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European Journal of Heart Failure 2008 10(1):14-21; doi:10.1016/j.ejheart.2007.11.007
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© 2008 European Society of Cardiology

Preservation of heart function in diabetic rats by the combined effects of muscle cell implantation and insulin therapy

Byung-Ok Kim, Subodh Verma, Richard D. Weisel, Shafie Fazel, Zhi-Qiang Jia, Tomohiro Mizuno and Ren-Ke Li*

Toronto General Research Institute, Division of Cardiovascular Surgery Toronto, Ontario, Canada
Toronto General Hospital, Division of Cardiac Surgery Toronto, Ontario, Canada
University of Toronto Ontario, Canada

* Corresponding author. Toronto General Hospital, MaRS Centre, Toronto Medical Discovery Tower, Room 3-702, 101 College Street, Toronto, ON, Canada M5G 1L7. Tel.: +1 416 581 7492; fax: +1 416 581 7493. E-mail address: renkeli{at}uhnres.utoronto.ca


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Background: Diabetic cardiomyopathy is a common cause of heart failure in diabetic patients, but current treatments do not directly improve ventricular function. Cell transplantation can prevent cardiac dilatation after injury, and may also prevent congestive heart failure in diabetic cardiomyopathy.

Aim: This study evaluated the functional effects of smooth muscle cells (SMCs) implanted into the myocardium of insulin- and non insulin-treated diabetic rats.

Methods: Four weeks after streptozotocin infusion, adult Wistar rats were implanted with BrdU-labelled SMCs or culture media (N=12/group). Six rats in each group were also treated with insulin. Echocardiograms were performed at 0, 4 and 8 weeks after streptozotocin injection, and histology and heart function were evaluated at 4 weeks after implantation.

Results: Blood glucose levels decreased after insulin treatment. Among cell-injected rats, histology indicated that those that did not receive insulin retained fewer surviving BrdU+ SMCs, and a smaller volume of myocardial tissue positive for {alpha}smooth muscle actin. Cardiac function was preserved in the insulin-treated groups relative to those that did not receive insulin. Among insulin-treated rats, the cell-injected group functioned better than the media-injected group.

Conclusions: Diabetic cardiomyopathy is partially treatable with insulin; however, a combination of SMC transplantation and insulin treatment produced the best functional result. Cell transplantation may prevent the progression of diabetic cardiomyopathy in patients whose glucose levels are controlled with insulin.

Key Words: Heart failure • Diabetic cardiomyopathy • Heart function • Cell transplantation • Diabetes

Received April 3, 2007; Revised October 12, 2007; Accepted November 6, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Among several factors that limit the survival and quality of life of patients with chronic diabetes, including coronary or peripheral artery disease, stroke, and diabetic retinopathy, nephropathy and neuropathy, cardiovascular complications account for up to 85% of mortalities in this group [1,2].

Diabetic cardiomyopathy is well documented, and may contribute to the increased incidence of treatment-resistant congestive heart failure in the diabetic population [2,3]. Diabetic patients are exquisitely sensitive to diastolic dysfunction, with an increased risk, relative to non-diabetic patients, of overt systolic heart failure despite excellent glucose control by insulin, and independent of cardiac risk factors [4,5]. The aetiology of diabetic cardiomyopathy remains obscure, but endothelial dysfunction, abnormal intracellular calcium handling [6,7] and accelerated cardiomyocyte apoptosis [8]. likely contribute to progressive cardiac dysfunction.

Because tight insulin control of blood glucose does not eliminate ventricular dysfunction, new treatments for diabetic cardiomyopathy are required. Cell transplantation has emerged as a strategy to prevent congestive heart failure following a myocardial infarction and in cardiomyopathic hearts [9]. Although it has been studied in various animal models, cell transplantation has not yet been evaluated in diabetic animals. Since hyperglycaemia is toxic to cardiomyocytes, it is unknown whether implanted cells could survive in the myocardium and improve heart function in diabetic animals. To investigate cell transplantation as a novel therapeutic approach for diabetic cardiomyopathy, we examined the changes in left ventricular function and histopathology after syngenic smooth muscle cell (SMC) implantation, with or without accompanying insulin treatment, in a diabetic rat model. Streptozotocin injection was used to generate diabetic rats exhibiting myocardial dysfunction similar to that seen in type I diabetes patients.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Experimental animals
Male Wistar rats (200-250 g, Harlan Company, Canada) were used in the study. All animal procedures were approved by the Animal Care Committee of the Toronto General Hospital, and carried out in compliance with the Canadian Council on Animal Care "Guide to the Care and Use of Experimental Animals" and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.

