AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H2196-H2203, 2006. First published February 10, 2006; doi:10.1152/ajpheart.01017.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2196    most recent
01017.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (28)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Berry, M. F.
Right arrow Articles by Sweeney, H. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berry, M. F.
Right arrow Articles by Sweeney, H. L.

CALL FOR PAPERS
Regulation and Function of Stem Cells in the Cardiovascular System

Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance

Mark F. Berry,1 Adam J. Engler,3 Y. Joseph Woo,1 Timothy J. Pirolli,2 Lawrence T. Bish,2 Vasant Jayasankar,1 Kevin J. Morine,2 Timothy J. Gardner,1 Dennis E. Discher,3 and H. Lee Sweeney2

1Division of Cardiothoracic Surgery, Department of Surgery, and 2Department of Physiology, University of Pennsylvania School of Medicine; and 3Institute for Medicine and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 26 September 2005 ; accepted in final form 30 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cellular therapy for myocardial injury has improved ventricular function in both animal and clinical studies, though the mechanism of benefit is unclear. This study was undertaken to examine the effects of cellular injection after infarction on myocardial elasticity. Coronary artery ligation of Lewis rats was followed by direct injection of human mesenchymal stem cells (MSCs) into the acutely ischemic myocardium. Two weeks postinfarct, myocardial elasticity was mapped by atomic force microscopy. MSC-injected hearts near the infarct region were twofold stiffer than myocardium from noninfarcted animals but softer than myocardium from vehicle-treated infarcted animals. After 8 wk, the following variables were evaluated: MSC engraftment and left ventricular geometry by histological methods, cardiac function with a pressure-volume conductance catheter, myocardial fibrosis by Masson Trichrome staining, vascularity by immunohistochemistry, and apoptosis by TdT-mediated dUTP nick-end labeling assay. The human cells engrafted and expressed a cardiomyocyte protein but stopped short of full differentiation and did not stimulate significant angiogenesis. MSC-injected hearts showed significantly less fibrosis than controls, as well as less left ventricular dilation, reduced apoptosis, increased myocardial thickness, and preservation of systolic and diastolic cardiac function. In summary, MSC injection after myocardial infarction did not regenerate contracting cardiomyocytes but reduced the stiffness of the subsequent scar and attenuated postinfarction remodeling, preserving some cardiac function. Improving scarred heart muscle compliance could be a functional benefit of cellular cardiomyoplasty.

cell transplantation; myocardial remodeling

MYOCARDIAL INFARCTION leads to myocardial loss due to necrosis as well as subsequent remodeling with progressive ventricular dilatation and fibrosis (11, 30). Cellular cardiomyoplasty is a technique in which exogenous cells are transplanted into dysfunctional areas of the heart in an attempt to replace damaged myocardium. Mesenchymal stem cells (MSCs) appear to be both multipotent and immune privileged, making them particularly attractive for this use (14, 18, 23, 28, 34). MSCs and other cell types have improved heart function in both animal models of acute myocardial injury as well as clinical studies of patients with heart failure (20, 26, 32, 35). However, the improvement observed in these studies has been limited to partial restoration of function, while engraftment of transplanted cells as contracting cardiomyocytes in native myocardium has not been rigorously demonstrated.

Fibrosis and ventricular remodeling after myocardial infarction starts with massive extracellular matrix (ECM) deposition, which in combination with the tissue necrosis stiffens the heart muscle (7, 29, 33, 36). Recently, ECM compliance has been demonstrated to regulate multiple cellular processes, including engraftment and differentiation (7, 8, 10, 15, 38). Myotubes grown on substrates of controlled ECM elastic moduli striate only on surfaces of optimal stiffness that mimic normal muscle elasticity (7). The stiffness of infarcted myocardium may therefore play a part in the postinfarction remodeling process that leads to heart failure. Furthermore, any intervention that softens the infarct area, including cell transplantation, may lessen deleterious remodeling. This study was undertaken to further understand and expand on the cellular and mechanical roles that MSCs have in the postinfarct remodeling process, focusing on hemodynamic, histological, and local elasticity measures.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. Animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The study was reviewed and approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Surgical preparation. Twenty-seven male Lewis inbred rats (250–300 g; Charles River, Wilmington, MA) were anesthetized with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg), intubated, and mechanically ventilated with 0.5% isoflurane. The proximal left anterior descending coronary artery (LAD) was encircled with a suture via a left thoracotomy. In two animals, the suture was removed without being tied. The suture was ligated in the other 25 animals, of which 11 received five MSC injections [2 x 106 total cells (Osiris Therapeutics, Baltimore, MD) diluted in 200 µl of Dulbecco's modified Eagle's medium (DMEM)] directly into central infarct and border zone areas of the myocardium, 12 received DMEM injections, and two received no injections. Injection of cells and DMEM was performed in a randomized and blinded fashion by a single investigator. All animals were closed and recovered.

