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
Manuscript received October 23, 2003;
revised manuscript received December 30, 2003,
accepted February 3, 2004.
* Reprint requests and correspondence: Dr. Seinosuke Kawashima, Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
kawashim{at}med.kobe-u.ac.jp
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Abstract
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OBJECTIVES: We investigated whether autologous transplantation of skeletal myoblasts (MB) transferred with cardiotrophin-1 (CT-1) gene could retard the transition to heart failure (HF) in Dahl salt-sensitive (DS) hypertensive rats.
BACKGROUND: Although MB is a therapeutic candidate for chronic HF, little is known about the efficiency of this strategy when applied in nonischemic HF. Cardiotrophin-1 has potent hypertrophic and survival effects on cardiac myocytes. We hypothesized that transplantation of CT-1–expressing myoblasts could provide cardioprotective effects against ventricular remodeling in DS hypertensive rats.
METHODS: The DS rats were fed a high salt diet for 6 weeks and developed left ventricular (LV) hypertrophy at 11 weeks. At this stage, animals underwent MB to the myocardium with skeletal myoblasts transferred with CT-1 gene using retrovirus (transplantation of CT-1–expressing myoblasts [MB + CT], n = 31) or myoblasts alone (MB, n = 31). The sham group rats were injected with phosphate-buffered saline (n = 24).
RESULTS: At 17 weeks, MB and MB + CT groups showed a significant alleviation of LV dilation and contractile dysfunction compared with the sham group. The degree of alleviation was significantly greater in the MB + CT group than the MB group (LV end-diastolic dimension: sham 7.06 ± 0.14 mm, MB 6.51 ± 0.16 mm, MB + CT 6.24 ± 0.07 mm; fractional shortening: sham 32.1 ± 1.4%, MB 38.5 ± 1.5%, MB + CT 43.2 ± 0.8%). Histological examination revealed that the myocyte size was 20% larger in the MB + CT group at 17 weeks than in the age-matched sham group. Upregulation of renin-angiotensin and endothelin systems during the transition to HF was attenuated by myoblast transplantation, and this effect was enhanced in the MB + CT group.
CONCLUSIONS: Transplantation of skeletal myoblasts combined with CT-1-gene transfer could be a useful therapeutic strategy for HF.
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Abbreviations and Acronyms
| | Ang II | = angiotensin II | | CT-1 | = cardiotrophin-1 | | DS | = Dahl salt-sensitive | | EDD | = end-diastolic dimension | | ET-1 | = endothelin-1 | | %FS | = percent fractional shortening | | HF | = heart failure | | LV | = left ventricle or left ventricular | | MB | = transplantation of skeletal myoblasts alone | | MB + CT | = transplantation of cardiotrophin-1–expressing myoblasts | | PWT | = posterior wall thickness |
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Despite medical and surgical advances, heart failure (HF) is still a major cause of death. Because mature cardiac myocytes cannot re-enter the cell cycle and the adult heart lacks functional repair mechanisms, myogenic cell transplantation into the damaged myocardium is a promising approach to the treatment for end-stage HF (1). Recent experimental studies have demonstrated that intramyocardial skeletal myoblast transplantation improves cardiac function after myocardial infarction (2–5). Clinical trials with skeletal myoblast transplantation are also on the way (6,7). However, little is known about the efficiency of this strategy when applied in global HF of nonischemic causes (8–10).
The Dahl salt-sensitive (DS) hypertensive rats undergo the transition from compensatory hypertrophy to congestive HF (11). When they are fed a high salt diet after the age of 6 weeks, they develop systemic hypertension and concentric left ventricular (LV) hypertrophy at 11 weeks, followed by marked LV dilation and contractile dysfunction at 15 to 20 weeks (11). Thus, they have been used as an animal model for nonischemic hypertension-based HF. We investigated whether autologous transplantation of skeletal myoblasts (MB) could retard the transition from compensatory hypertrophy to HF in DS hypertensive rats.
