Cardiac Function and Outcome in Patients with Cardio-Embolic Stroke

<div><p>Background</p><p>The relationship between whole spectrum of Ejection fraction (EF) and cardioembolic stroke (CES) outcome has not been fully described yet. Notably, it remains unclear whether borderline EF (41∼49%) is related with poor outcome after CES. We sought to evaluate whether lower ejection fraction and borderline EF could predict the outcome in patients with CES.</p><p>Method and Results</p><p>We evaluated the relationship between EF and functional outcome in 437 consecutive patients with CES. EF was introduced as continuous and categorical (EF≤40%, EF 41∼49%, EF≥50%) variable. Patients with CES and the subgroup with AF were evaluated separately. Poor short-term outcome (modified Rankin Score≥3at discharge or death within 90 days after stroke onset) and long-term mortality were evaluated. A total of 165 patients (37.8%) had poor short-term outcomes. EF tends to be lower in patients with poor short-term outcome (56.8±11.0 vs. 54.8±12.0, p-value 0.086). Overall cumulative death was136 (31.1%) in all CES patients and 106 (31.7%) in the AF subgroup. In a multivariable model adjusted for possible covariates, the hazard ratio for mortality significantly decreased by 3% for every 1% increase in ejection fraction in CES patients and 2% for every 1% increase in the AF subgroup. Reduced EF (EF≤40%) showed higher mortality (HR 2.61), and those with borderline EF (41∼49%) had a tendency of higher mortality (HR 1.65, p-value 0.067)compared with those with normal EF.</p><p>Conclusion</p><p>We found a strong association between lower EF and CES outcome. Echocardiographic evaluation helps to better determine the prognosis in CES patients, even in subgroup of patients with AF.</p></div

Basic demographics.

<p>Values are mean±SD or number of patients (percentage).</p><p>AF: Atrial fibrillation, NIHSS: National Institutes of Health Stroke Scale, IV: Intravenous, IA: Intraarterial,</p

EF according to short-term functional outcome (A), 90 days mortality (B), and mRS in CES patients (C).

<p>ns: not significant, ***: p-value <0.01. Abbreviations: ns: not significant, EF, Ejection fraction.mRS: modified Rankin Scale, CES: Cardioembolic stroke.</p

Multivariable model hazard ratios for long-term outcomes by EF compared with normal values.

<p>Adjusted for age, sex, history of stroke, hypertension, diabetes, dyslipidemia, smoking,</p><p>Admission NIHSS (<7, 7–14, >14), IV or IA thrombolysis,discharge warfarin, hemorrhagic transformation.</p><p>EF: Ejection Fraction, CES: cardioembolic stroke, AF: Atrial fibrillation, NIHSS: National Institutes of Health Stroke Scale,</p><p>IV: Intra-venous, IA: Intra-arterial.</p

Flow chart of patient enrollment.

<p>Abbreviations: SNUHSR: Seoul National University Hospital Stroke Registry, CES: Cardio-embolic stroke, TIA: Transient ischemic attack, EF: Ejection Fraction.</p

Distribution of EF in included CES patients.

<p>Abbreviations: EF:Ejection fraction, CES: Cardioembolic stroke.</p

Kaplan-Meier curves of long-term mortality by EF groups in CES patients (A) and AF subgroup (B).

<p>Abbreviations: EF, Ejection fraction. CES: Cardioembolic stroke, AF: Atrial fibrillation.</p

Swiprosin-1 dimerizes through its coiled-coil domain, and calcium ions are required for the dimer conformation.