Atomic force microscopy characterization. Eight of the animals that underwent the surgery described above (the two that did not undergo ligation, two that had cells injected, two that had DMEM injected, and the two that underwent ligation but no injections) were euthanized 2 wk later, after which the hearts were removed to obtain atomic force microscopy (AFM) measurements of elasticity in the infarct and border region. Infarcted and control tissue from these eight animals was sectioned parallel to the longitudinal axis of the left ventricle (LV) from ~5 mm above the LAD ligation point to a point ~15 mm downstream of the ligation (see Fig. 3A) to yield tissue samples 15-mm long, 1-mm wide, and 1-mm thick. The basal surface was mounted and immobilized on coverglass with adhesive tape, exposing the apical surface for probing. Tissue samples were submerged in DMEM, placed in an Asylum 1-D AFM (Asylum Research, Santa Barbara, CA), and indented with a blunted pyramid-tipped cantilever (Veeco, Santa Barbara, CA) having a nominal spring constant of 60 pN/nm, checked by a thermal calibration. Nine-hundred seventy-four total force-indentation measurements (see Fig. 3B, inset) were made on a spatially controlled stage (±0.1 mm) at 15–20 positions per animal along the length of the infarct (Fig. 3A, x-axis). At each of these lateral positions, measurements were varied along the width of the sample (Fig. 3A, y-axis) to obtain data over the entire sample surface. Each force-indentation plot (Fig. 3B, inset, black curve) was fit to a Hertz cone model (red curve) to determine an elastic modulus, E (6, 27). Elastic moduli were binned at 1- to 2-mm intervals and averaged for each animal (n = 2 animals for each sample group). Samples were indented at rates <2 µm/s, which is generally sufficient to explore elastic rather than viscoelastic properties of cells and ECM (16). Normal heart muscle from the noninfarcted animals was used to determine a baseline elastic modulus, whereas the "border zone slope modulus" was determined from data one deviation below the normal and stiff, infarcted regions and above the noninfarcted region.


Figure 3
View larger version (55K):
[in this window]
[in a new window]
  		Fig. 3. A: dashed white circle indicates ischemic area after left anterior descending coronary artery ligation. Black rectangle indicates section of tissue removed and mounted for atomic force microscopy (AFM) probing of elasticity (E). The x- and y-axes and asterisk indicate sample orientation on AFM stage and ligation point, respectively. Scale bar is 2 mm. B: AFM indentations were made as a function of length along tissue sections for normal, infarcted, DMEM-injected, and MSC-injected rats (n = 2 animals for each group). Elastic modulus for infarcted animals increased dramatically at point of ligation and plateaued at 55 ± 15 kPa in control infarcted animals and at 40 ± 10 kPa in MSC-injected animals. Gradients in elastic modulus resulting from border zone softening, represented by blue and black solid lines, were 8.5 kPa/mm for control infarcted animals, whereas the MSC-injected animals had a shallower gradient of 4.3 kPa/mm (dotted lines, extrapolated data). Gray shaded zone represents the normal stiffness required for differentiation of skeletal muscle reported by Engler et al. (7). Inset: sample force-indentation curve. MI, myocardial infarction.

 
Hemodynamic measurements. The remaining animals that underwent ligation and either cell (n = 8) or DMEM (n = 8) injection were recovered for 8 wk, when they were again anesthetized, intubated, and mechanically ventilated. A median sternotomy was performed, and a 2-Fr pressure-volume conductance catheter (Millar Instruments, Houston, TX) was inserted into the LV through the apex to obtain hemodynamic data, which were analyzed with ARIA 1 Pressure-Volume Analysis software (Millar Instruments) (4). The following parameters were chosen for analysis: maximum LV pressure, ejection fraction, maximum change in pressure over time (dP/dtmax), the dP/dtmax-end-diastolic volume (EDV) relationship, and maximal elastance as indexes of systolic function; minimum dP/dt, and the time constant of relaxation ({tau}) as indexes of diastolic function; and preload recruitable stroke work as an index of contractility. At the completion of the hemodynamic measurement, hearts were arrested in diastole by injection of 0.1 ml of KCl (1 meq/ml), and the LV cavity was filled with optimal cutting temperature (OCT)-embedding compound retrograde through the transected aortic root. The distended heart was placed in a container of OCT, frozen in liquid nitrogen, and stored at –80°C. Heart sections (10-µm thick) were prepared with a cryostat through the infarct region from the point of ligation to the apex of the heart at 2.5-mm intervals.

Histological and viability measurements. Sections were fixed with formaldehyde, permeabilized using 0.5% Triton X-100 in phosphate- buffered saline (PBS), and blocked with 5% bovine serum albumin in PBS. Sections were incubated with antibodies to either human lamin A/C or human cardiac muscle troponin T (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were then rinsed and incubated with secondary antibodies (Jackson ImmunoResearch; West Grove, PA) and mounted with Vectashield mounting medium with 4'-6-diamidino-2-phenylindole (Vector, Burlingame, CA).

Ventricular geometry was reconstructed from photomicrographs of hematoxylin and eosin-stained heart sections. Measurements were performed on two sections obtained from the midpoint between the LAD ligation and the apex of the heart for each animal and averaged for each geometric parameter. Wall thickness measurements were performed on two separate border zone areas, and ventricular diameter measurements were performed in both the anterior-posterior and septal-lateral axes for each section. For all analyses, the border zone was defined as the viable myocardial tissue immediately adjacent to the infarct scar. Infarct thickness was measured at the midpoint of the infarct region, which was the point in the infarct region that was equidistant from each infarct border. All thickness measurements were made in a radial direction, perpendicular to the ventricular wall. Infarct size in each heart was calculated by averaging the percentages of both inner and outer infarct scar circumferences relative to the LV free wall on two separate sections for each heart.

