SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells

<div><p>Background</p><p>Hemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1—transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca²⁺-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process.</p><p>Methods</p><p>Cerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied <i>in vitro</i>, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways.</p><p>Results</p><p>Cerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused <i>de novo</i> expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca²⁺-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion.</p><p>Conclusions</p><p>tPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.</p></div

SUR1-TRPM4 channel upregulation <i>in vitro</i>.

<p><b>A</b>: Activity of the <i>Abcc8</i> promoter, the <i>Trpm4</i> promoter and a positive control (four consecutive NF-κB consensus sequences), under basal conditions and after stimulation by TNF (20 ng/mL); 3 replicates; *, <i>P</i><0.05; **, <i>P</i><0.01. <b>B</b>: Murine BEC were exposed to TNF (20 ng/mL) and lysate was analyzed by RT-PCR 16 hours later; transcripts for <i>Abcc8</i> and <i>Trpm4</i> were upregulated compared to control (CTR); the induction of <i>Abcc8</i> mRNA by TNF was reduced by the NF-κB inhibitor, PDTC (pyrrolidinedithiocarbamate); β-actin mRNA was used as a loading control; representative of 3 replicates. <b>C</b>: Macroscopic and single channel (inside-out patch) currents induced by ATP depletion in activated but not in non-activated (CTR) murine BEC; currents were blocked by glibenclamide (Glib) and 9-phenanthrol (9-PHE). <b>D</b>: Single channel current in an inside-out patch with 4 single channel levels during changes in bath solution from 145 mM Cs⁺/1 μM Ca²⁺, 145 mM Cs⁺/0 μM Ca²⁺,145 mM Cs⁺/1 μM Ca²⁺, 0 mM Cs⁺/75 mM Ca²⁺, 145 mM Cs⁺/1 μM Ca²⁺. <b>E</b>: Quantification of macroscopic currents at –50 mV induced by ATP depletion or diazoxide in activated or non-activated (CTR) murine BEC; currents were blocked by glibenclamide and by gene deletion of <i>Abcc8</i> (SUR1KO) and gene suppression of <i>Trmp4</i> (siRNA); cells/condition for each bar: 35, 12, 14, 19, 11, 11; 24, 19; 9, 13; **, <i>P</i><0.01; ***, <i>P</i><0.001. <b>F</b>: Immunoblots for pCaMKII and CaMKII, with densitometric quantification of pCaMKII in murine BEC without activation, under control conditions (CT) and after exposure to the Ca²⁺ ionophore (CI), A23187 (5 μM × 10 minutes), and in activated BEC exposed to TNF in the absence and presence of glibenclamide (Glib); both bands shown had molecular masses of 50 kD, corresponding to pCaMKIIα and CaMKIIα; pCaMKII normalized to levels induced by A23187; n = 5.</p

SUR1-TRPM4 channel opening by tPA requires Ca²⁺ influx via TRPC3.

<p><b>A</b>: Macroscopic currents during 200-ms step pulses (#1,3,5) and ramp pulses (–100 to +100 mV; 4 mV/msec) (#2,4), induced by recombinant tPA (rtPA) in activated murine BEC, recorded initially using extracellular solution with 1.8 mM Ca²⁺, and after switching to extracellular solution containing 0 mM Ca²⁺; <i>right</i>: illustrative currents during ramp pulses after addition of rtPA, before and after switch to 0 Ca²⁺; the difference current is also shown (thick line). <b>B</b>: rtPA-induced current in activated BEC was not blocked by ruthenium red (RR), but was blocked by Gd⁺³ and Pyr3; illustrative currents during ramp pulses after addition of rtPA, before and after Gd⁺³ or Pyr3 are also shown; <i>bar graph</i>: rtPA-induced currents in activated BEC in the presence of 1.8 mM Ca²⁺, 0 mM Ca²⁺; 1.8 mM Ca²⁺ plus RR or SKF-96365 or Pyr3 or Gd³⁺; cells/condition for each bar: 25, 7, 5, 7, 5, 7. <b>C</b>: Change in intracellular Ca²⁺ concentration (ΔF/F₀) induced by rtPA in activated but not in non-activated (Ctr) BEC; the rtPA-induced increase in Ca²⁺ was blocked by pretreatment with SKF-96365 but not RR; <i>bar graph</i>: mean change at 10–12 minutes in intracellular Ca²⁺ concentration induced by rtPA in non-activated and activated BEC, in the presence of RR; SKF-96365; Gd³⁺; cells/condition for each bar: 10, 8, 10, 10, 10; ***, <i>P</i><0.001.</p

