Engraftment Is Optimal When Myoblasts Are Transplanted Early: The Role of Hepatocyte Growth Factor

Transcription

1 Engraftment Is Optimal When Myoblasts Are Transplanted Early: The Role of Hepatocyte Growth Factor Stacy B. O Blenes, MD, Audrey W. Li, PhD, Robert Chen, MD, Rakesh C. Arora, MD, PhD, and Magda Horackova, PhD IWK Health Centre, Departments of Surgery, Physiology and Biophysics, and Pediatrics, Dalhousie University, Halifax, Nova Scotia, and Cardiac Sciences Program, St. Boniface General Hospital, Winnipeg, Manitoba, Canada Background. In a recent clinical trial, skeletal myoblast (SKMB) transplantation performed late after myocardial infarction (MI) did not improve left ventricular function. We hypothesized that (1) delaying SKMB transplantation until a chronic infarct scar has developed reduces engraftment, and (2) hepatocyte growth factor (HGF), a main regulator of SKMBs, is present in acute but not chronic MI, potentially influencing engraftment. Methods. Rats underwent coronary artery ligation followed by SKMB transplantation immediately (n 12) or delayed by 5 weeks (n 11). The volume of engrafted SKMBs was quantified 6 weeks later. Hepatocyte growth factor was evaluated by computerized analysis of immunohistochemical labeling of rat heart sections 48 hours, 1 week, 2 weeks, and 5 weeks after coronary artery ligation. The impact of HGF on SKMB proliferation and its ability to protect against oxidative stress and hypoxia was evaluated in vitro. Results. Skeletal myoblast transplantation immediately after MI resulted in an engraftment volume of mm 3. However, delaying SKMB transplantation 5 weeks caused a 95% drop in engraftment ( mm 3 ; p < 0.001). Hepatocyte growth factor labeling in MIs 48 hours after coronary artery ligation was similar to control myocardium ( versus units). However, HGF declined progressively at 1, 2, and 5 weeks after MI ( , , and units, respectively; p < 0.05 versus 48 hours). Hepatocyte growth factor caused a dose-dependent increase in SKMB proliferation in vitro and protected against oxidative stress and hypoxia. Conclusions. These results demonstrate that engraftment of SKMBs is impaired when transplantation is delayed until a chronic infarct has developed. Hepatocyte growth factor in MI declines with time and may enhance engraftment of SKMBs transplanted early after MI. Delivery of exogenous HGF to enhance SKMB engraftment in chronic infarcts warrants further investigation. (Ann Thorac Surg 2010;89:829 36) 2010 by The Society of Thoracic Surgeons Cell transplantation is the focus of intensive investigation because it holds the promise of cardiac regeneration. Autologous cardiac progenitor cells [1, 2] and induced pluripotent stem cells [3, 4] are exciting candidates for cell transplantation under active investigation, but clinical trials have so far been limited to autologous mesenchymal stem cells and skeletal myoblasts (SKMBs). Results with mesenchymal stem cells delivered by catheter into the infarct-related artery were mixed [5, 6]. Skeletal myoblasts transplanted by direct injection improved ventricular function in animal models of myocardial injury [7, 8], but a randomized trial in patients with chronic myocardial infarction (MI) did not show improved ventricular function compared with placebo [9]. The primary aim of this study was to Accepted for publication Dec 2, Deceased. Address correspondence to Dr O Blenes, IWK Children s Heart Centre, IWK Health Centre, 5850/5980 University Ave, PO Box 9700, Halifax, Nova Scotia, Canada B3K6R8; stacy.oblenes@iwk.nshealth.ca. determine whether delaying SKMB transplantation until a chronic MI scar has developed, as was the case in the clinical trial [9], negatively impacts the extent of SKMB engraftment. Skeletal myoblasts are normally involved in repair of skeletal muscle by fusing to form multinucleated myotubes that incorporate into the injured fiber [10]. Satellite cells within skeletal muscle are activated and proliferate to produce SKMBs in response to injury [11]. This process is primarily regulated by hepatocyte growth factor (HGF) released from extracellular matrix stores and produced by infiltrating inflammatory cells and SKMBs themselves [10 13]. Although HGF in MI has not been extensively studied, it is elevated in the serum of patients with acute coronary syndromes, and HGF mrna levels are increased in rat hearts early after coronary artery ligation [14 16]. Therefore, our second aim was to evaluate the time course of HGF expression in MI and explore potential mechanisms by which HGF could enhance SKMB engraftment in early MI, including promotion of cell proliferation and protecting against oxidative stress and hypoxia by The Society of Thoracic Surgeons /10/$36.00 Published by Elsevier Inc doi: /j.athoracsur