Additionally, heart sections were stained with Masson Accustain Trichrome stain (Sigma, St. Louis, MO) to distinguish areas of connective tissue. The extent of fibrosis in the infarct region of each heart was measured as previously described (40). The percentage of blue staining, indicative of fibrosis, was measured (10 fields randomly selected from the infarct area on each section) from the infarct area on two sections from each heart and averaged. The value was expressed as the ratio of Trichrome-stained fibrosis area to total infarct area. Finally, TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed on heart sections with a TdT-FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, Boston, MA) to quantify apoptosis. Counterstaining with methyl green was performed to visualize normal nuclei. Measurements of the apoptotic nuclei percentage were obtained from the border zone area of two sections from each heart and averaged (4 fields randomly selected from the border zone of each heart).

Immunohistochemistry to assess angiogenesis in the border zone was performed by incubating sections with a mouse monoclonal antibody to the endothelial cell marker von Willebrand factor (Cedar Lane Laboratories, Hornby, ON, Canada), followed by a secondary antibody linked to alkaline phosphatase. Detection was performed with the 5-bromo-4-chloro-3-indolyl phosphate/Nitro Blue Tetrazolium Liquid Substrate System (Sigma). The sections were then counterstained with eosin, and quantitative assessment of the number of endothelial cells per high-powered field was determined in five representative border zone fields in a blinded fashion.

To compare matrix metalloproteinase (MMP) activity in cell-injected animals and control animals, myocardial biopsy specimens from the infarct border zone of three animals in each group were pulverized, homogenized in 10 vol of protein extraction buffer [50 mmol/l Tris (pH 7.5), 1 mmol/l CaCl2, and 0.5% Triton X-100], sheared with a 25-gauge needle, and centrifuged for 1 min at 6,000 rpm to remove cellular debris. The supernatants were assayed for total protein content (Bio-Rad Protein Assay), and 100 µg of each sample were assayed for MMP activity using the Type 1 Collagenase Activity Assay Kit (Chemicon) according to the manufacturer's instructions. Comparison of absorbance to p-aminophenylmercuric acetate-activated MMP-1 control samples (provided by the manufacturer) processed in parallel was performed to quantify the absorbance data, with levels of activity reported as nanograms of control MMP activity.

Statistical analysis. A single investigator blinded to the treatment group performed all hemodynamic and histological analyses and measurements. All values are expressed as means ± SE. The unpaired Student's t-test with the use of a two-tailed distribution was used to calculate the statistical significance between the means of the two groups. A P value of <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal mortality. Three of the animals in the study died in the immediate postoperative period; one had received cell injections and two had received DMEM injections. Animals that died had their chests immediately reopened, and none had an obvious cause of death or appeared qualitatively to have had significantly different infarct sizes from all other animals. Deaths were presumed to be from a malignant arrhythmia or acute respiratory insufficiency related to the thoracotomy and heart manipulation.

Cell engraftment and protein expression. Qualitative histological analysis of the infarct region showed increased cellularity in MSC-injected animals (Fig. 1, D and E) versus control animals (Fig. 1, A and B) 8 wk postinfarction. Immunofluorescent staining for human lamin, a nuclear structural protein, verified the presence of the injected human MSCs (Fig. 1F), although the engraftment pattern was not uniformly distributed. Some engrafted cells expressed the cardiomyocyte-specific protein troponin T (Fig. 2, A and B) but lacked morphological structures similar to cardiomyocytes and structural integration with native cardiomyocytes (Fig. 1, D and E).


Figure 1
View larger version (113K):
[in this window]
[in a new window]
  		Fig. 1. Mesenchymal stem cells (MSCs) engrafted into the infarct region 8 wk postinfarction. A, B, D, and E: hematoxylin and eosin staining qualitatively demonstrates presence of more cells in infarct region at 8 wk for MSC-injected rats vs. control animals (A and D, x100 magnification; B and E, x400). Infarct regions shown are approximately same distance from border zone for both MSC and control animals. C and F: merged images of double staining of sections for human lamin (red) and nonspecific nuclear stain 4'-6-diamidino-2-phenylindole (DAPI, blue) demonstrate the presence of human cells in infarct region of MSC-injected rats but not control animals (x400).

 

Figure 2
View larger version (60K):
[in this window]
[in a new window]
  		Fig. 2. A: human cardiomyocyte troponin T (green) and DAPI (blue) show that engrafted cells are expressing troponin T at 8 wk postinfarct. B: hearts lacking MSC injections do not have troponin T expression. All images are at x400. Note that control hearts do contain some nonspecific background green staining in region of fibrotic infarct; however, fluorescent signal intensity observed in control hearts was much less relative to MSC-injected hearts, and green staining in control hearts did not appear to be associated with an adjacent DAPI-stained nucleus.

