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

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

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

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