Introduction

Establishment of embryonic stem cell (ESC)-like cells (also know as induced pluripotent stem cells or iPSCs) by the reprogramming of adult somatic cells with a few defined transcription factors provides a fascinating route to generate patient-specific pluripotent cells as disease models and drug-testing systems1,2,3. Improvement of cardiac function by the transplantation of iPSC-derived cardiomyocytes (iPS-CMs) after myocardial infarction in animal models4 suggests a potential of using iPSCs in patient-specific cardiac regeneration5,6. However, to realize these application potentials, establishment of a highly efficient and easily practicable differentiation system is one of the prerequisites.

Cardiogenesis is a well-organized process tightly regulated by key developmental signals and extracellular microenvironment7,8. Although cardiomyocytes are successfully generated from mouse (m)9,10 and human (h)11,12 iPSCs in vitro, the cardiac differentiation efficiency remains very low5. Several attractive approaches focusing on the manipulation of critical signaling pathways to improve the cardiac differentiation efficiency of iPSCs have been reported currently13,14,15, while little is known about the contribution of manipulating extracellular microenvironments to the process of cardiac differentiation from iPSCs.

Another important obstacle hampering the utilization of iPSCs is the high interline variability in cardiac differentiation efficiency11,12,16, with some of the lines even showing no cardiac differentiation properties in vitro17. Therefore, a highly efficient and universal system must be developed to overcome or minimize such variations before the extensive use of iPSCs.

In addition, iPS-CMs have been proved to be less mature than those from ESCs or fetal hearts, reflected by the delayed development of sarcoplasmic reticulum and lower responses to β-adrenergic stimulus44.

To determine the apoptosis status of the cells, TUNEL staining was performed with the in situ Cell Death Detection kit (Roche, Mannheim, Germany) according to the manufacturer's instruction. Annexin V-PI double-stainings performed with PI (0.5 μg/ml) and APC-labeled Annexin V antibody (1:20; BD Biosciences) were further used to evaluate the apoptosis and necrosis levels. Cells were analyzed and quantified by flow cytometry.

Whole cell patch clamp

Whole cell patch clamps using EPC-10 amplifier (Heka Electronics, Bellmore, NY, USA) in current clamp mode were used to record APs in spontaneously beating iPS-CMs following the method described previously43. For AP recording, the pipette electrode (2∼6 MΩ) were filled with a solution containing (mmol/l): 50 KCl, 80 K-Asparate, 5 MgCl2, 5 EGTA, 10 Hepes, 5 Na2ATP (pH 7.2 adjusted with KOH); the extracellular bathing solution containing (mmol/l): 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 10.0 glucose and 10.0 HEPES (pH 7.4, adjusted with NaOH). The glass coverslips containing the cells were placed onto a temperature-controlled (35 °C) recording chamber and perfused continuously with extracellular solution.

Measurement of Ca2+ transients

Isolated mouse iPS-CMs were loaded with 5 μmol/l fura-2 AM and 0.45% pluronic F-127 (Molecular Probes, Eugene, OR, USA) for 10 min and washed in extracellular solution for 15 min at 35 °C room temperature. The cells were perfused continuously with extracellular solution at 35 °C. Fluorescence signals of fura-2 were detected by a Fluorescence System (IonOptix, Milton, MA). After subtraction of background fluorescence, the 340- to 380-nm fluorescence ratio (R) was recorded and analyzed by IonWizard 6.0 software (IonOptix).

Immunoblot analysis

Immunoblot analyses were performed according to the protocol described previously45. Protein samples were size fractionated by SDS-polyacrylamide gel electrophoresis and the separated proteins were electrophoretically transferred to polyvinylindene difluoride membranes (Bio-Rad, Hercules, CA, USA). Then the membrane was incubated with primary antibodies against p-ERK1/2 (1:1 000; Santa Cruz Biotechnology), total ERK1/2 (1:1 000; Cell Signaling), RyR2 (1:1 000; Abcam), SERCA2 (1:1 000; Santa Cruz Biotechnology), Phospholamban (1:2 000; Millipore), Connexin43 (1:500; Invitrogen), and GAPDH (1:1 000; Santa Cruz Biotechnology). Horseradish peroxidase-linked anti-rabbit (1:4 000; Santa Cruz Biotechnology) or anti-mouse antibodies (1:4 000; Sigma) were used as secondary antibodies.

Statistical analysis

Data were presented as means ± SEM. Statistical significance of differences was estimated by one way ANOVA or Student's t test by SigmaStat 3.5 software (Sigma). P < 0.05 was considered significant.