Skeletal myoblast transplantation is also useful as a tool for cell-mediated gene therapy, providing sustained local expression of recombinant proteins in the heart (12,13). Cardiotrophin-1 (CT-1), a member of the interleukin-6 superfamily, induces hypertrophy and prolongs survival of cardiac myocytes in vitro (14–16). It has been reported that CT-1 expression in the myocardium is upregulated in chronic HF (17,18). However, the pathophysiologic significance of CT-1 in the transition from compensated to decompensated HF is not fully elucidated (18). We accordingly analyzed the feasibility and efficiency of transplanting skeletal myoblasts that are transferred with CT-1 gene using retrovirus in preserving cardiac function of DS hypertensive rats. We hypothesized that this strategy could provide cardioprotective effects against ventricular remodeling in combination with functional benefits of skeletal myoblast-derived muscle.
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Methods
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Animal model and isolation of primary skeletal myoblasts.
All animal experiments were performed in accordance with the guidelines for animal experimentation at Kobe University Graduate School of Medicine. Male DS rats were obtained from Japan SLC Co. Ltd (Hamamatsu, Japan). After weaning, the rats were fed a low salt diet (0.3% NaCl) until the age of six weeks; thereafter, a high salt diet (8% NaCl) was started. The myoblast culture process was performed according to the method reported previously (2). In brief, skeletal muscles were harvested from hind limb skeletal muscle of DS rats, and then they were minced and seeded on polystyrene plates. Tissue pieces were incubated at 37°C for 48 h in Dulbecco's Modified Eagle's Medium (Sigma, St. Louis, Missouri) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin, and 500 µg/ml penicillin (all from Sigma) to allow primary satellite cell (myoblast) isolation. When cells began to migrate out of tissue, tissue pieces were removed. Myoblast growth densities were maintained at <70% to avoid the differentiation into the myotube (2).
Retrovirus-mediated CT-1–gene transfer.
The full-length complementary deoxyribonucleic acid of the rat CT-1 was obtained from total ribonucleic acid in neonatal rat ventricular cells using reverse transcriptase-polymerase chain reaction. Sense and antisense primers for rat CT-1 were 5'-TCTATGAGCCAGAGGGAGGGAAGTCTG-3' and 5'-TATTCAGGCAACGCCCCCTGGCAC-3', respectively (14). The polymerase chain reaction fragment was confirmed by deoxyribonucleic acid sequencing and then inserted into pLN plasmid (generously provided by Dr. Noriyuki Kasahara, USC Institute for Genetic Medicine, Los Angeles, California) (19) to construct a retroviral transfer vector encoding the rat CT-1 complementary deoxyribonucleic acid sequence (pLN-CT-1). Retroviral vector encoding CT-1 was produced as described previously, with slight modifications (19,20). Briefly, a 100-mm dish of nonconfluent 293T cells were transfected with 15 µg of pLN-CT-1, 15 µg of pHIT 60 (i.e., cytomegalovirus [CMV] gag pol encoding plasmid), and 10 µg pHIT 123 (CMV ecotropic envelope) using the calcium phosphate coprecipitation method (21); 16 h after transfer, the media were adjusted to a final concentration of 10 mM in sodium butyrate. After 8-h incubation, cells were washed and incubated in fresh medium without sodium butyrate. Conditioned medium containing retrovirus was harvested 16 h later and filtered through 0.45-µm Millipore-HA (Millipore Co., Bedford, Massachusetts). For transfer of CT-1 gene into the myoblasts, cells were infected overnight with a dilution of virus stock in cultured medium supplemented with 8 µg of polybrane per ml. Infections were performed twice for efficient transfer into myoblasts.
Cell transplantation.
At the age of 11 weeks, animals underwent thoracotomy under general anesthesia with intraperitoneal sodium pentobarbital (50 mg/kg) and autologous transplantation to the myocardium with CT-1-transferred myoblasts (MB + CT group, n = 31) or myoblasts alone (MB group, n = 31). Cells (1 x 106) were harvested and resuspended in 0.15 ml of phosphate-buffered saline, followed by intramyocardial injection 10 to 15 times into the anterior aspects of the LV free wall with a 26G needle. Accordingly, approximately 1 x 105 cells were injected in each site. In the sham group (n = 24), the same volume of phosphate-buffered saline was injected. The surgical wounds were repaired, and the rats were returned to the cages to recover.
