Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice

(1) Background: The c-Jun-NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase involved in regulating physiological processes in the central nervous system. However, the dual genetic deletion of Mkk4 and Mkk7 (upstream activators of JNK) in adult mice is not reported. The aim of this study was to induce the genetic deletion of Mkk4/Mkk7 in adult mice and analyze their effect in hippocampal neurogenesis. (2) Methods: To achieve this goal, Actin-CreERT2 (Cre+/−), Mkk4flox/flox, Mkk7flox/flox mice were created. The administration of tamoxifen in these 2-month-old mice induced the gene deletion (Actin-CreERT2 (Cre+/−), Mkk4∆/∆, Mkk7∆/∆ genotype), which was verified by PCR, Western blot, and immunohistochemistry techniques. (3) Results: The levels of MKK4/MKK7 at 7 and 14 days after tamoxifen administration were not eliminated totally in CNS, unlike what happens in the liver and heart. These data could be correlated with the high levels of these proteins in CNS. In the hippocampus, the deletion of Mkk4/Mkk7 induced a misalignment position of immature hippocampal neurons together with alterations in their dendritic architecture pattern and maturation process jointly to the diminution of JNK phosphorylation. (4) Conclusion: All these data supported that the MKK4/MKK7–JNK pathway has a role in adult neurogenic activity.

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The Adult Murine Intestine is Dependent on Constitutive Laminin-γ1 Synthesis

Scientific Reports ◽

10.1038/s41598-019-55844-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

British Fields ◽

Ann DeLaForest ◽

Mark Zogg ◽

Jennifer May ◽

Catherine Hagen ◽

...

Keyword(s):

Embryonic Development ◽

Mouse Model ◽

Gene Deletion ◽

Minimal Change ◽

Basal Rate ◽

Genetic Ablation ◽

Adult Mice ◽

Crypt Hyperplasia ◽

Small Intestines ◽

Induced Mutant

AbstractLaminin-γ1 is required for early embryonic development; however, the need for laminin-γ1 synthesis in adulthood is unknown. A global and inducible mouse model of laminin-γ1 deficiency was generated to address this question. Genetic ablation of the Lamc1 gene in adult mice was rapidly lethal. Despite global Lamc1 gene deletion in tamoxifen-induced mutant mice, there was minimal change in total cardiac, pulmonary, hepatic or renal laminin protein. In contrast, laminin-γ1 was significantly depleted in the small intestines, which showed crypt hyperplasia and dissociation of villous epithelium from adjacent mesenchyme. We conclude that the physiologic requirement for laminin-γ1 synthesis in adult mice is dependent on a tissue-specific basal rate of laminin-γ1 turnover that results in rapid depletion of laminin-γ1 in the intestine.

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Abstract 125: Increased Endothelial Endoglin Gene Deletion and Co-deletion of EphrinB2 Enhance Brain Arteriovenous Malformation in Mice

Stroke ◽

10.1161/str.48.suppl_1.125 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Rui Zhang ◽

Wan Zhu ◽

Li Ma ◽

Man Luo ◽

Lei Zhan ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Arteriovenous Malformation ◽

Gene Deletion ◽

Viral Vector ◽

Causative Gene ◽

Adult Mice ◽

Brain Avm ◽

Endothelial Growth Factor ◽

The Brain

Introduction: Endoglin (Eng) is an arteriovenous malformation (AVM) causative gene. In adult mice, global Eng deletion induced by a wild-type cre (cre) results in AVM formation in the brain angiogenic region. We hypothesize that using a codon-improved cre (icre) to increase Eng gene deletion in endothelial cells and co-deletion of the EphrinB2 gene, a determinant of arterial endothelial differentiation, will enhance brain AVM severity. Methods: Eng was globally deleted in adult Eng -floxed mice ( Eng 2f/2f ) using a rosa promoter driving estrogen inducible cre (Rosa-creER), or in endothelial cells using a platelet-derived growth factor b promoter driving estrogen inducible icre ( pdgfb- icreER). Pdgfb- icreER was also used to mediate endothelial deletion of EphrinB2 . An adeno-associated viral vector expressing vascular endothelial growth factor was injected into the brain to induce brain angiogenesis. Results: Compared with Rosa-CreER-mediated global Eng deletion, pdgfb- icreER-induced endothelial Eng deletion did not increase the number of abnormal vessels (P=0.39), but reduced vascular smooth muscle coverage (P=0.03) and increased hemorrhage (P=0.04) in the brain AVM lesion. Additional endothelial deletion of EphrinB2 gene increased the number of abnormal vessels in the brain (P=0.08). Conclusion: These data indicate that a positive correlation exists between the degree of gene mutation in the endothelial cells and brain AVM severity, and that dysregulation of endothelial arteriovenous specification enhances AVM formation and progression.