 
Local ventricular stiffness determined by AFM. The baseline elastic modulus for normal heart muscle from noninfarcted animals (EMyocardium) was 18 ± 2 kPa. Infarcted animals receiving no treatment or simply receiving DMEM injections formed significant fibrosis, with a similar threefold increase in the elastic modulus in both control groups (55 ± 15 kPa; P = 0.49) (Fig. 3). Tissue elasticity proximal to the occluded vessel was normal, but the tissue modulus was sharply increased from the LAD ligation point to a point ~6 mm distally. Fibrosis was less dramatic further away from the ligation point (6–9 mm) in the infarct border zone, and passive tissue stiffness softened at a rate of 8.5 kPa/mm until approaching normal EMyocardium. At the point of ligation, MSC-injected animals also exhibited a sharp increase but had a significantly softer tissue modulus (40 ± 10 kPa). Although the elevated stiffness of the infarct occurred over a similar area compared with control animals, softening of the border zone was more gradual (4.3 kPa/mm) and eases the modulus mismatch between stiff, infarcted regions and normal tissue. Overall, MSC injections resulted in a 30% reduction in modulus compared with controls (P < 0.001), and the myocardium was only twofold stiffer compared with normal. It is important to note the heterogeneity observed in both MSC-injected and control cases (illustrated by large error bars in the binned data), which is indicative of a very localized fibrosis-induced stiffening, effectively creating a "composite" tissue with less modulus mismatch.

Ventricular remodeling. Infarct size was not significantly different between the MSC-injected animals and the control animals [46 ± 2 (MSC) vs. 46 ± 4% (control) of LV free wall]. MSC-injected animals had greater preservation of normal LV geometry compared with control animals, including improved LV chamber diameter and wall thickness of both the border zone and infarct myocardium (Fig. 4). The geometrical improvements seen in the MSC-injected rats were associated with a 20% reduction in fibrosis compared with control hearts in the infarct region (Fig. 5). TUNEL staining demonstrated that MSC-injected hearts also had decreased apoptosis in the myocardial border zone (Fig. 6). Endothelial cell density was equivalent in the border zone myocardium of the MSC-injected and control animals [67.2 ± 4.1 (MSC) vs. 65.2 ± 4.5 (control) von Willebrand factor-positive cells per high-power field; P = 0.74, n = 7 in each group], as was MMP activity [19.6 ± 8.4 (MSC) vs. 14.2 ± 4.2 ng (control) of MMP-1 activity; P = 0.60, n = 3 in each group]. In vivo conductance catheter measurements of LV end-diastolic volume trended toward less ventricular dilation (Table 1). Therefore, cells remained embedded in the ventricular wall, softening it mechanically rather than differentiating or undergoing apoptosis.


Figure 4
View larger version (58K):
[in this window]
[in a new window]
  		Fig. 4. Tissue sections 8 wk postinfarct from control animals (A) and MSC-injected animals (B) (hematoxylin and eosin staining) are shown. Solid arrows indicate where infarct thickness was measured, and dashed arrows indicate where border zone thickness was measured. Average left ventricular (LV) chamber diameter, border zone wall thickness, and infarct wall thickness (C) are significantly improved in MSC-injected animals (n = 8) compared with controls (n = 8). *P = 0.009, {dagger}P = 0.0008, and {ddagger}P = 0.049 compared with control.

 

Figure 5
View larger version (63K):
[in this window]
[in a new window]
  		Fig. 5. Representative sections with Masson Trichrome staining 8 wk postinfarct from control animals (A and A') and MSC-injected animals (B and B') show that fibrotic blue areas are more prevalent in control animals. Magnification in A' and B' is x100. Average extent of fibrosis (expressed as percentage of infarct that is fibrotic) in infarct region (C) is shown for control (n = 6) and MSC (n = 8) groups. *P = 0.006.

 

Figure 6
View larger version (64K):
[in this window]
[in a new window]
  		Fig. 6. Representative TdT-mediated dUTP nick-end labeling (TUNEL)-stained sections in control (A) and MSC-injected (B) animals (x400) where apoptotic nuclei are stained dark brown (arrows) and normal nuclei are stained blue-green. Average percentage of TUNEL-positive cells in infarct border zone area (C) is shown for control (n = 8) and MSC-injected (n = 8) animals. HPF, high-powered field. *P = 0.006.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Rats injected with MSCs have improved hemodynamics and contractility versus control rats

 
Hemodynamics and cardiac function. MSC-injected rats had significant hemodynamic preservation compared with control animals (Table 1 and Fig. 7). Pressure-volume loops obtained during inferior vena cava (IVC) occlusion (Fig. 7A) show increased LV end-systolic pressure and decreased EDV for MSC-injected rats compared with controls. MSC-injected rats also showed improved LV systolic function in maximum pressure, dP/dtmax, and dP/dtmax-EDV relationship, as determined by pressure-volume loops during IVC occlusion (Table 1 and Fig. 7B). MSC-injected rats had a larger slope and decreased intercept for the dP/dtmax-EDV relationship. The relationship between stroke work and EDV, also determined from pressure-volume loops during IVC occlusion, demonstrated that MSC-injected rats had improved preload recruitable stroke work and cardiac contractility (Table 1 and Fig. 7C). In addition, MSC animals had higher maximal elastance (Table 1), which is another index of global cardiac contractility determined by the end-systolic pressure-volume relationship. Finally, MSC animals had better diastolic function, indicated by minimum dP/dt and a trend toward improvement in the relaxation time constant {tau} (Table 1).