2.2. Isolation, culture, and identification of smooth muscle cells
SMCs were isolated from the rat aorta as previously described [9]. In brief, the aorta was collected, and the outer layer of connective tissue was removed. After washing, the aorta was incised, and the endothelial cell layer and the underlying connective tissue were scraped away. Tissue from the smooth muscle layer was washed with phosphate-buffered saline (PBS), and minced. Single cells were isolated with protease solution (0.2% trypsin, 0.1% collagenase and 0.1% glucose in PBS), and then cultured in Iscove's modified Dulbecco's medium (Gibco Laboratory, Life Technologies, Grand Island, NY) containing 10% fetal bovine serum, 0.1 mmol/L β-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin.

The cultured SMCs were identified immunohistochemically prior to transplantation using a monoclonal antibody against {alpha}-smooth muscle actin (Sigma, St Louis, MO), as previously described [9]. Under the light microscope, more than 95% of cultured cells were {alpha}-smooth muscle actin positive. One third of the donor SMCs were labelled with 50 µl of 2.5% 5-bromo-2'-deoxyuridine (BrdU) (Zymed Lab Inc, South San Francisco, CA) for 24 hours before transplantation. Labelling efficiency was confirmed by staining for BrdU in 2 randomly-selected dishes. More than 80% of these cultured cells were BrdU positive.

2.3. Diabetic rats
Streptozotocin (STZ; 55 mg/Kg body weight, Sigma Chemical Company, St Louis, Missouri) was injected intravenously through the tail vein of adult rats to induce diabetes. Blood glucose was measured at 3 days following STZ administration, and thereafter weekly, in single blood drops obtained by tail vein puncture (Accu-Check Advantage test: Roche Diagnostics, Laval, Québec). Animals with non-fasting blood glucose levels greater than 16 mmol/L were included in the study.

2.4. SMC transplantation and insulin treatment
At 4 weeks following STZ injection, diabetic rats were randomly assigned to cell transplant and control groups (N=12 per group). All animals were anesthetized with ketamine (Ketalean, 20 mg/kg, intramuscular), followed by an intraperitoneal injection of pentobarbital (Somnotol, 30 mg/kg). Anesthetized animals were intubated, and ventilated with oxygen-supplemented room air using a Harvard ventilator (model683; Harvard Instruments, South Natick, MA), at a rate of 60 breaths per minute. In each rat, the heart was exposed through a 3 cm left lateral thoracotomy. Six million SMCs in 50 µl culture media (cell transplant group) or 50 µl media alone (control group) were injected into the left ventricular free wall with a tuberculin syringe. After chest closure, the animals were recovered from the procedure and treated with antibiotics (Duplocillin LA, 150,000 IU/kg body weight) and analgesics (buprenorphine, 0.01-0.05 mg/kg body weight); antibiotics were given once again 3 days postoperatively.

Half of the rats in each of the cell transplant and control groups (N=6 per group) were also treated with insulin (8-12 U/day, Humulin U Lilly) starting 3 days prior to transplantation, and continuing until the end of the study (4 weeks after transplantation) (groups: Cell + Insulin; Media + Insulin); the remaining rats in the cell transplant and control groups (N=6 per group) received no insulin (groups: Cell; Media).