Echocardiographic and hemodynamic studies.
Systolic blood pressure and heart rate were measured by a tail-cuff method (Muromachi Kikai, Japan).
At the age of 11, 15, and 17 weeks, transthoracic two-dimensional echocardiography (SONOS 5500, Philips Medical Systems Corp., Andover, Massachusetts) was performed under light anesthesia with sodium pentobarbital. A 12-MHz Ultraband Sector Transducer (Philips Medical Systems Corp.) probe was used. Left ventricular end-diastolic dimension (EDD), end-systolic dimension (ESD), and LV posterior wall thickness (PWT) were determined from the M-mode tracing based on the short-axis view of the LV at the papillary muscle level. Left ventricular percent fractional shortening (%FS) was calculated as: [(EDD – ESD)/EDD] x 100.
At the age of 17 weeks, animals underwent direct cardiac catheterization via subdiaphragmatic approach to measure LV pressure under light anesthesia. The catheter was connected to a pressure transducer, and continuous measurements of LV pressure and heart rate were recorded using a Maclab system (Bioresearch Center, Nagoya, Japan). Animals were allowed to breathe spontaneously during the pressure recording. Maximal rate of pressure rise (dP/dtmax) and LV end-diastolic pressure were determined from tracings of LV pressure, and averaged on 100 consecutive cardiac cycles.
Western blot analysis.
At the age of 13 weeks, three hearts from each group were collected to assess CT-1 expression in the heart. The isolated LV free wall was cut into small pieces and homogenized with a Polytron homogenizer (Kinematica Inc., Cincinnati, Ohio). Homogenates were centrifuged, and the supernatants were collected. For detection of secreted CT-1 from the transferred myoblasts, myoblasts were cultured in DMEM supplemented with 10% FBS, and conditioned media were collected after 72 h incubation. The expression of CT-1 protein was determined by Western blot analysis using an anti-human CT-1 polyclonal antibody (Pepro Tech EC Ltd., London, United Kingdom). The results were quantified by scanning densitometry.
Histological analysis.
Left ventricular specimens were obtained at the age of 17 weeks (n = 5 for each group). Specimens were frozen with liquid nitrogen and sectioned to 8-µm-thick slices. The slices were stained with hematoxylin-eosin. The slices also underwent immunohistochemical staining for skeletal-specific myosin heavy chain by MY-32 monoclonal antibody (Sigma-Aldrich Inc., St. Louis, Missouri), and for CT-1.
In the hematoxylin-eosin–stained sections, the cross-sectional area of cardiac myocytes that was cut transversely and showed nuclei in the center was measured in the free wall and the septum of the LV, respectively. In each side of LV wall, approximately 50 cells were counted per each animal. Before myoblast transplantation, the myocyte size was measured at the age of 6 and 11 weeks (n = 5 for each stage). A total of 100 cells in random areas of LV, including both the free wall and the septum, were counted per each animal, and the average was used for analysis.
Measurements of plasma angiotensin II (Ang II) and endothelin-1 (ET-1) levels.
At the age of 13 and 17 weeks, blood was collected in a polypropylene tube containing aprotinin (300 kallikrein-inhibiting units/ml) and ethylenediamine-tetraacetic acid (1 mg/ml) and then centrifuged at 3,000 rpm for 15 min at 4°C. The plasma thus obtained was stored at –80°C until assayed. The plasma levels of Ang II and ET-1 were measured by SRL, Inc. (Tokyo, Japan).
Effects of transferred-gene–derived CT-1 in vitro.