Figure 7
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 7. A: representative pressure-volume loops for control (left) and MSC-injected (right) animals. Loops for control animals are shifted downward and rightward compared with MSC-injected animals. B: maximum change in pressure over time (dP/dtmax)-end-diastolic volume (EDV) relationships show that MSC-injected animals have a leftward and upward shift compared with control animals. Each line on graph represents data points for dP/dtmax and EDV measured from individual pressure-volume loops obtained during reduction of preload volume by inferior vena cava (IVC) occlusion. C: representative stroke work (SW)-EDV relationships show that MSC-injected animals are shifted leftward and upward compared with control animals. Each line on graph represents data points for SW and EDV measured from individual pressure-volume loops obtained during reduction of preload volume by IVC occlusion.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Ventricular remodeling after myocardial infarction generally involves cardiomyocyte necrosis and apoptosis, expansion of the infarct border zone, ventricular dilation, wall thinning, progressive fibrosis, and ultimately, further loss of function and the clinical syndrome of heart failure (2, 3, 11, 13, 19, 21, 30, 31). Cellular cardiomyoplasty appears promising as a heart failure treatment, particularly with MSCs that could differentiate into cardiomyocytes (14, 23, 28, 34). In this study, MSCs engrafted and expressed muscle proteins but did not appear to form contractile elements that integrated with native tissue. Cell injection also did not stimulate significant angiogenesis because endothelial cell density was equivalent in the border zone myocardium of both the MSC and control groups, although it should be noted that interpreting endothelial cell density is limited in that vascularization could be different even with identical endothelial cell density if cells are organized differently in the two groups. Cell injection also did not change MMP activity in the border zone, though it is important to acknowledge that MMP activity could be impacted more in the infarct region where more dramatic effects on myocardial stiffness occur. Still, cellular engraftment attenuated postinfarction remodeling and preserved cardiac function, as seen in previous studies. Cellular engraftment appeared to provide functional benefit by effectively softening the tissue by increasing cellularity in the infarct scar and making it a more heterogeneous, "composite" tissue rather than by regenerating functional cardiomyocytes. Injecting cells significantly improved the subsequent elastic moduli of the infarct, as well as more gradually softening the border zone. Having a more compliant infarct scar early after infarction was subsequently associated with less remodeling, with reduced thinning of the infarct and border zone myocardium, border zone apoptosis, and dilation of the LV cavity. Whereas fibrosis of infarcted myocardium is needed to prevent myocardial rupture, the resultant dense scar that the remaining noninfarcted portions of the ventricle have to contract against may drive some of the subsequent detrimental ventricular remodeling. The results of this study appear to be the first observations that directly link changes in postinfarction remodeling to having a more compliant infarct scar.

The importance of matrix mechanics on cellular and tissue function is not restricted to cardiomyocytes and the heart. Similar phenomena have been reported with smooth muscle cells, epithelial cells, fibroblasts, endothelial cells, and neurons (5, 8, 9, 15, 22, 25, 37). A narrow optimum in matrix stiffness, recently shown to be required for myofibrillogenesis in myotubes, has also been extended to MSC differentiation, which shows matrix stiffness-dependent morphology (7). Interestingly, the baseline elasticity for normal heart muscle from noninfarcted animals is within the 95% confidence interval of elastic moduli found necessary for muscle striation, as indicated by the gray region of Fig. 3B. A dramatic example of the physical limitations that the ECM confers is that stem cells injected into fibrotic, dystrophic skeletal muscle differentiate into connective tissue rather than muscle (12).

Important questions remain regarding cellular cardiomyoplasty techniques. It is important to note that the success of cell engraftment was not quantified in this study; thus these results can only be interpreted as a qualitative correlation of functional changes with the presence of exogenous cells. Cell transplantation can confer some functional benefits with relatively modest amounts of engraftment, but longer-term studies are also needed to examine prolonged engraftment. Hearts may simply resume the postinfarction remodeling process if long-term cell survival is limited, in which case cell therapy simply delays the onset of heart failure. Survival of transplanted cells is limited by apoptosis and ischemia, so combining cell transplantation techniques with either revascularization or angiogenic techniques may be needed to improve engraftment and ultimately mechanical function (17, 39).