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Skeletal myofiber VEGF regulates contraction-induced perfusion and exercise capacity but not muscle capillarity in adult mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00533.2015 ◽

2016 ◽

Vol 311 (1) ◽

pp. R192-R199 ◽

Cited By ~ 11

Author(s):

Amy E. Knapp ◽

Daniel Goldberg ◽

Hamid Delavar ◽

Breanna M. Trisko ◽

Kechun Tang ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Contraction ◽

Exercise Capacity ◽

Fatigue Resistance ◽

Gene Deletion ◽

Endurance Capacity ◽

Force Frequency ◽

Vegf Gene ◽

Adult Mice ◽

Skeletal Myofiber

A single bout of exhaustive exercise signals expression of vascular endothelial growth factor (VEGF) in the exercising muscle. Previous studies have reported that mice with life-long deletion of skeletal myofiber VEGF have fewer capillaries and a severe reduction in endurance exercise. However, in adult mice, VEGF gene deletion conditionally targeted to skeletal myofibers limits exercise capacity without evidence of capillary regression. To explain this, we hypothesized that adult skeletal myofiber VEGF acutely regulates skeletal muscle perfusion during muscle contraction. A tamoxifen-inducible skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF−/−) was used to reduce skeletal muscle VEGF protein by 90% in adult mice. Three weeks after inducing deletion of the skeletal myofiber VEGF gene, skmVEGF−/− mice exhibited diminished maximum running speed (−10%, P < 0.05) and endurance capacity (−47%; P < 0.05), which did not persist after 8 wk. In skmVEGF−/− mice, gastrocnemius complex time to fatigue measured in situ was 71% lower than control mice. Contraction-induced perfusion measured by optical imaging during a period of electrically stimulated muscle contraction was 85% lower in skmVEGF−/− than control mice. No evidence of capillary rarefication was detected in the soleus, gastrocnemius, and extensor digitorum longus (EDL) up to 8 wk after tamoxifen-induced VEGF ablation, and contractility and fatigue resistance of the soleus measured ex vivo were also unchanged. The force-frequency of the EDL showed a small right shift, but fatigue resistance did not differ between EDL from control and skmVEGF−/− mice. These data suggest myofiber VEGF is required for regulating perfusion during periods of contraction and may in this manner affect endurance capacity.

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Abstract 305: Pressure Overload-Induced Myocardial Hypertrophy Causes an Electrical Remodeling of CLC-3 Chloride Channels in Mouse Heart

Circulation Research ◽

10.1161/res.115.suppl_1.305 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Dayue D Duan ◽

Ying Yu ◽

Guanlei Wang ◽

Lingyu L Ye ◽

Yi-gang Li

Keyword(s):

Heart Failure ◽

Chloride Channels ◽

Myocardial Hypertrophy ◽

Pressure Overload ◽

Left Ventricular ◽

Cell Swelling ◽

Protective Mechanism ◽

Electrical Remodeling ◽

Mouse Heart ◽

Constitutive Activation

Backgrand: Myocardial hypertrophy causes an increase in myocyte volume and constitutive activation of a volume-sensitive outwardly-rectifying anion channel (VSORAC). The underlying molecular mechanisms and function of VSORAC in the electrical remodeling during myocardial hypertrophy and heart failure remain undefined. We tested whether cardiac CLC-3 chloride channels play a role in the hypertrophy-induced electrophysiological remodeling. Methods and Results: The age-matched CLC-3 knockout (Clcn3-/-) mice and their wild-type (Clcn3+/+) littermates were subjected to minimally-invasive transverse aortic banding (MTAB). In 77% (44/57) left ventricular (LV) myocytes isolated from MTAB-Clcn3+/+ mice a large VSORAC current was activated under isotonic conditions. Hypotonic cell-swelling caused no changes in the VSORAC but hypertonic cell-shrinkage significantly inhibited it. This constitutively-activated VSORAC had an anion selectivity of I->Cl->Asp-, and was inhibited by tamoxifen, PKC activation and intracellular application of anti-CLC-3 antibody. In the age-matched MTAB-Clcn3-/- mice, a significantly smaller outwardly-rectifying current was present in only 38% (36/94, P<0.05 vs MTAB-Clcn3+/+) LV myocytes. This current was neither increased by hypotonic stress nor inhibited by tamoxifen, PKC or anti-CLC-3 antibody, indicating not a VSORAC or CLC-3 current. Expression of CLC-3 protein was significantly increased in the LV tissues of MTAB-Clcn3+/+ mice but not in Sham-Clcn3+/+ and MTAB-Clcn3-/- mice. Both surface and intracardiac electrophysiological recordings revealed more atrial or ventricular arrhythmias in MTAB-Clcn3-/- mice than in MTAB- and Sham-Clcn3+/+ mice. Conclusions: Pressure-overload-induced myocardial hypertrophy causes an upregulation of CLC-3 expression and constitutive activation of CLC-3 may serve as a novel protective mechanism against the electrical remodeling during myocardial hypertrophy and heart failure.