2.5. Cardiac functional measurements
2.5.1. Echocardiography
Echocardiography was performed at 0, 4 and 8 weeks after STZ injection using a SEQUOIA Echocardiography System (ACUSON Corporation, USA) with a 12-MHz transducer. The rats were anaesthetized by isoflurane (3-5%) inhalation of and maintained by mask ventilation of isoflurane (1-2%). The left side of the chest was shaved to gain a clear image. All rats were studied in the left lateral decubitus position. Parasternal long- and short-axis views were obtained with both M-mode and two-dimensional echo images. Left ventricular (LV) dimensions (end-diastolic and end-systolic) were measured perpendicular to the long axis of the ventricle at the mid-chordal level. Fractional shortening was calculated as the end-diastolic dimension (LVEDD) minus the end-systolic dimension (LVESD) divided by the end-diastolic dimension. Ejection fraction (LVEF) was calculated automatically by the echocardiography system, using the equation: LVEF(%)=(LVEDV-LVESV)/LVEDVx100, where LVEDV=7.0xLVEDD3/(2.4+LVEDD) and LVESV=7.0xLVESD3/(2.4+LVESD).

2.5.2. Langendorff assessment
At 4 weeks after transplantation (8 weeks after STZ injection), the rats were anesthetized with isoflurane, and heparin sodium (200 U) was administered intravenously. The hearts were quickly excised and perfused in a Langendorff apparatus with filtered Krebs-Henseleit buffer (NaCl, 118 mmol/L; KCL, 4.7 mmol/L; KH2PO4, 1.2 mmol/L; CaCl2, 2.5 mmol/L; MgSO4 1.2 mmol/L; NaHCO3, 25 mmol/L; glucose, 11 mmol/L; pH 7.4) equilibrated with 5% carbon dioxide and 95% oxygen. A latex balloon was passed into the LV across the mitral valve and connected to a pressure transducer (model p10EZ; Vigo-Spectramed, Oxnard, CA) and differentiator amplifier (model 11-G4113-01; Gould Instrument System Inc, Valley View, OH). Coronary flow was measured in triplicate by timed collection in the empty, beating heart. After 30 min of stabilization, the balloon volume was increased in 0.02 ml increments by the stepwise addition of saline, from an initial 0.04 ml, until the end-diastolic pressure exceeded 25 mmHg. Heart rate, systolic and diastolic LV pressure, and maximum rates of increase or decrease of the LV pressure (+dP/dt, –dP/dt) were recorded at each balloon volume. Developed pressure was calculated as the difference between the systolic and diastolic pressures. After completion of all measurements, the hearts were arrested in diastole and perfused with 10 ml of 20% KCL solution.

2.6. Histology and immunohistochemistry
The perfused hearts were fixed in LV distension (30 mmHg) for 3 days with a 10% phosphate-buffered formalin solution, and then cut into 3-mm slices (5 to 6 slices per heart). Heart slices were fixed in 5% glacial acetic acid in methanol, embedded in paraffin, and cut into 10 µm thick sections. The sections were stained with haematoxylin and eosin, following the manufacturer's specifications (Sigma Diagnostics, St. Louis, MO), in order to reveal basic histological structures within the myocardial tissue, or underwent immunohistochemical staining using antibodies against BrdU (to assess implanted cell survival) or {alpha}-smooth muscle actin (to identify implanted SMCs), according to previously described protocols [9].

2.7. In vivo assessment of implanted cell survival
Engrafted SMCs were identified in the implanted area as BrdU positive cells, and quantified in 5 randomly selected fields from single heart sections from each of 6 hearts per cell transplanted group. The counts were expressed as the average number of BrdU positive nuclei per 0.2 mm2.

2.8. Statistical Analyses
Data are presented as mean±SEM. Statistical analyses were performed using the SPSS software package (SPSS Inc. Chicago, IL). Comparison of multiple variables among the 4 groups was performed with a one-way analysis of variance in a general linear model analysis. Differences between groups over time were assessed using a two-way analysis of variance. If the F ratio associated with the main effect or the interaction between the main effect and time was significant (p<0.05), then the difference was specified by a post hoc one-way analysis of variance at each time point. LV function was evaluated by analysis of covariance (covariant: intraventricular balloon volume; dependent variables: systolic, diastolic, and developed pressures, dP/dt; main effects: group, volume, and interaction between group and volume). When the F ratio associated with a main effect or the interaction was significant (p<0.05), then the difference was specified by post hoc multiple pairwise comparisons.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Metabolic characteristics
Blood glucose levels in all rats were greater than 20 mmol/L at 3 days after STZ injection. Hyperglycaemia persisted for the first 4 weeks of the study. Three days prior to cell or media transplantation, 12 rats (N=6 per group) were treated with insulin. On the day of cell or media transplantation, fasting blood glucose levels for the insulin-treated animals decreased to between 6 and 16 mmol/L, and remained elevated at between 20 and 32 mmol/L in the non insulin-treated animals.

