kn-93 and Fibrosis

This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-β administration.. The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-β1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal-regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic-reperfusion injury.. NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dextromethorphan; Dizocilpine Maleate; Epithelial-Mesenchymal Transition; Excitatory Amino Acid Antagonists; Fibrosis; Gene Knockdown Techniques; Humans; In Vitro Techniques; Kidney; Kidney Tubules, Proximal; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Protein Kinase Inhibitors; Receptors, N-Methyl-D-Aspartate; Renal Insufficiency, Chronic; Reperfusion Injury; Sulfonamides; Transforming Growth Factor beta; Ureteral Obstruction

A Pitx2c deficiency increases the risk of atrial fibrillation (AF). Atrial structural remodelling with fibrosis blocks electrical conduction and leads to arrhythmogenesis. A Pitx2c deficiency enhances profibrotic transforming growth factor (TGF)-β expression and calcium dysregulation, suggesting that Pitx2c may play a role in atrial fibrosis. The purposes of this study were to evaluate whether a Pitx2c deficiency modulates cardiac fibroblast activity and study the underlying mechanisms.. A migration assay, proliferation analysis, Western blot analysis and calcium fluorescence imaging were conducted in Pitx2c-knockdown human atrial fibroblasts (HAFs) using short hairpin (sh)RNA or small interfering (si)RNA.. Compared to control HAFs, Pitx2c-knockdown HAFs had a greater migration but a similar proliferative ability. Pitx2c-knockdown HAFs had a higher calcium influx with enhanced phosphorylation of calmodulin kinase II (CaMKII), α-smooth muscle actin and matrix metalloproteinase-2. In the presence of a CaMKII inhibitor (KN-93, 0.5 μmol/L), control and Pitx2c-knockdown HAFs exhibited similar migratory abilities.. These findings suggest that downregulation of Pitx2c may regulate atrial fibrosis through modulating calcium homeostasis, which may contribute to its role in anti-atrial fibrosis, and Pitx2c downregulation may change the atrial electrophysiology and AF occurrence through modulating fibroblast activity.

Topics: Actins; Arrhythmias, Cardiac; Atrial Fibrillation; Atrial Remodeling; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Movement; Cell Proliferation; Down-Regulation; Fibroblasts; Fibrosis; Gene Knockdown Techniques; Heart Atria; Homeobox Protein PITX2; Homeodomain Proteins; Humans; In Vitro Techniques; Matrix Metalloproteinase 2; Optical Imaging; Phosphorylation; Protein Isoforms; Protein Kinase Inhibitors; RNA, Small Interfering; Sulfonamides; Transcription Factors

Activation of Wnt signaling results in maladaptive cardiac remodeling and cardiomyopathy. Recently, calcium/calmodulin-dependent protein kinase II (CaMKII) was reported to be a pivotal participant in myocardial remodeling. Because CaMKII was suggested as a downstream target of noncanonical Wnt signaling, we aimed to elucidate the role of CaMKII in dishevelled-1-induced cardiomyopathy and the mechanisms underlying its function. Dishevelled-1-induced cardiomyopathy was reversed by deletion of neither CaMKIIδ nor CaMKIIγ. Therefore, dishevelled-1-transgenic mice were crossed with CaMKIIδγ double-knockout mice. These mice displayed a normal cardiac phenotype without cardiac hypertrophy, fibrosis, apoptosis, or left ventricular dysfunction. Further mechanistic analyses unveiled that CaMKIIδγ couples noncanonical Wnt signaling to histone deacetylase 4 and myosin enhancer factor 2. Therefore, our findings indicate that the axis, consisting of dishevelled-1, CaMKII, histone deacetylase 4, and myosin enhancer factor 2, is an attractive therapeutic target for prevention of cardiac remodeling and its progression to left ventricular dysfunction.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Benzylamines; beta Catenin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dishevelled Proteins; Fibrosis; Heart Failure; Histone Deacetylases; Hypertrophy, Left Ventricular; MAP Kinase Signaling System; MEF2 Transcription Factors; Mice; Mice, Knockout; Myocardium; Phenotype; Phosphoproteins; Protein Kinase C; Sulfonamides; Ultrasonography; Ventricular Dysfunction, Left; Ventricular Remodeling; Wnt Proteins; Wnt Signaling Pathway

Oxidative stress with hydrogen peroxide (H(2)O(2)) readily promotes early afterdepolarizations (EADs) and triggered activity (TA) in isolated rat and rabbit ventricular myocytes. Here we examined the effects of H(2)O(2) on arrhythmias in intact Langendorff rat and rabbit hearts using dual-membrane voltage and intracellular calcium optical mapping and glass microelectrode recordings. Young adult rat (3-5 mo, N = 25) and rabbit (3-5 mo, N = 6) hearts exhibited no arrhythmias when perfused with H(2)O(2) (0.1-2 mM) for up to 3 h. However, in 33 out of 35 (94%) aged (24-26 mo) rat hearts, 0.1 mM H(2)O(2) caused EAD-mediated TA, leading to ventricular tachycardia (VT) and fibrillation (VF). Aged rabbits (life span, 8-12 yr) were not available, but 4 of 10 middle-aged rabbits (3-5 yr) developed EADs, TA, VT, and VF. These arrhythmias were suppressed by the reducing agent N-acetylcysteine (2 mM) and CaMKII inhibitor KN-93 (1 microM) but not by its inactive form (KN-92, 1 microM). There were no significant differences between action potential duration (APD) or APD restitution slope before or after H(2)O(2) in aged or young adult rat hearts. In histological sections, however, trichrome staining revealed that aged rat hearts exhibited extensive fibrosis, ranging from 10-90%; middle-aged rabbit hearts had less fibrosis (5-35%), whereas young adult rat and rabbit hearts had <4% fibrosis. In aged rat hearts, EADs and TA arose most frequently (70%) from the left ventricular base where fibrosis was intermediate ( approximately 30%). Computer simulations in two-dimensional tissue incorporating variable degrees of fibrosis showed that intermediate (but not mild or severe) fibrosis promoted EADs and TA. We conclude that in aged ventricles exposed to oxidative stress, fibrosis facilitates the ability of cellular EADs to emerge and generate TA, VT, and VF at the tissue level.

Topics: Acetylcysteine; Action Potentials; Age Factors; Aging; Animals; Antioxidants; Benzylamines; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Communication; Computer Simulation; Disease Models, Animal; Electrocardiography; Enzyme Activation; Fibrosis; Heart Ventricles; Hydrogen Peroxide; Male; Models, Cardiovascular; Myocytes, Cardiac; Oxidants; Oxidative Stress; Perfusion; Protein Kinase Inhibitors; Rabbits; Rats; Rats, Inbred F344; Sulfonamides; Tachycardia, Ventricular; Time Factors; Ventricular Fibrillation