Connexin 43

Western Blot analysis was used to quantify the expression of Cx43 in neuronal tissue. Therefore, neuronal tissue was homogenized, and RIPA-Buffer was added. After 5 min incubation in an ultrasound bath, samples were incubated another 10 min on ice. Protein levels were measured determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). For denaturation, sodium dodecyl sulfate buffer (SDS-buffer) and mercaptoethanol (9:1) were added and samples were incubated at 95 °C for 5 min. For gel electrophoresis, 10% SDS gels were prepared and samples were loaded into lanes using 40 µg protein/lane. After adequate separation (85 min at 100 V), proteins were transferred onto a polyvinylidene difluoride membrane applying 220 mA for 1 h. Further, the membrane was blocked with TBS-T for 2 h. After the administration of the primary antibody, the membrane was incubated overnight at 4 °C. As primary antibodies, Connexin 43 (Abcam, 1:10,000) and Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH Abcam, 1:50,000) were used. After elimination of unbound antibodies via washing three times with TBS-T, the secondary antibody was added and incubated for 2 h. After disposing the unbound antibodies, membrane-bound antibodies were detected by using the enhanced chemiluminescent detection method (cool snap HQ2; Photometrics^®, Tuscon, USA)

CSCs were isolated from the hearts of neonatal SD rats (3–5 days old) according to the previous methods1 with a minor modification. Briefly, the hearts were harvested from rats and minced into 1‐ to 2‐mm³ pieces, washed with phosphate‐buffered solution (PBS), and digested 3 times for 15 minutes at 37°C with 0.1% II collagenase (Worthington). The cell suspension after enzymatic digestion was collected and filtered with a strainer. Then, the mixed cells were incubated with anti–c‐kit biotin antibody (Sigma) and isolated through the use of anti‐biotin immunomagnetic microbeads (Miltenyi). The newly sorted c‐kit⁺ cells were grown in the fibronectin‐coated 60‐mm culture dish at 37°C and 5% CO₂. The expansion medium contained DMEM‐high glucose (Hyclone), 10% FBS (Gibco), 10 ng/mL basic fibroblast growth factor (Sigma), 10 ng/mL leukemia inhibitory factor (Sigma), and 5 U/L human erythropoietin (Sigma). When passaged, CSCs was digested by using Accutase (Millipore), which caused less damage to the cells. To detect the purity of the isolated CSCs, they (P₀) were identified by flow cytometry with antibody against c‐kit. To identify the ability of differentiation, the CSCs (P₃) were stained with immunofluorescence antibodies against desmin, Connexin‐43, α–smooth muscle actin, and CD31 (Abcam). In addition, the CSCs (P₀) were stained with immunofluorescence antibody against Ki‐67 (Abcam).