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Atrial fibrillation (AF) patients experience inflammation and vascular dysfunction and have elevated levels of cytokines that promote vascular leak and edema, such as vascular endothelial growth factor (VEGF). We previously identified edema-induced disruption of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains as a novel arrhythmia mechanism. Therefore, we hypothesized that: (i) VEGF-induced vascular leak acutely slows action potential propagation in the atria and increases arrhythmia risk by disrupting ID nanodomains, and (ii) protection of the vascular barrier can prevent vascular leak-induced atrial arrhythmias. Electron microscopy revealed ID nanodomain swelling, near both gap junctions (GJ) and mechanical junctions (MJ) following VEGF treatment (60 minutes) in mouse hearts. Super-resolution STORM and STED microscopy both revealed Na V 1.5 enrichment at GJ and MJ relative to other ID sites in control hearts. VEGF reduced Na V 1.5 enrichment at both sites, consistent with Na V 1.5 translocation from ID nanodomains. VEGF increased distance from GJs to 90% of Na V 1.5 signal (3.17μm vs. 0.47 μm in vehicle controls), measured by a distance transformation-based analysis of 3D confocal images of IDs. VEGF slowed atrial conduction (optical mapping) and increased atrial arrhythmia incidence (ECG) relative to vehicle controls in both ex vivo (80 vs 0%) and in vivo (70 vs 20%) studies. Overall, in vivo arrhythmia burden was higher in VEGF-treated mice (7.5±11 vs. 0±6s/hr in vehicle controls). Preserving the vascular barrier by blocking endothelial Panx1 channels (PxIL2P peptide) decreased VEGF-induced in vivo arrhythmia burden (0 ± 6.09 s/hr with 1.6 μM PxIL2P). Concurrently, distance from GJs to 90% of Na V 1.5 was restored to control levels (0.57μm) in these hearts. In summary, these results highlight inflammation-induced vascular leak as a novel AF mechanism, and suggest vascular barrier protection as an anti-arrhythmic strategy.

Objective— Several physiological stimuli activate smooth muscle cell (SMC) G q PCRs (G q protein–coupled receptors) to cause vasoconstriction. As a protective mechanism against excessive vasoconstriction, SMC G q PCR stimulation invokes endothelial cell vasodilatory signaling. Whether Ca 2+ influx in endothelial cells contributes to the regulation of G q PCR-induced vasoconstriction remains unknown. Ca 2+ influx through TRPV4 (transient receptor potential vanilloid 4) channels is a key regulator of endothelium-dependent vasodilation. We hypothesized that SMC G q PCR stimulation engages endothelial TRPV4 channels to limit vasoconstriction. Approach and Results— Using high-speed confocal microscopy to record unitary Ca 2+ influx events through TRPV4 channels (TRPV4 sparklets), we report that activation of SMC α 1 ARs (alpha 1 -adrenergic receptors) with phenylephrine or thromboxane A 2 receptors with U46619 stimulated TRPV4 sparklets in the native endothelium from mesenteric arteries. Activation of endothelial TRPV4 channels did not require an increase in Ca 2+ as indicated by the lack of effect of L-type Ca 2+ channel activator or chelator of intracellular Ca 2+ EGTA-AM. However, gap junction communication between SMCs and endothelial cells was required for phenylephrine activation or U46619 activation of endothelial TRPV4 channels. Lowering inositol 1,4,5-trisphosphate levels with phospholipase C inhibitor or lithium chloride suppressed phenylephrine activation of endothelial TRPV4 sparklets. Moreover, uncaging inositol 1,4,5-trisphosphate profoundly increased TRPV4 sparklet activity. In pressurized arteries, phenylephrine-induced vasoconstriction was followed by a slow, TRPV4-dependent vasodilation, reflecting activation of negative regulatory mechanism. Consistent with these data, phenylephrine induced a significantly higher increase in blood pressure in TRPV4 −/− mice. Conclusions— These results demonstrate that SMC G q PCR stimulation triggers inositol 1,4,5-trisphosphate–dependent activation of endothelial TRPV4 channels to limit vasoconstriction.

Objective— In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. Approach and Results— We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. Conclusions— Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.

Objective— Hemoglobin α (Hb α) and endothelial nitric oxide synthase (eNOS) form a macromolecular complex at myoendothelial junctions; the functional role of this interaction remains undefined. To test if coupling of eNOS and Hb α regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb α to disrupt this interaction. Approach and Results— In silico modeling of Hb α and eNOS identified a conserved sequence of interaction. By mutating portions of Hb α, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb α sequence (Hb α X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb α and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb α X significantly decreased interaction between eNOS and Hb α. Fluorescein isothiocyanate labeling of Hb α X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb α X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb α X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb α X, which caused a significant decrease in blood pressure; injection of Hb α X into eNOS -/- mice had no effect. Conclusions— These results identify a novel sequence on Hb α that is important for Hb α/eNOS complex formation and is critical for nitric oxide signaling at myoendothelial junctions.