Another important question is whether multipotent MSCs will truly differentiate into cardiomyocytes after transplantation. The mechanical properties and fibrotic microenvironment of the infarct area may limit striation and development of a contractile myocardium, as seen in myotubes (7, 29). Further matrix softening with other antifibrotic therapies may be required before a proper microenvironment for MSC differentiation will exist such that preinfarct ventricle function can be restored. In addition, cardiomyocyte contact has been shown to induce MSCs to express a cardiomyocyte phenotype (24). More recently, however, it has been shown that bone marrow cells fuse with cardiac muscle cells and do not truly "transdifferentiate" into cardiomyocytes (1). The cells in our study that expressed cardiac muscle proteins were located in the infarct region and isolated from viable myocardium, making it unlikely that they were expressing a cardiomyocyte phenotype due to cell fusion. However, even with the expression of cardiac proteins, the engrafted cells did not receive the appropriate cues to fully differentiate into cardiomyocytes. Additional signaling in the form of genetic modification or from extracellular mediators may have to be provided to the engrafted cells to completely push them toward a cardiomyocyte phenotype. Also, whether cell fusion or simply cell contact is needed, transplantation of enough cells to completely fill the infarct region to ensure a bridge of cells from border zone to border zone may be needed to result in the engrafted cells eventually becoming cardiomyocytes that effectively participate in the contractile process.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grants HL-59407 (to H. L. Sweeney), HL-007843 (to M. F. Berry), and HL-07281201 (to Y. J. Woo) from National Heart, Lung, and Blood Institute, a National Institutes of Health-Bioengineering Research Partnerships grant (to A. J. Engler and D. E. Discher) from National Institute of Arthritis and Musculoskeletal and Skin Diseases, and by a grant from the Thoracic Surgery Foundation for Research and Education (to Y. J. Woo).