<p>(A) HEK293T cells (2×10⁶) were transiently transfected with 4 µg of GFP, GFP_Swip-1, and myc or increasing concentrations (4, 7, and 11 µg) of myc_Swip-1. The cell lysates were immunoprecipitated with anti-GFP-conjugated beads. Immune complexes were resolved on by SDS-PAGE and blotted with anti-GFP or anti-myc antibodies. (B) HEK293T cells (2×10⁶) were transiently transfected with GFP, GFP_Swip-1, or mutant GFP_SW1s (M1, M2, and M3). The cell lysates were incubated on ice with glutaraldehyde (GA) at the indicated concentrations (0.001–0.01%) for 20 min. The samples were resolved on SDS-PAGE and blotted with anti-GFP antibodies. The positions of monomer (M), dimer (D), tetramer (T), and GFP alone (G) are indicated by arrows. (C) The purified wild-type His_Swip-1 or wild-type GST_Swip-1 (a) and coiled-coil domain deletion mutants (l, m, and n) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right). (D) The cells from (A) were incubated for 1 h with 20 µM BAPT-AM or 2 µM ionomycin. The cell lysates were immunoprecipitated in the presence of 2 mM EGTA (BAPTA-treated cells) or 1 mM CaCl2 (ionomycin-treated cells), and the amount of binding protein as well as the expression of the indicated proteins were then evaluated by western blotting. All the procedures were performed in the presence of 10 µM cytochalasin D to exclude the effect of actin polymerization. (E) The purified wild-type His_Swip-1 and wild-type GST_Swip-1 (a) or coiled-coil domain containing mutants (h, i) were co-incubated with glutathione (GSH)-Sepharose 4B beads for 2 h at 4°C in the presence or absence of 2 mM EGTA, and the samples were then resolved by SDS-PAGE and blotted with anti-His antibodies (left). Each sample was compared to a loading control (right).</p

Swiprosin-1 is located in the F-actin-rich region and mediates cell spreading and lamellipodium formation.

<p>(A) (A–a) Localization of endogenous swiprosin-1 and actin in Jurkat T cells conjugated with superantigen SEE-pulsed Raji B cells. White arrows show the contact region of T and B cells. Scale bars: 10 µm. (A–b) Localization of GFP_Swip-1 or GFP_actin in Jurkat T cells or human PBLs. Jurkat T cells or PBLs were transfected with GFP, GFP_Swip-1, or GFP_actin. After 24 h of incubation, the cells were incubated with anti-CD3/CD28-coated beads or SEE-pulsed Raji B cells for 30 min. The fluorescence signals were analyzed by confocal microscopy. (A–c) Jurkat T cells were infected with GFP or GFP_Swip-1 lentiviral vector and the expression efficiency was evaluated using a flow cytometer. The cells were plated on FN-coated coverslips and treated with SDF-1α. After 20 min, images were captured using a confocal microscope, and the degree of spreading T cells was quantitated. White arrows indicate spreading cells. Scale bars: 20 µm. NT = no treatment. <i>*P</i><0.05 <i>vs.</i> GFP-infected cells. (B) Western blot analysis of swiprosin-1 expression in Jurkat T, 293T, HeLa, and CHO-K1 cells. The cell lysates were resolved on by SDS-PAGE and blotted with anti-Swip-1 antibodies. (C-a) CHO-K1 cells were transfected with GFP or GFP_Swip-1. After 48 h of incubation, the indicated cells were placed on PLL- or FN-coated coverslips for 1 h. F-actin was stained with phalloidin- TRITC. The cells were imaged using confocal microscopy with reconstitution in the z-axis. White arrow indicates the area of lamellipodium. The average area of the cells (C–b) and lamellipodia formation observed by scores (C–c) were quantitated as described in the Materials and Methods. The results are expressed as the mean ± SD of triplicate experiments <i>*P</i><0.05 <i>vs.</i> GFP-transfected cells. (C–d) GFP or GFP_Swip-1-transfected CHO-K1 cells were plated on FN for 1 h. Cells were then lysed and subjected to the Rac1 activity assay.</p

Swiprosin-1 mediates cell spreading and migration.

<p>(A) HeLa cells were transfected with scrambled siRNA or siRNA targeting <i>swiprosin-1</i> for 48 h, and the efficiency of siRNA transfection was then determined by RT-PCR (A–a, top) and Western blotting (A–a, bottom). The cells were plated on FN-coated coverslips for 30 and 60 min. The average cell area (A–b) and percentage of fully spread cell (cell area >1500 µm²) (A–c) were then determined. (A–d) F-actin was stained with phalloidin-Alexa 488, and the fluorescence signals were analyzed by confocal microscopy. Scale bars: 20 µm. (B) The cells from (A) were subjected to a wound-healing assay. The average rates of wound closure were determined 10 h after the wounding (B–a) and visualized by phase contrast microscopy (B–b). The results are expressed as the mean ± SD of triplicate experiments. Sc, scrambled. <i>*P</i><0.05 <i>vs.</i> scrambled siRNA-transfected cells.</p