ABSTRACT
We have shown previously that during myocardial ischemia/reperfusion (MI/R), toll-like receptor 2 (TLR2) signaling regulates connexin 43 (Cx43) subcellular localization and function and dampens arrhythmia formation. We aimed to identify sites capable of TLR2-dependent redox modification within Cx43. Post-ischemic TLR2-/- or wild-type (WT) mouse hearts were analyzed by OxICAT. Cx43 was mutated to exclude redox modification and transfected into HL-1 cardiomyocytes (CM) that were challenged with a TLR2 agonist. We identified Cys260 of Cx43 to be susceptible to reversible oxidation MI/R; TLR2-/- leads to reduced H2O2 production in post-ischemic isolated mitochondria and subsequently reduced oxidation of Cx43 at Cys260. Cx43 was dephosphorylated in WT, while phosphorylation was preserved in TLR2-/-. Mutation of Cx43 (C260A) and lentiviral transfection in HL-1 CM accelerated pacemaker activity and reduced activity after TLR2 ligand stimulation. We here provide evidence for TLR2-dependent reversible oxidation of Cx43 at Cys260, which led to decreased Cx43 phosphorylation and affected CM pacemaker frequency and intercellular communication.
Subject(s)
Arrhythmias, Cardiac/metabolism , Connexin 43/metabolism , Heart Rate , Mitochondria, Heart/metabolism , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/metabolism , Toll-Like Receptor 2/metabolism , Action Potentials , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/pathology , Arrhythmias, Cardiac/physiopathology , Cell Communication , Cell Line , Connexin 43/deficiency , Connexin 43/genetics , Cysteine , Disease Models, Animal , Hydrogen Peroxide/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/pathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Myocytes, Cardiac/pathology , Oxidation-Reduction , Phosphorylation , Signal Transduction , Toll-Like Receptor 2/deficiency , Toll-Like Receptor 2/geneticsABSTRACT
RATIONALE: Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and micro-computed tomography (CT) was studied to establish potential alternatives to histology. METHODS: The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for in vivo labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent. RESULTS: Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving in vivo injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM. CONCLUSION: UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.