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Runx1 deletion or dominant inhibition reduces Cebpa transcription via conserved promoter and distal enhancer sites to favor monopoiesis over granulopoiesis

Blood ◽

10.1182/blood-2011-12-397091 ◽

2012 ◽

Vol 119 (19) ◽

pp. 4408-4418 ◽

Cited By ~ 62

Author(s):

Hong Guo ◽

Ou Ma ◽

Nancy A. Speck ◽

Alan D. Friedman

Keyword(s):

Myeloid Leukemia ◽

Gene Deletion ◽

Myeloid Cells ◽

Myeloid Differentiation ◽

Distal Enhancer ◽

Leukemic Transformation ◽

Adult Mice ◽

Runx1 Gene ◽

P300 Binding ◽

Marrow Cells

Abstract Deletion of Runx1 in adult mice produces a myeloproliferative phenotype. We now find that Runx1 gene deletion increases marrow monocyte while reducing granulocyte progenitors and that exogenous RUNX1 rescues granulopoiesis. Deletion of Runx1 reduces Cebpa mRNA in lineage-negative marrow cells and in granulocyte-monocyte progenitors or common myeloid progenitors. Pu.1 mRNA is also decreased, but to a lesser extent. We also transduced marrow with dominant-inhibitory RUNX1a. As with Runx1 gene deletion, RUNX1a expands lineage−Sca-1+c-kit+ and myeloid cells, increased monocyte CFUs relative to granulocyte CFUs, and reduced Cebpa mRNA. Runx1 binds a conserved site in the Cebpa promoter and binds 4 sites in a conserved 450-bp region located at +37 kb; mutation of the enhancer sites reduces activity 6-fold in 32Dcl3 myeloid cells. Endogenous Runx1 binds the promoter and putative +37 kb enhancer as assessed by ChIP, and RUNX1-ER rapidly induces Cebpa mRNA in these cells, even in cycloheximide, consistent with direct gene regulation. The +37 kb region contains strong H3K4me1 histone modification and p300-binding, as often seen with enhancers. Finally, exogenous C/EBPα increases granulocyte relative to monocyte progenitors in Runx1-deleted marrow cells. Diminished CEBPA transcription and consequent impairment of myeloid differentiation may contribute to leukemic transformation in acute myeloid leukemia cases associated with decreased RUNX1 activity.

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AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure

Basic Research in Cardiology ◽

10.1007/s00395-021-00880-w ◽

2021 ◽

Vol 116 (1) ◽

Author(s):

Anca Remes ◽

Andreas H. Wagner ◽

Nesrin Schmiedel ◽

Markus Heckmann ◽

Theresa Ruf ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Hypertrophy ◽

Myocardial Hypertrophy ◽

Cardiomyocyte Hypertrophy ◽

Activated T Cells ◽

Specific Expression ◽

Adult Mice ◽

Before And After ◽

A Cell

AbstractPrevious studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

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Inducible Fli-1 gene deletion in adult mice modifies several myeloid lineage commitment decisions and accelerates proliferation arrest and terminal erythrocytic differentiation

Blood ◽

10.1182/blood-2010-02-270405 ◽

2010 ◽

Vol 116 (23) ◽

pp. 4795-4805 ◽

Cited By ~ 25

Author(s):

Joëlle Starck ◽

Michèle Weiss-Gayet ◽

Colette Gonnet ◽

Boris Guyot ◽

Jean-Michel Vicat ◽

...

Keyword(s):

Cell Sorting ◽

Gene Deletion ◽

Liquid Cultures ◽

Drastic Decrease ◽

Differentiated Cells ◽

Adult Mice ◽

Myeloid Progenitor ◽

Ets Transcription Factor ◽

Late Stages

Abstract This study investigated the role of the ETS transcription factor Fli-1 in adult myelopoiesis using new transgenic mice allowing inducible Fli-1 gene deletion. Fli-1 deletion in adult induced mild thrombocytopenia associated with a drastic decrease in large mature megakaryocytes number. Bone marrow bipotent megakaryocytic-erythrocytic progenitors (MEPs) increased by 50% without increase in erythrocytic and megakaryocytic common myeloid progenitor progeny, suggesting increased production from upstream stem cells. These MEPs were almost unable to generate pure colonies containing large mature megakaryocytes, but generated the same total number of colonies mainly identifiable as erythroid colonies containing a reduced number of more differentiated cells. Cytological and fluorescence-activated cell sorting analyses of MEP progeny in semisolid and liquid cultures confirmed the drastic decrease in large mature megakaryocytes but revealed a surprisingly modest (50%) reduction of CD41-positive cells indicating the persistence of a megakaryocytic commitment potential. Symmetrical increase and decrease of monocytic and granulocytic progenitors were also observed in the progeny of purified granulocytic-monocytic progenitors and common myeloid progenitors. In summary, this study indicates that Fli-1 controls several lineages commitment decisions at the stem cell, MEP, and granulocytic-monocytic progenitor levels, stimulates the proliferation of committed erythrocytic progenitors at the expense of their differentiation, and is a major regulator of late stages of megakaryocytic differentiation.

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