By 4 weeks after cell transplantation, blood glucose levels in the insulin-treated animals had increased (Cell+Insulin 22.1±1.1; Media+Insulin 21.4±1.1 mmol/L), but remained significantly lower (p<0.05 for all groups) than those in the non insulin-treated rats (Cell 28.6±1.1 mmol/L; Media 27.3 ±1.0). Following transplantation, the mean change in body weight was significantly greater (p<0.05) in the insulin-treated rats than in the non insulin-treated rats (Table 1), suggesting that the diabetic rats had a severe metabolic disorder.


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  		Table 1 Body and ventricular weights (wt.) of cell transplanted (TX) and control rats

 
3.2. Cardiac function
3.2.1. Echocardiography
There were no significant differences among the 4 groups in ventricular weights or the ratio of ventricular weight to body weight (Table 1). Echocardiography demonstrated no differences in LVEDD among groups, but significantly (p<0.05) smaller LVESD values in the insulin-treated groups than in the non insulin-treated groups (Fig. 1) at the end of the study. At 4 weeks after cell/media transplantation (8 weeks after STZ injection), both ejection fraction and fractional shortening values (Fig. 2) were significantly greater (p<0.05 for all groups) in the insulin-treated hearts than in either cell- or media-implanted hearts without insulin treatment (with no significant differences between these 2 groups). However, these values were greatest (p<0.05 compared to all other groups) in the cell and insulin-treated hearts.


Figure 01
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  		Fig. 1 Left ventricular dimensions. Left ventricular end-systolic diameter (LVESD), measured by echocardiography at 0 (baseline), 4, and 8 weeks after streptozotocin treatment in rats receiving media or cell transplantation alone (at 4 weeks), and those that also received insulin beginning 3 days prior to transplantation (cell+insulin, media+insulin). LVESD increased following streptozotocin injection (p<0.05). At 8 weeks (4 weeks after cell/media transplantation), LVESD was significantly smaller (p<0.05) in the insulin treated groups than in the non-insulin treated groups. *p<0.05; data are mean±SEM (n=6 per group).

 


Figure 02
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  		Fig. 2 Cardiac function by echocardiography. Ejection fraction (A) and fractional shortening (B) measured at 0 (baseline), 4, and 8 weeks after streptozotocin treatment in rats receiving media or cell transplantation alone (at 4 weeks), and those that also received insulin beginning 3 days prior to transplantation (cell+insulin, media+insulin). Ejection fraction and fractional shortening decreased following cell/media transplantation in the media and cell groups, but not in either insulin-treated group (p<0.05). At 8 weeks, values for both measures of cardiac function remained significantly higher in the cell+insulin group than the other 3 groups (p<0.05 for all groups). *p<0.05; data are mean±SEM (n=6 per group).

 
3.2.2. Langendorff assessment
Heart function was also evaluated at 4 weeks after cell/media transplantation using a Langendorff perfusion technique to provide load independent indices of ventricular performance. SMC transplantation did not improve heart function when the rats were not treated with insulin (Fig. 3). However, among those that received insulin, cell implantation improved peak systolic and developed pressures more than media (p<0.01 for both groups). Similarly, +dP/dt values were significantly greater and –dP/dt values were smaller (p<0.01) at 4 weeks after cell/media transplantation in cell and insulin-treated rats compared to those treated with media and insulin, while rats that did not receive insulin exhibited the poorest ventricular functioning on these measures (p<0.01) (Fig. 4). The relation between diastolic pressure and balloon volume was not significantly different among the four groups.


