(A) Reverse transcriptase-polymerase chain reaction. In the single mesenchymal stem cell (MSC) culture condition, treatment of MSCs with cocktails of growth factors (GFs) does not affect the expression of cardiac specific genes except for the alpha-sarcomeric actin. But in coculture condition with cardiomyocytes (CMCs), treatment of GFs significantly augmented the induction of cardiac specific genes in MSCs, such as atrial natriuretic peptide, connexin-43 (Cx43), cardiac troponin-I (cTnI), alpha and beta myosin heavy chain (MHC) genes. (B) Western blot analysis in the single MSC culture condition. The expression of GATA-4 and NKx-2.5 were distinctively induced only in GF-treated MSCs. (C) Western blot analysis after 2 days of coculture with CMCs. The GF treatment augmented the induction of cTnI and Cx43 protein expression in MSC by coculture with CMCs. (D) The percentage of MSCs expressing Cx43 was significantly higher in the GF-treated MSCs than in naïve ones. Data were derived from 5 randomly chosen microscopic fields of 3 separate experiments. (E) Representative images after 2 days of coculture of MSCs with CMCs. The MSCs were labeled with DAPI (blue). The CMCs were stained with TnI (red) without DAPI. Connexin-43 (green) was expressed more frequently in the treated MSCs compared with the untreated ones. Connexin-43, which indicates the formation of gap junction (arrows), was expressed linearly between MSCs and CMCs (TnI positive cells without DAPI). Arrowheads indicate MSCs expressing both TnI (red) and Cx43. *The MSCs expressing only Cx43 (magnification x200). T = treated; UT = untreated.

(A and B) The MSCs labeled with 1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (red) and calcein-AM (green) were cocultured with unlabeled CMCs. Only green calcein-AM is transmittable through functioning gap junction. Cells having green fluorescence without red denote CMCs that are connected with labeled MSCs through functioning gap junction. These cells were scarcely observed in the coculture with untreated MSCs (A), whereas they were frequently observed in the coculture with GF-treated MSCs (B). (C) After addition of heptanol into coculture condition with treated MSC and CMCs, there are no CMCs having green fluorescence alone, corroborating that calcein transfer was mediated through gap junction (magnification x200). (D) Treatment with GFs significantly increased intercellular communication between MSCs and CMCs. Data were derived from 5 randomly chosen microscopic fields of 3 separate experiments. HPF = high power field; other abbreviations as in Figure 1.

(A to D) Fluorescence-activated cell sorter (FACS) for evaluating apoptosis. The CMCs were exposed to hypoxia alone as a control (A), under coculture with untreated MSCs (B), and GF-treated MSCs (C). Hypoxia-induced apoptosis of CMCs was reduced by coculture with MSCs, which was more notable in coculture condition with GF-treated MSCs. Heptanol partially inhibited cytoprotective effects of GF-treated MSCs on CMCs (D). (E) Western blot analysis. The expression of phospho-Akt (p-Akt) and phospho-c-AMP response element binding protein (p-CREB) increased in CMCs cocultured with GF-treated MSCs under hypoxia, which was reversed by gap junctional blocker. (F) Enzyme-linked immunosorbent assay (ELISA) for insulin-like GF (IGF)-1 and hepatocyte GF (HGF). There was no significant difference in secretion of IGF-1 and HGF between untreated and GF-treated MSCs. Abbreviations as in Figure 1.

(A) Representative images of infarcted rat heart. Heart obtained 8 weeks after myocardial infarction (MI), and transplantation of GF-treated MSC showed smaller infarct than that with transplantation of untreated MSCs (Masson’s trichrome staining: blue indicates infarcted scar tissue). (B) The area of fibrosis was significantly smaller in the infarcted heart transplanted with GF-treated MSCs compared with that with untreated MSCs (n = 7 in each group). (C) Representative figures of echocardiographic finding. The anterior wall motion was preserved better in the GF-treated MSC group. (D and E) Although there was no significant difference at baseline, percent fractional shortening (%FS) was higher in the group transplanted with GF-treated MSC than with untreated MSC or with media at 8 weeks after MI. Left ventricular end-diastolic dimension (LVEDD) was significantly smaller in groups with MSC transplantation than with media at 4 weeks, but there was no significant difference at 8 weeks. The MI with media injection (n = 8), MI with transplantation of untreated MSC (n = 10), and MI with GF-treated MSC (n = 9). *p < 0.05 versus media injection group; #p < 0.05 versus untreated MSC group. Abbreviations as in Figure 1.

(A and B) Representative figures of immunostaining at 2 and 8 weeks after MI. Transplanted MSCs, labeled with DiI (red) before injection and with DAPI (blue) at nuclei, were engrafted in the myocardium (unlabeled CMCs with DAPI nuclear staining only). There was significantly higher connexin-43 expression in the infarcted heart transplanted with the GF–pre-treated MSCs than with the untreated MSCs. Connexin-43 (green) was expressed between MSCs (arrows) or between MSCs and CMCs (arrowheads). (C) The percentage of MSCs expressing connexin-43 was significantly higher in the GF–pre-treated MSCs than in the untreated ones. (D) DiI-positive area, which might reflect the engraftment area of transplanted MSCs, was significantly greater in the heart transplanted with GF–pre-treated MSCs than with the untreated ones at 2 weeks after MI. The statistical significance was lost at 8 weeks. (E) Low-power views, which showed that DiI-positive area at 2 weeks was greater in the infarcted myocardium transplanted with GF–pre-treated MSCs than with naïve ones. (F) Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. (G) Immunohistochemistry for caspase-3. At 2 weeks after MI, apoptosis (arrows) was significantly lower in the infarcted heart transplanted with GF-pretreated MSCs than with untreated ones. (H) Three randomly selected fields in 2 separate slides/animal (n = 5 in each group) were counted. Abbreviations as in Figures 1, 2, and 5.

(A) Representative figures of electrocardiographic recording in rats. (B) The arrhythmia score according to Lambeth Convention showed a tendency to be lower in the infarcted heart transplanted with GF–pre-treated MSCs than in the heart with the untreated MSCs (1.1 ± 0.6 vs. 2.4 ± 0.7, p = 0.09). (C) The difference of premature ventricular complex counts between the GF-treated MSC and the untreated group did not reach statistical significance (402 ± 288 vs. 753 ± 305, p = 0.11). PVC = premature ventricular contraction.; other abbreviations as in Figure 1.