Human BEC upregulate SUR1-TRPM4 channels that are involved in tPA-induced phasic secretion of MMP-9.

<p><b>A–D</b>: Macroscopic currents induced by recombinant tPA (rtPA) in activated but not in non-activated (CTR) human BEC; currents were blocked by glibenclamide (Glib) and 9-phenanthrol (9-PHE); n = 6–10 cells/condition; **, <i>P</i><0.01. <b>E</b>: Gelatin zymography showing that after activation, rtPA and PAR1-agonist TFLLR induce <i>phasic</i> secretion of MMP-9 in human BEC that is inhibited by glibenclamide; the bar graphs represent densitometric measurements of total MMP-9; the lanes and the bars in the graph are aligned for the different conditions: PAR1-agonist (TFLLR), rtPA and glibenclamide (Glib); n = 4; *, <i>P</i><0.05.</p

Plasmin opens the SUR1-TRPM4 channel.

<p><b>A</b>: Control experiment showing requirement for proteolytic activity; change in intracellular Ca²⁺ concentration (ΔF/F₀) induced by recombinant tPA (rtPA) in activated murine BEC, in the absence and presence of the non-specific serine threonine protease inhibitors (PI’s), aprotinin and leupeptin; representative of 5–7 cells per condition. <b>B</b>: Control experiment showing non-involvement of NMDA (N-methyl-D-aspartate) receptor; change in intracellular Ca²⁺ induced by glutamate plus glycine (Glu+Gly) in non-activated (CTR) and in activated BEC; representative of 7 cells per condition. <b>C</b>: In activated murine BEC, rtPA fails to induce SUR1-TRPM4 current in the presence of tranexamic acid (TXA), although Ca²⁺ influx via A23187 activates the channel; exogenous plasmin induces SUR1-TRPM4 current blocked by glibenclamide (Glib); bar graph showing SUR1-TRPM4 currents under the conditions indicated; illustrative currents during ramp pulses are also shown; cells/condition for each bar: 25, 11, 8, 5, 5. <b>D</b>: In activated murine BEC, rtPA fails to induce SUR1-TRPM4 current in the presence of PAR1-antagonist RWJ56110, although Ca²⁺ influx via A23187 activates the channel; PAR1-agonist SFLLRN induces SUR1-TRPM4 current that is blocked by glibenclamide; bar graph showing SUR1-TRPM4 currents under the conditions indicated; illustrative currents during ramp pulses are also shown; cells/condition for each bar: 25, 12, 11, 5, 5. <b>E</b>: Plasmin induces Ca²⁺ influx (ΔF/F₀) in activated but not non-activated (CTR) murine BEC; 5–8 cells per condition. <b>F</b>: rtPA induces phosphorylation of ERK1/2 (p42/44 MAPK); bar graph showing >20% increase in p-ERK42 and p-ERK44 due to rtPA; n = 3; **, <i>P</i><0.01.</p

Primer sequences for RT-PCR.

<p>Primer sequences for RT-PCR.</p

rtPA and PAR1-agonist cause secretion of MMP-9 from activated endothelial cells that is reduced by SUR1 inhibition.