Myoblasts were cultured in DMEM supplemented with 0.1% bovine serum albumin, ITS (10 µg/ml insulin, 10 µg/ml transferrin, and 10 ng/ml selenious acid), and conditioned media were collected after 72-h incubation. Primary culture of neonatal rat cardiac myocytes was prepared as previously described (21). Cardiac myocytes were treated with either DMEM supplemented with 0.1% bovine serum albumin and ITS (control), the media from myoblasts with or without CT-1–transfer, or 1 nM recombinant human CT-1 (Pepro Tech EC Ltd.) for 48 h. Then cellular morphology was examined and photographed under light microscopy.
Proliferation and survival tests in myoblasts were performed by use of C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia). Briefly, 5,000 cells were plated in 96-well dishes and grown for 24-h in media containing 10% FBS. The media were then changed to 0% or 10% serum-media, and cell number was determined by the absorbance of the WST-8 reagent (Dojindo Co., Kumamoto, Japan) at 0, 8, 24, and 48 h after media replacement.
Statistical procedures.
All values were expressed as mean ± SEM. The serial measurements of echocardiography were assessed using two-way analysis of variance for repeated measures. The differences at specific stages among groups were analyzed by one-way analysis of variance, followed by Bonferroni's multiple-comparison t test. Paired t test was used to assess significant differences in myocyte size between areas in each group. Statistical analyses were performed using StatView (version 5.0, SAS Institute Inc., Cary, North Carolina). Values were considered statistically significant at p < 0.05.
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Results
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Functional assessment after cell transplantation.
The DS rats who were fed a high salt diet developed systemic hypertension (>220 mm Hg) at the age of 11 weeks, which continued until the age of 17 weeks. There was no significant difference in systolic blood pressure among three groups throughout the experiment (Table 1). At the age of 14 to 17 weeks, two rats in the sham group and one rat in the MB group deceased. These animals showed labored respiration with a loss of activity before they died. Therefore, the cause of their death seemed to be congestive heart failure. At the age of 17 weeks, LV end-diastolic pressure increased, and LV dP/dtmax decreased in the sham group, reflecting congestive HF (Table 2). In contrast, LV end-diastolic pressure was not increased, and LV dP/dt was relatively preserved in both the MB and MB + CT groups. Moreover, LV dP/dtmax was significantly higher in the MB + CT group than in the MB group (p < 0.01) (Table 2).
Representative M-mode echocardiograms of the LV at the papillary muscle level were shown in Figure 1A. At the age of 11 weeks, the DS rats developed concentric LV hypertrophy, and there were no differences in preoperative data among the three groups (Fig. 1). At the age of 17 weeks, the sham group showed marked LV dilation and global hypokinesis (Fig. 1A). From the age of 11 to 17 weeks, a marked decrease in %FS and an increase in EDD occurred in sham group, which was associated with a reduction in PWT (%FS, 50.9 ± 0.4 vs. 32.1 ± 1.4; EDD, 5.68 ± 0.02 vs. 7.06 ± 0.14 mm; PWT, 2.12 ± 0.05 vs. 1.68 ± 0.02 mm) (Fig. 1B). In contrast, LV dilation was attenuated, and contractile function was maintained significantly in both the MB and MB + CT groups at the age of 17 weeks compared with the age-matched sham group (Fig. 1). Moreover, %FS, EDD, and PWT were more preserved in the MB + CT group than in the MB group at the age of 17 weeks (%FS 43.2 ± 0.8 vs. 38.5 ± 1.5; EDD 6.24 ± 0.07 vs. 6.51 ± 0.16 mm; PWT 1.79 ± 0.02 vs. 1.73 ± 0.02 mm, p < 0.05, respectively) (Fig. 1B). On the other hand, B-mode echocardiogram did not exhibit asymmetrical LV wall motion after cell transplantation in both the MB and MB + CT groups (data not shown).
We also performed Holter electrocardiogram in some animals at the age of 17 weeks, which revealed no lethal arrhythmias after cell transplantation (n = 3 in the MB group and n = 2 in the MB + CT group, data not shown).
Grafted myoblasts in the myocardium.