   ACKNOWLEDGMENTS
 
The mesenchymal stem cells used in this study were supplied by Osiris Therapeutics (Baltimore, MD).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. L. Sweeney, Dept. of Physiology, Univ. of Pennsylvania School of Medicine, A700 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6085 (e-mail: lsweeney{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, and Alvarez-Buylla A. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425: 968–973, 2003.[CrossRef][Medline]
  2. Beltrami CA, Finato N, Rocco M, Feruglio GA, Puricelli C, Cigola E, Quaini F, Sonnenblick EH, Olivetti G, and Anversa P. Structural basis of end-stage failure in ischemic cardiomyopathy in humans. Circulation 89: 151–163, 1994.[Abstract/Free Full Text]
  3. Chatterjee S, Stewart AS, Bish LT, Jayasankar V, Kim EM, Pirolli T, Burdick J, Woo YJ, Gardner TJ, and Sweeney HL. Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation 106: I212–I217, 2002.
  4. Cingolani OH, Yang XP, Cavasin MA, and Carretero OA. Increased systolic performance with diastolic dysfunction in adult spontaneously hypertensive rats. Hypertension 41: 249–254, 2003.[Abstract/Free Full Text]
  5. Deroanne CF, Lapiere CM, and Nusgens BV. In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. Cardiovasc Res 49: 647–658, 2001.[Abstract/Free Full Text]
  6. Domke J and Radmacher M. Measuring the elastic properties of thin polymer films with the atomic force microscope. Langmuir 14: 3320–3325, 1998.[CrossRef]
  7. Engler AJ, Griffin MA, Sen S, Bonneman C, Sweeney HL, and Discher DE. Myotubes differentiate optimally on substrates with tissue-like stiffness: implications for soft or stiff microenvironments. J Cell Biol 166: 877–887, 2004.[Abstract/Free Full Text]
  8. Engler A, Bacakova L, Newman C, Hategan A, Griffin M, and Discher D. Substrate compliance versus ligand density in cell on gel responses. Biophys J 86: 617–628, 2004.[Web of Science][Medline]
  9. Flanagan LA, Ju YE, Marg B, Osterfield M, and Janmey PA. Neurite branching on deformable substrates. Neuroreport 13: 2411–2415, 2002.[CrossRef][Web of Science][Medline]
  10. Gaudet C, Marganski WA, Kim S, Brown CT, Gunderia V, Dembo M, and Wong JY. Influence of type I collagen surface density on fibroblast spreading, motility, and contractility. Biophys J 85: 3329–3335, 2003.[Web of Science][Medline]
  11. Gheorghiade M and Bonow R. Chronic heart failure in the United States: a manifestation of coronary artery disease. Circulation 97: 282–289, 1998.[Free Full Text]
  12. Huard J, Cao B, and Qu-Petersen Z. Muscle-derived stem cells: potential for muscle regeneration. Birth Defects Res C Embryo Today 69: 230–237, 2003.[CrossRef][Medline]
  13. Hunter JJ and Chien KR. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 341: 1276–1283, 1999.[Free Full Text]
  14. Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AM, Deans R, Marshak DR, and Flake AW. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 6: 1282–1286, 2000.[CrossRef][Web of Science][Medline]
  15. Lo CM, Wang HB, Dembo M, and Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J 79: 144–152, 2000.[Web of Science][Medline]
  16. Mahaffy RE, Shih CK, MacKintosh FC, and Kas J. Scanning probe-based frequency-dependent microrheology of polymer gels and biological cells. Phys Rev Lett 85: 880–883, 2000.[CrossRef][Web of Science][Medline]
  17. Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, and Dzau VJ. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 9: 1195–1201, 2003.[CrossRef][Web of Science][Medline]
  18. Min JY, Sullivan MF, Yang Y, Zhang JP, Converso KL, Morgan JP, and Xiao YF. Significant improvement of heart function by cotransplantation of human mesenchymal stem cells and fetal cardiomyocytes in postinfarcted pigs. Ann Thorac Surg 74: 1568–1575, 2002.[Abstract/Free Full Text]
  19. Olivetti G, Quaini F, Sala R, Lagrasta C, Corradi D, Bonacina E, Gambert SR, Cigola E, and Anversa P. Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol 28: 2005–2016, 1996.[CrossRef][Web of Science][Medline]
  20. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, Mckay R, Nadal-Ginard B, Bodine DM, Leri A, and Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature 410: 701–705, 2001.[CrossRef][Medline]
  21. Pan J, Fukuda K, Saito M, Matsuzaki J, Kodama H, Sano M, Takahashi T, Kato T, and Ogawa S. Mechanical stretch activates the JAK/STAT pathway in rat cardiomyocytes. Circ Res 84: 1127–1136, 1999.[Abstract/Free Full Text]
  22. Pelham RJ and Wang YL. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA 94: 13661–13665, 1997.[Abstract/Free Full Text]
  23. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, and Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147, 1999.[Abstract/Free Full Text]
  24. Rangappa S, Entwistle JW, Wechsler AS, and Kresh JY. Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg 126: 124–132, 2003.[Abstract/Free Full Text]
  25. Reinhart-King CA, Dembo M, and Hammer DA. Endothelial cell traction forces on RGD-derivatized polyacrylamide substrata. Langmuir 19: 1573–1579, 2003.[CrossRef]
  26. Roell W, Lu ZJ, Bloch W, Siedner S, Tiemann K, Xia Y, Stoecker E, Fleischmann M, Bohlen H, Stehle R, Kolossov E, Brem G, Addicks K, Pfitzer G, Welz A, Hescheler J, and Fleischmann BK. Cellular cardiomyoplasty improves survival after myocardial injury. Circulation 105: 2435–2441, 2002.[Abstract/Free Full Text]
  27. Rotsch C, Jacobson K, and Radmacher M. Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy. Proc Natl Acad Sci USA 96: 921–926, 1999.[Abstract/Free Full Text]
  28. Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, Pittenger MF, and Martin BJ. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 73: 1919–1925, 2002.[Abstract/Free Full Text]
  29. Stedman HH, Sweeney HL, Shrager JB, Maguire HC, Panettieri RA, Petrof B, Narusawa M, Leferovich JM, Sladky JT, and Kelly AM. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 352: 536–539, 1991.[CrossRef][Medline]
  30. St. John Sutton M, Pfeffer MA, Moye L, Plappert T, Rouleau JL, Lamas G, Rouleau J, Parker JO, Arnold MO, Sussex B, and Braunwald E. Cardiovascular death and left ventricular remodeling two years after myocardial infarction. Circulation 96: 3294–3299, 1997.[Abstract/Free Full Text]
  31. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev 79: 215–262, 1999.[Abstract/Free Full Text]
  32. Taylor DA, Atkins BZ, Hungspreugs P, Jones TR, Reedy MC, Hutcheson KA, Glower DD, and Kraus WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 4: 929–933, 1998.[CrossRef][Web of Science][Medline]