<p><b>A</b>: RT-PCR showing that activation of murine BEC upregulates MMP-9 but not MMP-2 mRNA; n = 5. <b>B</b>: Gelatin zymography showing that non-activated murine BEC secrete minimal MMP-9, and that <i>tonic</i> secretion of MMP-9 and MMP-2, both pro and active forms, following overnight activation is not affected by glibenclamide; n = 5. <b>C</b>: Gelatin zymography showing that after activation, rtPA and PAR1-agonist TFLLR induce <i>phasic</i> secretion of MMP-9 that is inhibited by glibenclamide; n = 5; **, <i>P</i><0.01. <b>D</b> <i>left</i>: Immunoblot showing suppression of SUR1 after infection with shAbcc8 lentiviral vector; CTR, untreated cells; Scr, lentiviral vector with scrambled shRNA. <b>D</b> <i>right</i>: Gelatin zymography showing that after overnight activation, <i>phasic</i> secretion of MMP-9 induced by TFLLR is inhibited by pretreatment of the cells with shRNA against <i>Abcc8</i> (*); n = 3.</p

Primer sequences for qRT-PCR.

<p>Primer sequences for qRT-PCR.</p

Proposed mechanism for tPA-induced phasic secretion of MMP-9 by NF-κB-activated brain endothelium.

<p>tPA catalyzes the cleavage of plasminogen, yielding plasmin. Plasmin activates the G-protein coupled receptor, PAR1, by proteolytic cleavage of its N-terminal domain at Arg 41, allowing its tethered ligand to bind intramolecularly to activate the receptor. Activated PAR1 signals via the G-protein, Gαq, activating phospholipase Cβ (PLCβ), which catalyzes the cleavage of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP₂) into the second messengers, inositol (1,4,5) trisphosphate (IP₃) and diacylglycerol (DAG). DAG activates TRPC3, resulting in Ca²⁺ influx. DAG-induced Ca²⁺ influx triggers phasic secretion of MMP-9, and causes activation of SUR1-TRPM4, which results in Na⁺ influx. Cell depolarization due to Na⁺ influx reduces the inward electrochemical driving force for Ca²⁺, consistent with SUR1-TRPM4 functioning as a negative regulator of Ca²⁺ influx.</p

Right atrial volume by cardiovascular magnetic resonance predicts mortality in patients with heart failure with reduced ejection fraction

<div><p>Background</p><p>Right Atrial Volume Index (RAVI) measured by echocardiography is an independent predictor of morbidity in patients with heart failure (HF) with reduced ejection fraction (HFrEF). The aim of this study is to evaluate the predictive value of RAVI assessed by cardiac magnetic resonance (CMR) for all-cause mortality in patients with HFrEF and to assess its additive contribution to the validated Meta-Analysis Global Group in Chronic heart failure (MAGGIC) score.</p><p>Methods and results</p><p>We identified 243 patients (mean age 60 ± 15; 33% women) with left ventricular ejection fraction (LVEF) ≤ 35% measured by CMR. Right atrial volume was calculated based on area in two- and four -chamber views using validated equation, followed by indexing to body surface area. MAGGIC score was calculated using online calculator. During mean period of 2.4 years 33 patients (14%) died. The mean RAVI was 53 ± 26 ml/m²; significantly larger in patients with than without an event (78.7±29 ml/m² vs. 48±22 ml/m², p<0.001). RAVI (per ml/m²) was an independent predictor of mortality [HR = 1.03 (1.01–1.04), p = 0.001]. RAVI has a greater discriminatory ability than LVEF, left atrial volume index and right ventricular ejection fraction (RVEF) (C-statistic 0.8±0.08 vs 0.55±0.1, 0.62±0.11, 0.68±0.11, respectively, all p<0.02). The addition of RAVI to the MAGGIC score significantly improves risk stratification (integrated discrimination improvement 13%, and category-free net reclassification improvement 73%, both p<0.001).</p><p>Conclusion</p><p>RAVI by CMR is an independent predictor of mortality in patients with HFrEF. The addition of RAVI to MAGGIC score improves mortality risk stratification.</p></div