Objective— Plasminogen activator inhibitor-1 (PAI-1) has previously been shown to be key to the formation of myoendothelial junctions (MEJs) in normal and pathological states (eg, obesity). We therefore sought to identify the mechanism whereby PAI-1 could be selectively accumulated at the MEJ. Methods and Results— We identified PAI-1 protein enrichment at the MEJ in obese mice and in response to tumor necrosis factor (TNF-α) with a vascular cell coculture. However, PAI-1 mRNA was also found at the MEJ and transfection with a PAI-1–GFP with TNF-α did not demonstrate trafficking of the protein to the MEJ. We therefore hypothesized the PAI-1 mRNA was being locally translated and identified serpine binding protein-1, which stabilizes PAI-1 mRNA, as being enriched in obese mice and after treatment with TNF-α, whereas Staufen, which degrades PAI-1 mRNA, was absent in obese mice and after TNF-α application. We identified nicotinamide phosphoribosyl transferase as a serpine binding protein-1 binding partner with a functional τ-like microtubule binding domain. Application of peptides against the microtubule binding domain significantly decreased the number of MEJs and the amount of PAI-1 at the MEJ. Conclusion— We conclude that PAI-1 can be locally translated at the MEJ as a result of a unique mRNA binding protein complex.

Arterial vascular reactivity is a fundamental process to the regulation of blood pressure, blood flow and oxygen delivery and is governed in part by endothelial nitric oxide (NO) synthase (eNOS). We recently demonstrated that monomeric hemoglobin α (Hb α) is 1) expressed in arterial endothelial cells, 2) enriched at the myoendothelial junction, 3) forms a complex with eNOS and 4) regulates the effect of eNOS signaling during vascular reactivity. Based on these results, we hypothesized that a macromolecular complex may form between Hb α and eNOS to regulate NO signaling. In silico modeling of potential Hb α and eNOS protein-protein interaction sites identified a discreet pocket and sequence where Hb α and eNOS may interface. Therefore, we created a TAT-linked peptide identical to the Hb α sequence (Hb α-X) along with a scrambled control (Hb α-scr). Using recombinant eNOS and Hb α protein, we demonstrate by coimmunoprecipitation a loss of protein-protein interaction in the presence of the Hb α-X peptide but not the Hb α-scr. Consistent with these results, the Hb α-X peptide incubated with thoracodorsal arteries exhibited disruption of the Hb α and eNOS complex measured by coimmunoprecpitation and by a proximity ligation technique. Next, we performed vascular reactivity studies, which resulted in significantly altered responses to both phenylephrine and acetylcholine with the Hb α-X peptide, which were reversed with a NOS inhibitor. To test the in vivo effect of these peptides on blood pressure, we administered the peptides to mice via intraperitoneal injection and performed real-time measurements using radio telemetry. Mice injected with the Hb α-X peptide showed a significant decrease in blood pressure (MAP= -10 mmHg). Collectively, these results identify a novel Hb α sequence that is critical for Hb α/eNOS interaction, control of arterial vascular reactivity and regulation of blood pressure.

Intro Connexins are integrally involved in the process of cell division and atherosclerosis. Recently we identified that connexin 43 (Cx43) could significantly alter smooth muscle cell proliferation in vitro and in vivo. We now investigate the involvement of Cx43 in human neointimal formation and the mechanisms through which it controls cell cycle. Methods Protein interactions between Cx43 were identified in smooth muscle cells through co-immunoprecipitation, immuno-transmission electron microscopy and proximity ligation assays. Novel mice with alanine substitutions at MAPK phosphorylated serines (Cx43-MK4A) were investigated. Samples from diseased and non-diseased human coronary arteries were assessed for level of Cx43 phosphorylation and interactions. Ex vivo saphenous vein explant cultures were used to assess the contribution of Cx43 phosphorylation to human neointimal formation. In vitro studies were performed in human coronary artery smooth muscle cells and in mouse aortic smooth muscle cells from wild type and knockout mice. Results We demonstrate that levels of Cx43 are increased in human atherosclerotic plaques and that neointimal formation in human tissues corresponds to a significant increase in Cx43 phosphorylation and cyclin E interactions. In vitro, Cx43 facilitates trafficking of cyclin E to the nucleus in a manner that is dependent on its MAPK phosphorylation. Discussion Our studies suggest that in human vascular disease and neointimal formation Cx43 interactions with cyclin E are elevated. Further, Cx43 facilitates the transport of the cell cycle promoter cyclin E to the nucleus suggesting that this is a critical component of VSMC proliferation and therefore could be a novel target in the prevention of neointimal formation in human vascular disease.

Objective— In endothelial cells (EC) and vascular smooth muscle cells (VSMC) from atherosclerotic mice, connexin (Cx) expression becomes distorted. Lipoprotein-derived phospholipid oxidation products (OxPAPC) play a critical role in atherosclerosis, and we hypothesized that they may act as trigger molecules causing the changes in connexin expression. Methods and Results— We applied OxPAPC to murine carotid arteries in vivo and vascular cell cocultures. OxPAPC applied to carotids induced an upregulation of both Cx37 and Cx43 in the VSMC. In EC, Cx43 was upregulated and Cx37 was downregulated, whereas Cx40 in EC remained constant. In the vascular cell coculture, OxPAPC caused similar changes in Cx37 and Cx43 but caused a decrease in Cx40 in EC and an elevation of Cx40 in VSMC. In the coculture model, OxPAPC treatment led to the selective disappearance of Cx40 at the myoendothelial junction. Biocytin dye transfer between EC and VSMC coupling was dramatically reduced by OxPAPC. The decrease in dye transfer after OxPAPC treatment was correlated with an increase in tyrosine 265 phosphorylation of Cx43, especially at the in vitro myoendothelial junction. Conclusions— We conclude that OxPAPC could be responsible for the changes in connexin expression previously reported in atherosclerosis.