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Transplantation of cardiotrophin-1–expressing myoblasts to the left ventricular wall alleviates the transition from compensatory hypertrophy to congestive heart failure in Dahl salt-sensitive hypertensive rats

Ryuji Toh, MD^*, Seinosuke Kawashima, MD, PhD^*,*, Miki Kawai, MD, PhD^*, Tsuyoshi Sakoda, MD, PhD, Tomomi Ueyama, MD, PhD^*, Seimi Satomi-Kobayashi, MD^*, Sonoko Hirayama, MD, PhD^* and Mitsuhiro Yokoyama, MD, PhD^*

^* Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
Department of Internal Medicine, Cardiovascular Division, Hyogo College of Medicine, Nishinomiya, Japan

View larger version (87K): [in a new window]   Figure 1 (A) Representative tracings of left ventricular M-mode echocardiograms of the sham group at the age of 11 weeks (upper left) and 17 weeks (upper right), the myoblast transplantation (MB) group (lower left), and the transplantation of CT-1–expressing myoblasts (MB + CT) group (lower right) at the age of 17 weeks. Left ventricular dilation and contractile dysfunction were attenuated in both MB and MB + CT groups at the heart failure stage (six weeks after transplantation). (B) Serial measurements of echocardiography in the sham, MB, and MB + CT groups. A p value by two-way analysis of variance: group <0.001; time course <0.001; group/time course interaction <0.001 for each parameter. *p < 0.05 and **p < 0.01 vs. sham group; p < 0.05, and p < 0.01 vs. MB group at same stage by Bonferroni's multiple-comparison t test. Values are means ± SEM. EDD = end-diastolic diameter; FS = fractional shortening; PWT = posterior wall thickness; W = the age (weeks).

View larger version (70K): [in a new window]   Figure 2 Serial sections of transplanted area at the age of 17 weeks (six weeks after transplantation). Left panel shows hematoxylin-eosin (H-E) staining. Multinuclear elongated structure is typical of myotubes (arrows). Right panel shows immunohistochemical staining for skeletal-specific myosin heavy chain by MY-32. Positive staining with MY-32 demonstrates the presence of myotubes (arrows). Original magnification, x400.

View larger version (90K): [in a new window]   Figure 3 (A) Detection of secreted cardiotrophin-1 (CT-1) in culture media by Western blot analysis. Human CT-1 was detected as a 26-kDa band. Only nonspecific staining was seen in 10% FBS-DMEM and the conditioned media from parental myoblasts. In the media from CT-1-transferred myoblasts, CT-1 was detected in addition to nonspecific staining. Twice, infections of retrovirus augmented the CT-1 expression. (B) Transplanted area stained for CT-1 in the myoblast (MB) group (left) and the MB + CT group (right) at the age of 17 weeks (six weeks after transplantation). Grafted cells in the MB + CT group showed positive staining for CT-1 (arrows). Original magnification, x400. (C) Western blot analysis for CT-1 in the left ventricular free wall. Each protein level of CT-1 was normalized to a mean value of sham group (n = 5 for each). *p < 0.05 vs. sham group. Values are means ± SEM.

View larger version (88K): [in a new window]   Figure 4 (A) Photomicrographs of cardiac myocytes in the anterior aspect of left ventricular (LV) free wall at the age of 17 weeks in sham (left), myoblast (MB) (center), and MB + cardiotrophin (CT) (right) groups. The bar indicates 50 µm. (B) Relative cross-sectional area of cardiac myocytes in the LV (open bars), the free wall (solid bars), and the septum (striped bars) of the LV. Myocyte size was normalized to a mean value of Dahl salt-sensitive rats at the age of six weeks. *p < 0.05 vs. sham group by analysis of variance and Bonferroni's multiple-comparison t test. p < 0.05 versus the septum wall by paired t test. Values are means ± SEM. (C) Phase-contrast photomicrographs of cultured neonatal rat ventricular cardiac myocytes (CM). Cardiac myocytes were treated as described for 48 h. The media from parental myoblasts showed no morphologic changes compared with control. The media from CT-1–transferred myoblasts induced myocardial cell hypertrophy, similar to that seen in 1 nM CT-1–incubated CMs. MyoblastCT-1 = CT-1–transferred myoblasts.

View larger version (12K): [in a new window]   Figure 5 Plasma levels of angiotensin II (Ang II) (A) and endothelin-1 (ET-1) (B) at the age of 13 weeks (open bars) and 17 weeks (solid bars). Values are means ± SEM. n = 5 per group. *p < 0.05 vs. sham group; p < 0.05 vs. myoblast (MB) group at same stage by analysis of variance and Bonferroni's multiple-comparison t test. CT = cardiotrophin.

View larger version (13K): [in a new window]   Figure 6 Proliferation and survival of parental and cardiotrophin-1 (CT-1)–transferred myoblasts in media containing 10% (A) or 0% (B) serum. Cell number was determined by the absorbance of the WST-8 reagent. Optical density values were normalized to the mean value of each group at 0 h. Data are presented as mean ± SEM of four measurements. *p < 0.05 and **p < 0.01 vs. parental C2C12 cells at same time point by analysis of variance and Bonferroni's multiple-comparison t test. C2C12CT-1 = CT-1–transferred C2C12 cells.