Figure 03
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  		Fig. 3 Cardiac function by Langendorff assessment. Peak systolic pressures (A) and developed pressures (B) measured at 8 weeks after streptozotocin treatment in rats receiving media or cell transplantation alone (at 4 weeks), and those that also received insulin beginning 3 days prior to transplantation (cell+insulin, media+insulin). Both measures were significantly higher (p<0.01 for all groups) in the cell+insulin group than in all other groups by an analysis of covariance. *p<0.01; data are mean±SEM (n=6 per group).

 


Figure 04
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  		Fig. 4 Cardiac function by Langendorff assessment. Maximum rates of change in left ventricular pressure, +dP/dt (A) and –dP/dt (B), measured at 8 weeks after streptozotocin treatment in rats receiving media or cell transplantation alone (at 4 weeks), and those that also received insulin beginning 3 days prior to transplantation (cell+insulin, media+insulin). +dP/dt values were significantly higher, and –dP/dt values were lower (p<0.01 for all groups) in the cell+insulin group than in all other groups by an analysis of covariance. *p<0.01; data are mean±SEM (n=6 per group).

 
3.3. Histology
At 4 weeks after SMC transplantation, the engrafted cells (those that stained positive for BrdU), were identified in the myocardium of both cell-transplanted groups (with and without insulin treatment), but not in the media transplanted groups (Figs. 5 and 6). In animals that received SMCs, the implanted cells appeared to form muscle tissue within the myocardium, which stained positive for {alpha}-smooth muscle actin. However, the group that received the combination of SMCs and insulin retained substantially more (p<0.01) implanted cells (BrdU positive) than the group that received SMCs alone (Fig. 7). The volume of muscle tissue formed ({alpha}-smooth muscle actin positive) also appeared to be much greater in labelled sections from the animals that received SMCs and insulin (Figs. 5-6Go). No muscle-like tissue was observed in the hearts from either group injected with culture media rather than SMCs.


Figure 05
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  		Fig. 5 Myocardial histology in insulin-treated rats. Micrographs illustrating basic histological structures (haematoxylin and eosin staining, H&E; A, D) and immunohistochemical staining for {alpha}-smooth muscle actin (B, E) and BrdU (C, F) in myocardial tissue sections from streptozotocin-treated rats that received insulin beginning 3 days prior to transplantation with cells (cell+insulin; A,B,C) or media (media+insulin; D,E,F). At 4 weeks after transplantation, implanted cells were identified only in the cell+insulin group by positive staining for {alpha}-smooth muscle actin (arrows in B) and BrdU (arrowheads in C). Open arrows in E = smooth muscle cells within blood vessel structures. Mag. = 40X in A-F.

 


Figure 06
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  		Fig. 6 Myocardial histology in non insulin-treated rats. Micrographs illustrating basic histological structures (haematoxylin and eosin staining, H&E; A, D) and immunohistochemical staining for {alpha}-smooth muscle actin (B, E) and BrdU (C, F) in myocardial tissue sections from streptozotocin-treated rats that received no insulin treatment prior to transplantation with cells (cell; A,B,C) or media (D,E,F). At 4 weeks after transplantation, implanted cells were identified only in the cell group by positive staining for {alpha}-smooth muscle actin (arrows in B) and BrdU (arrowheads in C). Open arrows in E=smooth muscle cells within blood vessel structures. Mag. = 40X in A-F.

 


Figure 07
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  		Fig. 7 Engrafted cell survival in cell-treated rats at 4 weeks after transplantation. Survival of implanted smooth muscle cells (expressed as the average number of BrdU positive nuclei per 0.2 mm2) in the myocardial tissue of rats that received transplanted cells alone (cell), or in combination with insulin beginning 3 days prior to transplantation (cell+insulin). BrdU positive cells were significantly more numerous in the cell+insulin group. *p<0.01 compared to cell group; data are mean±SEM (n=6 per group).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In patients, diabetic cardiomyopathy results in mortality at nearly twice the normal rate [3,10]. The current study may provide the first report describing the effects of implanted muscle cells on cardiac function in experimental type I diabetes mellitus, and their augmentation by insulin therapy. While improved glycaemic control is by far the principal means of decreasing the secondary complications of diabetes [11], insulin treatment alone could not normalize heart function in experimental chronic diabetes [12]. or postprandial hyperglycaemia [13], or in clinical studies [14]. Based on previous work in alternative models of heart failure indicating that muscle or progenitor cells implanted into the damaged heart can delay the onset of ischaemic cardiomyopathy [15,16], we hypothesized that cell therapy - not previously evaluated in experimental diabetes - might improve the prognosis for diabetic cardiomyopathy.

