deficits in rotorod performance, aberrant pain responses, and abnormal myelin sheath folding. These data indicate that dystroglycan is crucial for both myelination and nodal architecture. Dystroglycan may be required for the normal maintenance of voltage-gated sodium channels at nodes of Ranvier, possibly by mediating trans interactions between Schwann cell microvilli and the nodal axolemma.


Subject(s)
Cytoskeletal Proteins/deficiency , Membrane Glycoproteins/deficiency , Myelin Sheath/metabolism , Peripheral Nerves/growth & development , Ranvier's Nodes/metabolism , Schwann Cells/metabolism , Sodium Channels/metabolism , Animals , Animals, Newborn , Cell Membrane/metabolism , Cell Membrane/pathology , Cell Membrane/ultrastructure , Cells, Cultured , Cytoskeletal Proteins/genetics , Dystroglycans , Laminin/genetics , Laminin/metabolism , Macromolecular Substances , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Movement Disorders/genetics , Movement Disorders/metabolism , Movement Disorders/physiopathology , Mutation/genetics , Myelin Sheath/pathology , Myelin Sheath/ultrastructure , Neural Conduction/genetics , Peripheral Nerves/pathology , Peripheral Nerves/ultrastructure , Protein Binding/genetics , Ranvier's Nodes/pathology , Ranvier's Nodes/ultrastructure , Schwann Cells/ultrastructure , Wallerian Degeneration/genetics , Wallerian Degeneration/metabolism , Wallerian Degeneration/physiopathology
9.
Nature ; 423(6936): 168-72, 2003 May 08.
Article in English | MEDLINE | ID: mdl-12736685

ABSTRACT

Muscular dystrophy includes a diverse group of inherited muscle diseases characterized by wasting and weakness of skeletal muscle. Mutations in dysferlin are linked to two clinically distinct muscle diseases, limb-girdle muscular dystrophy type 2B and Miyoshi myopathy, but the mechanism that leads to muscle degeneration is unknown. Dysferlin is a homologue of the Caenorhabditis elegans fer-1 gene, which mediates vesicle fusion to the plasma membrane in spermatids. Here we show that dysferlin-null mice maintain a functional dystrophin-glycoprotein complex but nevertheless develop a progressive muscular dystrophy. In normal muscle, membrane patches enriched in dysferlin can be detected in response to sarcolemma injuries. In contrast, there are sub-sarcolemmal accumulations of vesicles in dysferlin-null muscle. Membrane repair assays with a two-photon laser-scanning microscope demonstrated that wild-type muscle fibres efficiently reseal their sarcolemma in the presence of Ca2+. Interestingly, dysferlin-deficient muscle fibres are defective in Ca2+-dependent sarcolemma resealing. Membrane repair is therefore an active process in skeletal muscle fibres, and dysferlin has an essential role in this process. Our findings show that disruption of the muscle membrane repair machinery is responsible for dysferlin-deficient muscle degeneration, and highlight the importance of this basic cellular mechanism of membrane resealing in human disease.


Subject(s)
Cell Membrane/pathology , Gene Deletion , Membrane Fusion , Membrane Proteins , Muscle Proteins/deficiency , Muscular Dystrophies/genetics , Muscular Dystrophies/pathology , Animals , Calcium/metabolism , Dysferlin , Dystrophin/metabolism , Mice , Mice, Knockout , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophies/metabolism , Muscular Dystrophies/physiopathology , Regeneration , Stress, Mechanical
10.
Brain Res ; 958(2): 227-50, 2002 Dec 27.
Article in English | MEDLINE | ID: mdl-12470859

ABSTRACT

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.


Subject(s)
Chloride Channels/deficiency , Hippocampus/pathology , Nerve Degeneration/pathology , Neurodegenerative Diseases/pathology , Receptors, GABA-A/metabolism , Animals , Chloride Channels/genetics , Female , Glutamate Decarboxylase/metabolism , Hippocampus/metabolism , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Retinal Degeneration/pathology
11.
Cell ; 110(5): 639-48, 2002 Sep 06.
Article in English | MEDLINE | ID: mdl-12230980

ABSTRACT

Striated muscle-specific disruption of the dystroglycan (DAG1) gene results in loss of the dystrophin-glycoprotein complex in differentiated muscle and a remarkably mild muscular dystrophy with hypertrophy and without tissue fibrosis. We find that satellite cells, expressing dystroglycan, support continued efficient regeneration of skeletal muscle along with transient expression of dystroglycan in regenerating muscle fibers. We demonstrate a similar phenomenon of reexpression of functional dystroglycan in regenerating muscle fibers in a mild form of human muscular dystrophy caused by disruption of posttranslational dystroglycan processing. Thus, maintenance of regenerative capacity by satellite cells expressing dystroglycan is likely responsible for mild disease progression in mice and possibly humans. Therefore, inadequate repair of skeletal muscle by satellite cells represents an important mechanism affecting the pathogenesis of muscular dystrophy.


Subject(s)
Cytoskeletal Proteins/physiology , Membrane Glycoproteins/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Muscular Dystrophy, Animal/genetics , Aging , Animals , Cell Differentiation , Cytoskeletal Proteins/genetics , Dystroglycans , Hypertrophy , Membrane Glycoproteins/genetics , Mice , Mice, Inbred mdx , Muscle, Skeletal/pathology , Recombination, Genetic , Regeneration
12.
Nature ; 418(6896): 422-5, 2002 Jul 25.
Article in English | MEDLINE | ID: mdl-12140559

ABSTRACT

Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), and Walker-Warburg syndrome are congenital muscular dystrophies (CMDs) with associated developmental brain defects. Mutations reported in genes of FCMD and MEB patients suggest that the genes may be involved in protein glycosylation. Dystroglycan is a highly glycosylated component of the muscle dystrophin-glycoprotein complex that is also expressed in brain, where its function is unknown. Here we show that brain-selective deletion of dystroglycan in mice is sufficient to cause CMD-like brain malformations, including disarray of cerebral cortical layering, fusion of cerebral hemispheres and cerebellar folia, and aberrant migration of granule cells. Dystroglycan-null brain loses its high-affinity binding to the extracellular matrix protein laminin, and shows discontinuities in the pial surface basal lamina (glia limitans) that probably underlie the neuronal migration errors. Furthermore, mutant mice have severely blunted hippocampal long-term potentiation with electrophysiologic characterization indicating that dystroglycan might have a postsynaptic role in learning and memory. Our data strongly support the hypothesis that defects in dystroglycan are central to the pathogenesis of structural and functional brain abnormalities seen in CMD.


Subject(s)
Brain/metabolism , Brain/pathology , Cytoskeletal Proteins/metabolism , Gene Deletion , Membrane Glycoproteins/metabolism , Muscular Dystrophies/congenital , Muscular Dystrophies/genetics , Animals , Brain/abnormalities , Brain/physiopathology , Cell Movement , Cytoskeletal Proteins/genetics , Dystroglycans , Electrophysiology , Glycosylation , Hippocampus/abnormalities , Hippocampus/physiopathology , Laminin/metabolism , Learning/physiology , Long-Term Potentiation , Membrane Glycoproteins/genetics , Memory/physiology , Mice , Mice, Knockout , Muscular Dystrophies/metabolism , Muscular Dystrophies/physiopathology , Neurons/metabolism , Neurons/pathology , Organ Specificity , Syndrome
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