Calcium handling property of the cardiomyocytes derived from empty construct- and apoA-I-transduced ESCs.

<p>(A) Representative tracings of rhythmic spontaneous Ca2+ transients in cardiomyocytes derived from empty construct- and apoA-I- transduced cells. (B): Amplitude, (C) Maximal upstroke velocity (Vmax upstroke), (D) Maximal decay velocity (Vmax decay) of Ca2+ transients in the mESC-derived cardiomyocytes. (E) Representative tracings of caffeine-induced Ca2+ release from sarcoplasmic reticulum in cardiomyocytes derived from wild type, empty construct and apoA-I-1α transduced cells (right), demonstrating caffeine-sensitive Ca2+ stores and fractional release of total sarcoplasmic reticulum Ca2+ load during spontaneous activation. (F): Amplitude, (G) Maximal upstroke velocity (Vmax upstroke), (H) Maximal decay velocity (Vmax decay) of Ca2+ transients in the ESC-derived cardiomyocytes. Data shown as mean ± SEM from the recordings of 20–30 cells from 3–5 independent experiments, * p<0.05; ** p<0.005.</p

Inhibition of the BMP4 signaling pathway abolished the pro-cardiogenic effects of apoA-I gene transfer.

<p>The BMP4 signaling pathway was inhibited by the application of noggin (1 µ/ml) to the apoA-I- transduced ESCs upon plating. (A) The phosphorylation status of the differentiating cells was evaluated 24 hr after noggin treatment. (B) The appearance of beating clusters during the 8-day period of differentiation. (C) The percentage of troponin-T positive cells as determined by flow cytometry analysis. Data shown as mean ± SEM from 3 independent experiments, ^# p<0.05 comparing to the LV-apoA-I group; *p<0.05 and ** p<0.005 comparing to the LV-GFP group.</p

Primers used in the real-time quantitative RT-PCR analysis.

<p>Abbreviation: ALK1: ; ALK2: ; α-MHC: alpha-myosin heavy chain; β-MHC: beta-myosin heavy chain; MLC2v: myosin light chain 2v; GATA4: GATA binding protein 4; Nkx2.5: NK2 transcription factor related, locus 5; NCX: sodium calcium exchanger; SERCA-2a: sarcoplasmic reticulum Calcium ATPase 2a; RyR2: ryanodine receptor 2.</p

apoA-I gene transfer activates the BMP4-SMAD1/5 signaling cascade in undifferentiated ESCs.

<p>(A) The mRNA levels of bone morphogenic protein 4 (BMP4), activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 2 (ALK2) in the undifferentiated ESCs transduced with empty construct or apoA-I were evaluated by real-time quantitative PCR analysis using ribosomal protein S16 as internal control. (B) The phosphorylation status and total protein levels of the SMAD proteins were evaluated with Western blot analysis using β-actin as loading control. (C) The densitometry quantification of the Western blot results. Data shown as mean ± SEM from 3 independent experiments,* p<0.05; ** p<0.005.</p

Synergistic effect of recombinant apoA-I and BMP4 on the cardiac differentiation of D3 ESCs.

<p>Untransduced D3 ESCs were subjected to cardiac differentiation in the presences or absence of recombinant apoA-1 (100 nM) and/or BMP4 (0.5 ng/ml). The appearance of beating clusters during the 8-day period of differentiation were recorded. Data shown as mean ± SEM from 3 independent experiments, ** p<0.005 comparing to the control group.</p

Expression of human apoA-I in D3 mouse ESCs.

<p>Undifferentiated ESCs were cultured in the absence of a feeder layer and transduced with lentiviral particles containing the full-length wild type apoA-I cDNA under the control of CMV promoter. This expression cassette was linked to the IRES-GFP reported cassette for the identification of transduced cells, while the lentiviral particles containing the empty construct served as a control. To evaluate the success of gene transfer, undifferentiated empty construct- and apoA-I-transduced cells were examined for expression of GFP (A). The secretion of apoA-I in the concentrated spent medium was determined by Western blot analysis using an antibody specific to apoA-I.</p

Primary Antibodies used for Western Blot analysis.

<p>Primary Antibodies used for Western Blot analysis.</p

Effect of apoA-I gene transfer on generation of beating embryoid body and cardiac cells.

<p>(A) Empty construct and apoA-I-transduced mouse ESCs were differentiated using the conventional “hanging-drop” method, and the resultant embryoid bodies (EBs) were plated onto gelatin coated plates. The occurrence of beating areas within the EBs was observed and counted for 8 days starting from the day of plating. (B) Percentage of ESC-derived cardiomyocytes (troponin-T positive cells) on day 8 as determined by flow cytometry. (C) Individual cardiomyocytes were isolated from the beating area of the EBs and identified with immunnohistochemistry using antibody specific to the cardiac troponin-T. (D) Cardiac maker gene expression in the embryoid bodies derived from empty construct- and apoA-I-transduced ESCs as revealed by real-time quantitative PCR analysis. MHCA: α-myosin heavy chain; MHCB: β-myosin heavy chain; MLC2V: myosin light chain 2 ventricular transcripts. Data shown as mean ± SEM from at least 3 independent experiments, n = 3–5, *p<0.05; **p<0.005.</p

The expression of calcium handling components in cardiomyocytes.

<p>The mRNA levels of sodium/calcium exchanger (NCX1), sarcoplasmic reticulum Ca2+ ATPase (SERCA2A) and ryanodine receptor 2 (RYR2) of embryoid bodies (at d8 after plating) were evaluated by real-time quantitative PCR analysis using ribosomal protein S16 as internal control. Data shown as mean ± SEM from 3 independent experiments, ** p<0.005.</p

Additional file 1: of PR interval prolongation in coronary patients or risk equivalent: excess risk of ischemic stroke and vascular pathophysiological insights

Table S1. Univariable and Multivariable Predictors for Carotid IMT. Table S2. Univariable and Multivariable Predictors for Cardiovascular Death. Table S3. Univariable and Multivariable Predictors for New-Onset Ischemic Stroke. Table S4. Univariable and Multivariable Predictors for New-Onset Myocardial Infarction. Table S5. Univariable and Multivariable Predictors for Combined Cardiovascular Endpoints of New-Onset Myocardial Infarction, Ischemic Stroke, Congestive Heart Failure and Cardiovascular Death. Table S6. Estimates of Sensitivity (Se), Specificity (Sp), Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of PR Interval in the Prediction for Cardiovascular Events. (DOC 444 kb