In this study, SMCs were chosen for transplantation because they may be ideally suited to improve function in diabetic cardiomyopathy. Skeletal myoblasts augment cardiac function following implantation into the infarcted myocardium [17]; studies have demonstrated that these cells retain their skeletal muscle characteristics for at least 17 months after implantation [17,18] and do not beat synchronously with the heart. Therefore, the contractility of the implanted cells is not essential to improve heart function, and non-contractile SMCs may be preferred for cell transplantation because they respond to haemodynamic stresses with hypertrophy and hyperplasia. We previously demonstrated that SMCs prevented cardiac dilatation and preserved systolic function after myocardial injury [9,19].

In experimental diabetes, abnormalities in the calcium pump [7,20], diminished creatine kinase activity [21], and changes in the structure or function of myosin heavy chain and thin filaments [22,23] contribute to the development of cardiomyopathy. Several animal models of diabetes are available for cardiovascular research. For this study, STZ was used to induce in rats a hyperglycaemic state similar to that characteristic of type I diabetes. Although this particular model does not reproduce the chronic effects of hyperglycaemia seen in diabetic patients and does not induce associated comorbidities such as hypertension and autonomic dysfunction, it does accurately reproduce many of the cardiac effects of diabetic hyperglycaemia [8]. Animals treated with STZ display numerous cardiac metabolic defects observed in clinical diabetes, and treatment with insulin lowers their blood glucose to levels characteristic of patients with diabetes. Further, the STZ model produces a more homogenous and reproducible model of heart failure than does the standard model of coronary ligation, which results in a heterogeneous infarct and variable regional dysfunction.

We found that rats exposed to hyperglycaemia for 8 weeks without accompanying insulin treatment demonstrated progressive functional deterioration, with increasing LVESD and decreasing LV fractional shorting and LVEF. Insulin therapy alone had a dramatic effect on functional recovery. The hearts of the insulin-treated animals functioned better, and displayed smaller end-systolic dimensions than those of the animals that did not receive insulin. These in vivo echocardiographic findings were confirmed by the load independent assessment of function performed during ex vivo perfusion with the Langendorff apparatus.

Cell transplantation alone did not prevent the deterioration of cardiac function in STZ-treated rats. However, when SMC implantation was accompanied by insulin therapy, the improvement in cardiac function was more significant than that observed with insulin therapy alone. The fact that BrdU labelled SMCs were significantly more numerous at 4 weeks after cell transplantation in the group that also received insulin suggests that the functional advantage in this group may have resulted from the effects of insulin on implanted cell survival. We previously showed that the functional benefits of cell transplantation were directly proportional to the number of cells implanted into the infarcted myocardium [24], since only about 20% of implanted SMCs survived to engraft in the injured heart [25]. Hyperglycaemia is known to decrease cell survival in vitro [26], and the current results indicate that uncontrolled hyperglycaemia also decreases the survival of implanted cells. We believe that SMCs implanted alone did not engraft in numbers sufficient to induce the paracrine effects (angiogenesis, matrix remodelling, progenitor recruitment) we have previously observed after SMC implantation in alternative models (ischaemic cardiomyopathy) [27]. Therefore, SMCs alone did not induce significant muscle tissue formation or prevent ventricular dilatation. When insulin was given in combination with the implanted cells, the resulting normalization of glucose levels boosted the survival and engraftment of the SMCs, thereby enhancing their effects on functional restoration.