Serial sections of the transplanted area after cell transplantation were shown in Figure 2. Graft survival was identified at six weeks after transplantation (the age of 17 weeks) by hematoxylin-eosin staining and immunohistochemical staining for skeletal-specific myosin heavy chain, by MY-32 mAb. Multinuclear elongated structure was identified in H-E staining, which indicates that myoblasts had differentiated into myotubes (Fig. 2). These muscular structures were positively stained with MY-32 (Fig. 2), whereas no cells were stained in the PBS-injected hearts. Positive staining for skeletal myosin heavy chain revealed the presence of myotubes. Surviving cells aligned with the cardiac fiber axis within the native myocardium. On the other hand, accumulation of inflammatory cells was hardly detected around the transplanted area at day 0 and two weeks, four weeks, and six weeks after transplantation in both MB and MB + CT groups. As reported previously, the fibrosis was found mainly in perivascular regions of the arterioles (11), and there were no differences in the extent of fibrosis among the three groups.
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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.
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CT-1 expression in the myocardium after cell transplantation.
The secretion of transferred gene-derived CT-1 was confirmed in vitro by Western blot analysis (Fig. 3A). Immunohistochemical staining of the transplanted area for CT-1 is shown in Fig. 3B. Myotubes positively stained for CT-1 were detected in the MB + CT group at six weeks after transplantation, whereas no myotubes were stained in the MB group (Fig. 3B). These data suggest that local expression of CT-1 in CT-1–transfected cells was sustained. Positive staining of myocardium for CT-1, although slightly, was also detected, indicating that endogenous CT-1 was expressed in the myocardium. Western blot analysis revealed that tissue expression of CT-1 in the LV free wall of the MB + CT group significantly increased compared with sham group at two weeks after transplantation (Fig. 3C) (2.3 ± 0.5-fold, p < 0.05).
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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.
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Morphometry of the ventricular myocytes.
The cross-sectional area of LV myocytes markedly increased from the age of 6 to 11 weeks (Fig. 4B). Then the sham group exhibited a slight decrease of myocyte size, although statistically not significant, from the age of 11 to 17 weeks (Fig. 4B). In contrast, the myocyte size in both the free wall and the septum wall of LV increased in the MB + CT group, from the age of 11 to 17 weeks (Fig. 4B). At the age of 17 weeks, although no difference in myocyte size was found between the sham and MB groups, the myocyte size in the free wall was 20% larger in the MB + CT group than in sham group (p < 0.05) (Figs. 4A and 4B). Furthermore, the myocyte size in the free wall at the cell-injected site was significantly larger than that of septum wall at the remote site of cell injection in the MB + CT group at this stage (p < 0.05) (Fig. 4B). On the other hand, the myocyte size in the free wall did not significantly differ from that of the septum wall in both the sham and MB groups. We also demonstrated that the conditioned media from CT-1–transferred myoblasts induced cardiac myocyte hypertrophy in vitro (Fig. 4C). These data suggest that CT-1 secreted from grafted cells in the MB + CT group induced hypertrophy of the adjacent myocardial cells in a paracrine manner.
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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.
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Neurohumoral regulation during the transition to congestive HF.
In this DS rat model, it has been demonstrated that the activation of local renin-angiotensin and endothelin systems in the heart contributes to the transition to heart failure (22,23). Indeed, serum Ang II levels increased at the congestive heart failure stage compared with the LV hypertrophy stage in sham group (Fig. 5A). However, these changes were attenuated in both the MB and MB + CT groups, and the degree of attenuation was greater in MB + CT group than the MB group (p < 0.05) (Fig. 5A). The serum ET-1 levels were also upregulated during the transition to congestive heart failure in sham group, but remained unchanged in both the MB and MB + CT groups (Fig. 5B). We also found that upregulation of angiotensinogen, angiotensin-converting enzyme, prepro-ET-1, and ET-converting enzyme messenger ribonucleic acid in the LV during the transition to congestive heart failure were all attenuated by myoblast transplantation, using semiquantitative reverse transcriptase-polymerase chain reaction (data not shown).
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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.
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The effect of CT-1 on myoblast survival in vitro.