  33. Thai HM, Van HT, Gaballa MA, Goldman S, and Raya TE. Effects of AT1 receptor blockade after myocardial infarct on myocardial fibrosis, stiffness, and contractility. Am J Physiol Heart Circ Physiol 276: H873–H880, 1999.[Abstract/Free Full Text]
  34. Toma C, Pittenger MF, Cahill KS, Byrne BJ, and Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105: 93–98, 2002.[Abstract/Free Full Text]
  35. Tomita S, Li RK, Weisel RD, Mickle DAG, Kim EJ, Sakai T, and Jia ZQ. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 100: II247–II256, 1999.
  36. Tsuda T, Gao E, Evangelisti L, Markova D, Ma X, and Chu ML. Post-ischemic myocardial fibrosis occurs independent of hemodynamic changes. Cardiovasc Res 59: 926–933, 2003.[Abstract/Free Full Text]
  37. Wong JY, Velasco A, Rajagopalan P, and Pham Q. Directed movement of vascular smooth muscle cells on gradient-compliant hydrogels. Langmuir 19: 1908–1913, 2003.[CrossRef]
  38. Zaari N, Rajagopalan P, Kim SK, Engler AJ, and Wong JY. Hydrogels photopolymerized in a microfluidics gradient generator: tuning substrate compliance at the microscale to control cell response. Adv Mat 16: 2133–2137, 2004.[CrossRef]
  39. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, and Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 33: 907–921, 2001.[CrossRef][Web of Science][Medline]
  40. Zou Y, Takano H, Mizukami M, Akazawa H, Qin Y, Toko H, Sakamoto M, Minamino T, Nagai T, and Komuro I. Leukemia inhibitory factor enhances survival of cardiomyocytes and induces regeneration of myocardium after myocardial infarction. Circulation 108: 748–753, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Eur J Heart FailHome page
K. Yao, R. Huang, A. Sun, J. Qian, X. Liu, L. Ge, Y. Zhang, S. Zhang, Y. Niu, Q. Wang, et al.
Repeated autologous bone marrow mononuclear cell therapy in patients with large myocardial infarction
Eur J Heart Fail, July 1, 2009; 11(7): 691 - 698.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
D. E. Discher, D. J. Mooney, and P. W. Zandstra
Growth Factors, Matrices, and Forces Combine and Control Stem Cells
Science, June 26, 2009; 324(5935): 1673 - 1677.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
S.-W. Song, W. Chang, B.-W. Song, H. Song, S. Lim, H.-J. Kim, M.-J. Cha, E. Choi, S.-H. Im, B.-C. Chang, et al.
Integrin-Linked Kinase Is Required in Hypoxic Mesenchymal Stem Cells for Strengthening Cell Adhesion to Ischemic Myocardium
Stem Cells, June 1, 2009; 27(6): 1358 - 1365.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
L. Herbots, J. D'hooge, E. Eroglu, D. Thijs, J. Ganame, P. Claus, C. Dubois, K. Theunissen, J. Bogaert, J. Dens, et al.
Improved regional function after autologous bone marrow-derived stem cell transfer in patients with acute myocardial infarction: a randomized, double-blind strain rate imaging study
Eur. Heart J., March 2, 2009; 30(6): 662 - 670.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Gnecchi, Z. Zhang, A. Ni, and V. J. Dzau
Paracrine Mechanisms in Adult Stem Cell Signaling and Therapy
Circ. Res., November 21, 2008; 103(11): 1204 - 1219.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. J. Engler, C. Carag-Krieger, C. P. Johnson, M. Raab, H.-Y. Tang, D. W. Speicher, J. W. Sanger, J. M. Sanger, and D. E. Discher
Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating
J. Cell Sci., November 15, 2008; 121(22): 3794 - 3802.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
R. A. Rose, H. Jiang, X. Wang, S. Helke, J. N. Tsoporis, N. Gong, S. C.J. Keating, T. G. Parker, P. H. Backx, and A. Keating
Bone Marrow-Derived Mesenchymal Stromal Cells Express Cardiac-Specific Markers, Retain the Stromal Phenotype, and Do Not Become Functional Cardiomyocytes In Vitro
Stem Cells, November 1, 2008; 26(11): 2884 - 2892.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. A. Carr, D. J. Stuckey, L. Tatton, D. J. Tyler, S. J. M. Hale, D. Sweeney, J. E. Schneider, E. Martin-Rendon, G. K. Radda, S. E. Harding, et al.
Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: an in vivo cine-MRI study
Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H533 - H542.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
R. W. Grauss, J. van Tuyn, P. Steendijk, E. M. Winter, D. A. Pijnappels, B. Hogers, A. C. Gittenberger-De Groot, R. van der Geest, A. van der Laarse, A. A.F. de Vries, et al.
Forced Myocardin Expression Enhances the Therapeutic Effect of Human Mesenchymal Stem Cells After Transplantation in Ischemic Mouse Hearts
Stem Cells, April 1, 2008; 26(4): 1083 - 1093.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
R. G. Wells and D. E. Discher
Matrix Elasticity, Cytoskeletal Tension, and TGF-{beta}: The Insoluble and Soluble Meet
Sci. Signal., March 11, 2008; 1(10): pe13 - pe13.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. W. Grauss, E. M. Winter, J. van Tuyn, D. A. Pijnappels, R. V. Steijn, B. Hogers, R. J. van der Geest, A. A. F. de Vries, P. Steendijk, A. van der Laarse, et al.
Mesenchymal stem cells from ischemic heart disease patients improve left ventricular function after acute myocardial infarction
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2438 - H2447.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
W. Li, N. Ma, L.-L. Ong, C. Nesselmann, C. Klopsch, Y. Ladilov, D. Furlani, C. Piechaczek, J. M. Moebius, K. Lutzow, et al.
Bcl-2 Engineered MSCs Inhibited Apoptosis and Improved Heart Function
Stem Cells, August 1, 2007; 25(8): 2118 - 2127.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
S. Schenk, N. Mal, A. Finan, M. Zhang, M. Kiedrowski, Z. Popovic, P. M. McCarthy, and M. S. Penn
Monocyte Chemotactic Protein-3 Is a Myocardial Mesenchymal Stem Cell Homing Factor
Stem Cells, January 1, 2007; 25(1): 245 - 251.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
G. R. Gaudette and I. S. Cohen
Cardiac Regeneration: Materials Can Improve the Passive Properties of Myocardium, but Cell Therapy Must Do More
Circulation, December 12, 2006; 114(24): 2575 - 2577.
[Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
L. C. Amado, K. H. Schuleri, A. P. Saliaris, A. J. Boyle, R. Helm, B. Oskouei, M. Centola, V. Eneboe, R. Young, J. A.C. Lima, et al.
Multimodality Noninvasive Imaging Demonstrates In Vivo Cardiac Regeneration After Mesenchymal Stem Cell Therapy
J. Am. Coll. Cardiol., November 21, 2006; 48(10): 2116 - 2124.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2196    most recent
01017.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (28)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Berry, M. F.
Right arrow Articles by Sweeney, H. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berry, M. F.
Right arrow Articles by Sweeney, H. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.