The clinical significance of this study will require confirmation in other models of diabetes and in large animal models of cell transplantation. If the functional benefits of muscle cell transplantation in combination with insulin therapy are confirmed in subsequent studies, then cell therapy may represent a treatment to reduce cardiac dysfunction following a myocardial infarction in diabetic patients with tight glucose control. Cell transplantation could also be effective in patients experiencing diabetic cardiomyopathy without coronary occlusions, as demonstrated in other models of non-ischaemic cardiomyopathy [19,28]. The feasibility of this approach has been demonstrated in clinical trials of skeletal myoblasts implanted into patients with global cardiomyopathy who require LV assistance [29].

In summary, this study reports the novel finding that a treatment combining muscle cell transplantation and insulin therapy prevents cardiac dilatation and functional deterioration in experimental diabetes. Diabetic cardiomyopathy in rats is partially treatable with insulin; however, cell transplantation may further improve the functional outcome in patients whose glucose levels are controlled with insulin.


   Acknowledgements
 
R-KL is a Career Investigator of the Heart and Stroke Foundation of Canada, and holds a Canada Research Chair in Cardiac Regeneration. This research was supported by grants to R-KL from the Canadian Institutes for Health Research (MOP 62698, MOP 14795), and the Heart and Stroke Foundation of Ontario (NA 5294, T 5206).