To examine the effect of CT-1 on myoblast survival in vitro, we used C2C12 myoblasts rather than rat myoblasts (harvested from rats) to avoid contamination of other type cells such as fibroblasts. Proliferation rates of cells were measured in 10% serum-media. Although statistically not significant, the number of C2C12 cells transferred CT-1 by retrovirus (C2C12CT-1) tended to be higher than that of parental C2C12 cells at 48 h (Fig. 6A). Then we assessed whether C2C12CT-1 cells were resistant to serum-deprivation-induced cell death. In the absence of serum, C2C12 cells rapidly died, whereas C2C12CT-1 cells not only survived but also proliferated (Fig. 6B). This protective effect was also observed by addition of CT-1 in culture medium (Fig. 6B). Based on these findings, it is likely that CT-1 augments the graft survival ratio in the myocardium.
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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.
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Discussion
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In the present study, we examined the effect of skeletal myoblast transplantation with and without CT-1 gene transfer on global heart failure of nonischemic cause.
We demonstrated that the transplantation of skeletal myoblasts alleviated the transition from compensatory hypertrophy to congestive heart failure in DS hypertensive rats. So far, only a few animal studies showed that direct intramyocardial injection of fetal cardiac myocytes and smooth muscle cells could improve cardiac function in global heart failure due to nonischemic causes (8–10). Suzuki et al. (24) showed the efficient transplantation of skeletal myoblasts via the intracoronary route to doxorubicin-induced HF. Indeed, intracoronary injection is a reasonable method for global dissemination of cells into the heart, but there might be a danger of myocardial infarction. Intramyocardial injection is the most practical method for cell delivery in human patients at the moment. In this study, we injected skeletal myoblasts directly into the myocardium of DS hypertensive rats at the LV hypertrophy stage (the age of 11 weeks). Six weeks after transplantation, the transplanted myoblasts survived and formed myotubes, which aligned with cardiac fiber axis within the native myocardium.
Echocardiographic examination demonstrated reduced %FS, increased EDD, and decreased PWT at the congestive HF stage (the age of 17 weeks) in all groups. Therefore, ventricular dilation and thinning of the ventricular wall were associated with reduced cardiac contractility. Myocardial muscle fiber slippage and realignment are implicated in the ventricular remodeling of DS hypertensive rats, particularly in the thinning of the myocardium (25). Myoblast transplantation attenuated these morphologic changes associated with ventricular remodeling and served to preserve ventricular function in DS hypertensive rats. Although the precise mechanisms underlying the beneficial effect of myoblast transplantation have not been fully elucidated, it is likely that their passive girdling effect against mechanical stretching prevented LV dilation and remodeling (1). In DS hypertensive rats, activations of local renin-angiotensin or ET systems and matrix metalloproteinases in the heart contribute to LV remodeling and contractile dysfunction during the transition to heart failure (22,23,25). We found that myoblast transplantation attenuated upregulation of renin-angiotensin and ET systems during the transition to congestive HF. Although a causal relationship between the alteration of these neurohumoral regulations and the functional outcome remains elusive, it is possible that a decrease in wall stress due to the elastic property of engrafted cells attenuated the expression of such neurohumoral factors and alleviated the LV remodeling. On the other hand, there is a possibility that inflammation by direct injection induced paracrine effects, such as secretion of growth factors, and improved the cardiac function (8), but accumulation of inflammatory cells was hardly detected around the transplanted area throughout the experiment.
Active force generation by grafted cells is another conceivable mechanism (1). After engraftment, myoblasts merge into myotubes, and this graft might contract in synchrony with the host tissue. Because gap junction proteins, such as N-cadherin and connexin-43, have been shown to be downregulated after differentiation into myotubes, the presence of electromechanical coupling seems unlikely (26,27). On the other hand, simple stretch might initiate contraction of myotubes (28).
The efficiency of the cell-mediated gene delivery, which employs skeletal myoblasts expressing transforming growth factor-beta1 or vascular endothelial growth factor, has been reported (12,13). We examined the effect of prolonged overexpression of CT-1 in the myocardium by myoblast-mediated gene transfer using retrovirus.
Cardiotrophin-1 has hypertrophic and cardioprotective properties and acts through LIF receptor beta/glycoprotein 130 (gp130)-coupled signaling pathway (14–16). Cardiotrophin-1 promotes cardiac myocyte hypertrophy by directing sarcomere assembly in series in vitro (15). On the other hand, CT-1 prolongs survival of cardiac myocytes (16,29). Cardiac myocyte-restricted knockout of gp130 in adult mice develops LV dilation and induces apoptosis in the myocardium when the LV is subjected to increased wall stress, suggesting that gp130 signaling has the protective effect on cardiac myocytes (30). In chronic HF, CT-1 expression in the myocardium is upregulated corresponding to the severity (17,18). In this study, we demonstrated that the transplantation of skeletal myoblasts expressing CT-1 provides further benefits in preserving cardiac function compared with myoblast transplantation alone, suggesting that CT-1 has the protective effects against ventricular remodeling.
Western blot analysis showed the CT-1 secretion from CT-1–gene transferred myoblasts in vitro and the increased expression of CT-1 in the LV free wall of the MB + CT group in vivo. Immunohistochemical staining exhibited that, in the MB + CT group, overexpression of CT-1 in grafted cells within myocardium sustained until six weeks after transplantation. The plasma level of CT-1 was not evaluated in this study. Intravenous injection of CT-1 has been reported to elicit systemic hypotension via a nitric oxide-dependent mechanism (31). Because there were no significant differences in systolic blood pressure among three groups throughout the experiment, the plasma level of CT-1 was probably not elevated in the MB + CT group compared with the other two groups. We speculate that transferred gene-derived CT-1 operated in an autocrine/paracrine manner at the stage of transition from compensated to decompensated HF.
Transferred gene-derived CT-1 induced myocardial cell hypertrophy in vitro, and histological examination revealed the increased cross-sectional area of myocytes in the MB + CT group in congestive heart failure stage compared with the MB group. Echocardiographic examination also revealed that the wall thickness was more preserved at the congestive HF stage in the MB + CT group compared with the MB group. Excessive hypertrophy may induce contractile dysfunction, but the cardiac function of the MB + CT group was preserved more effectively than the MB groups. Although no definite mechanism has been proven in the relationship between contractile function and myocardial hypertrophy, the present findings suggest that CT-1 from the transplanted cells serves to maintain cardiac function by inducing hypertrophy of the adjacent cardiac myocytes. Cardiotrophin-1 is known to be a survival factor for cardiac myocytes (16). In the present study, we revealed that CT-1 also has a survival effect on myoblast in vitro. Therefore, it is also possible that CT-1 augmented the graft survival ratio in the myocardium and, thus, enhanced the beneficial effects of cell transplantation. To elucidate this issue, quantitative assessment of the area of injected myoblasts is needed. Also, it is important to verify that myoblasts were injected to the equivalent extent of areas between rats in the MB group and those in the MB + CT group. We tried to measure the graft survival ratio by use of a retroviral vector encoding CT-1–IRES (internal ribosomal entry site)-green fluorescent protein gene; however, the system did not work in the transplanted hearts. Those are the limitations of the present study, and recently reported new methods may help to resolve this issue (32,33).
In conclusion, we demonstrated that MB myoblasts alleviated the transition from compensatory hypertrophy to congestive heart failure in DS hypertensive rats. Transplantation of CT-1–expressing skeletal myoblasts by retroviral-mediated gene transfer resulted in prolonged overexpression of CT-1 within myocardium and preserved cardiac function more effectively. The results of this study suggest that CT-1 has the protective effects against ventricular remodeling. Transplantation of skeletal myoblasts combined with CT-1–gene transfer could be a useful strategy for the treatment of HF.
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Acknowledgments
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The authors are grateful to Ms. Kiyoko Matsui for her skillful technical assistance. Dr. Toh thanks Takami T. for her encouragement.
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Footnotes
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Supported by grants-in-aid for the research from the Ministry of Health and Welfare of Japan (2002 to 2003) and from the Ministry of Education, Science, Sports, and Culture of Japan (2001 to 2002).
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