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Dries D.L., Sweitzer N.K., Drazner M.H., Stevenson L.W., Gersh B.J. Prognostic impact of diabetes mellitus in patients with heart failure according to the etiology of left ventricular systolic dysfunction. J Am Coll Cardiol (2001) 38:421–428.[Abstract/Free Full Text]
  2. Kannel W.B., Hjortland M., Castelli W.P. Role of diabetes in congestive heart failure: the Framingham study. Am J Cardiol (1974) 34:29–34.[CrossRef][Web of Science][Medline]
  3. Aronson D., Rayfield E.J., Chesebro J.H. Mechanisms determining course and outcome of diabetic patients who have had acute myocardial infarction. Ann Intern Med (1997) 126:296–306.[Abstract/Free Full Text]
  4. Stone P.H., Muller J.E., Hartwell T., et al. The effect of diabetes mellitus on prognosis and serial left ventricular function after acute myocardial infarction: contribution of both coronary disease and diastolic left ventricular dysfunction to the adverse prognosis. The MILIS Study Group. J Am Coll Cardiol (1989) 14:49–57.[Abstract]
  5. Gottdiener J.S., Arnold A.M., Aurigemma G.P., et al. Predictors of congestive heart failure in the elderly: the Cardiovascular Health Study. J Am Coll Cardiol (2000) 35:1628–1637.[Abstract/Free Full Text]
  6. Verma S., Maitland A., Weisel R.D., et al. Increased endothelin-1 production in diabetic patients after cardioplegic arrest and reperfusion impairs coronary vascular reactivity: reversal by means of endothelin antagonism. J Thorac Cardiovasc Surg (2002) 123:1114–1119.[Abstract/Free Full Text]
  7. Temsah R.M., Kawabata K., Chapman D., Dhalla N.S. Modulation of cardiac sarcoplasmic reticulum gene expression by lack of oxygen and glucose. FASEB J (2001) 15:2515–2517.[Abstract/Free Full Text]
  8. Shiomi T., Tsutsui H., Ikeuchi M., et al. Streptozotocin-induced hyperglycemia exacerbates left ventricular remodeling and failure after experimental myocardial infarction. J Am Coll Cardiol (2003) 42:165–172.[Abstract/Free Full Text]
  9. Li R.K., Jia Z.Q., Weisel R.D., Merante F., Mickle D.A. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol (1999) 31:513–522.[CrossRef][Web of Science][Medline]
  10. Jacoby R.M., Nesto R.W. Acute myocardial infarction in the diabetic patient: pathophysiology, clinical course and prognosis. J Am Coll Cardiol (1992) 20:736–744.[Abstract]
  11. Iribarren C., Karter A.J., Go A.S., et al. Glycemic control and heart failure among adult patients with diabetes. Circulation (2001) 103:2668–2673.[Abstract/Free Full Text]
  12. Tahiliani A.G., Lopaschuk G.D., McNeill J.H. Effect of insulin treatment on long-term diabetes-induced alteration of myocardial function. Gen Pharmacol (1984) 15:545–547.[Web of Science][Medline]
  13. Regan T.J., Wu C.F., Yeh C.K., Oldewurtel H.A., Haider B. Myocardial composition and function in diabetes. The effects of chronic insulin use. Circ Res (1981) 49:1268–1277.[Abstract/Free Full Text]
  14. Dhalla N.S., Pierce G.N., Innes I.R., Beamish R.E. Pathogenesis of cardiac dysfunction in diabetes mellitus. Can J Cardiol (1985) 1:263–281.[Medline]
  15. Yau T.M., Tomita S., Weisel R.D., et al. Beneficial effect of autologous cell transplantation on infarcted heart function: comparison between bone marrow stromal cells and heart cells. Ann Thorac Surg (2003) 75:169–176.[Abstract/Free Full Text]
  16. Szmitko P.E., Fedak P.W., Weisel R.D., Stewart D.J., Kutryk M.J., Verma S. Endothelial progenitor cells: new hope for a broken heart. Circulation (2003) 107:3093–3100.[Free Full Text]
  17. Menasche P., Hagege A.A., Vilquin J.T., et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol (2003) 41:1078–1083.[Abstract/Free Full Text]
  18. Hagege A.A., Carrion C., Menasche P., et al. Viability and differentiation of autologous skeletal myoblast grafts in ischaemic cardiomyopathy. Lancet (2003) 361:491–492.[CrossRef][Web of Science][Medline]
  19. Yoo K.J., Li R.K., Weisel R.D., Mickle D.A., Li G., Yau T.M. Autologous smooth muscle cell transplantation improved heart function in dilated cardiomyopathy. Ann Thorac Surg (2000) 70:859–865.[Abstract/Free Full Text]
  20. Netticadan T., Temsah R.M., Kent A., Elimban V., Dhalla N.S. Depressed levels of Ca2+-cycling proteins may underlie sarcoplasmic reticulum dysfunction in the diabetic heart. Diabetes (2001) 50:2133–2138.[Abstract/Free Full Text]
  21. Popovich B.K., Boheler K.R., Dillmann W.H. Diabetes decreases creatine kinase enzyme activity and mRNA level in the rat heart. Am J Physiol (1989) 257:E573–E577.[Web of Science][Medline]
  22. Dillmann W.H. Diabetes mellitus induces changes in cardiac myosin of the rat. Diabetes (1980) 29:579–582.[Web of Science][Medline]
  23. Malhotra A., Reich D., Reich D., et al. Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade. Circ Res (1997) 81:1027–1033.[Abstract/Free Full Text]
  24. Pouzet B., Vilquin J.T., Hagege A.A., et al. Factors affecting functional outcome after autologous skeletal myoblast transplantation. Ann Thorac Surg (2001) 71:844–850.[Abstract/Free Full Text]
  25. Liu T.B., Fedak P.W., Weisel R.D., et al. Enhanced IGF-1 expression improves smooth muscle cell engraftment after cell transplantation. Am J Physiol Heart Circ Physiol (2004) 287:H2840–H2849.[Abstract/Free Full Text]
  26. Rao V., Merante F., Weisel R.D., et al. Insulin stimulates pyruvate dehydrogenase and protects human ventricular cardiomyocytes from simulated ischemia. J Thorac Cardiovasc Surg (1998) 116:485–494.[Abstract/Free Full Text]
  27. Fedak P.W., Szmitko P.E., Weisel R.D., et al. Cell transplantation preserves matrix homeostasis: a novel paracrine mechanism. J Thorac Cardiovasc Surg (2005) 130:1430–1439.[Abstract/Free Full Text]
  28. Pouly J., Hagege A.A., Vilquin J.T., et al. Does the functional efficacy of skeletal myoblast transplantation extend to nonischemic cardiomyopathy? Circulation (2004) 110:1626–1631.[Abstract/Free Full Text]
  29. Pagani F.D., DerSimonian H., Zawadzka A., et al. Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation. J Am Coll Cardiol (2003) 41:879–888.[Abstract/Free Full Text]

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