2 830 O BLENES ET AL Ann Thorac Surg HGF AND TIMING OF MYOBLAST TRANSPLANT 2010;89: Coronary Artery Ligation Rats were anesthetized with ketamine hydrochloride (26 mg/kg intraperitoneally), xylazine (4.8 mg/kg intraperitoneally), and isoflurane (approximately 2%), intubated, and ventilated. The mid- left anterior descending coronary artery was ligated through a left thoracotomy. Persistent ventricular arrhythmias were treated (lidocaine, 1 mg/kg intravenously). Animals were extubated when breathing spontaneously. Ketoprofen (5 mg/kg subcutaneously) and buprenorphine (0.03 mg/kg subcutaneously, every 8 to 12 hours 4 doses) were used for analgesia. Fig 1. Characterization of cells used for transplantation. Representative merged image of desmin immunofluorescence and phase-contrast photomicrographs of cultured skeletal myoblasts used for transplantation. Cells are labeled with antibodies against desmin (red). Desmin-positive cells are identified by red labeling of the cytoplasm (arrows). Negative cells are identified by absence of red labeling in the cytoplasm (arrowheads). (Scale bar 40 m.) Material and Methods Animals Inbred Lewis rats (approximately 300 g, Charles River Canada, St Constant, Canada) were cared for in accordance with the Canadian Council on Animal Care guidelines. The Dalhousie University Committee on Laboratory Animals approved the experimental protocol. Skeletal Myoblast Isolation and Culture Rats were anesthetized with pentobarbital sodium (160 mg/kg intraperitoneally). The soleus muscles were removed and dissociated in Dulbecco s modified Eagle s Medium (Invitrogen, Grand Island, NY) containing protease (4 U/mL, Sigma Chemical Co, St Louis, MO), and collagenase (200 U/mL, Worthington, Lakewood, NJ). Cells isolated by centrifugation were resuspended in Ham s F-12 media (Invitrogen) containing 20% fetal bovine serum (FBS, Invitrogen) and plated in laminin- (1 g/cm 2, Sigma) coated dishes. Before transplantation, samples of the SKMBs were characterized according to a previously described method [17] using immunofluorescence labeling with antibodies against desmin, a cytoskeletal protein expressed in myoblasts and myocytes. We found that 87% 1% of the cells used for transplantation were desmin positive (Fig 1). During our initial experience, viability was verified with trypan blue exclusion, which was regularly found to be more than 95%. The SKMBs for transplantation were harvested from the primary cultures on day 7 by trypsinization and resuspended in minimum essential medium (Sigma) at a final concentration of cells/85 L. Skeletal Myoblast Transplantation Skeletal myoblast transplantation was performed either 5 minutes after coronary artery ligation (immediate) or 5 weeks later (delayed) during a repeat thoracotomy. Skeletal myoblasts were injected using 27-gauge needles equipped with calibrated flanges to prevent penetration into the ventricular chamber, confirmed by inability to aspirate blood and by observing infiltration of the ventricular wall. Four injections of SKMBs in 85 Lof minimum essential medium were performed in a diamond pattern within each infarct (total of cells per animal). Tissue Collection and Fixation Rats were euthanized 6 weeks after SKMB transplantation. The left ventricle was opened longitudinally opposite to the MI, pinned to a dish with the endocardium exposed, and fixed in 2% paraformaldehyde followed by 30% sucrose. Sections (40 m thick) were cut parallel to the endocardium using a freezing microtome. Immunofluorescence Staining, Quantification of Infarct Size, and Skeletal Myoblast Engraftment Every fifth section through the ventricular wall was double-labeled with antibodies against skeletal myosin to identify SKMBs and connexin 43 to identify surviving myocardium. Floating sections were incubated with primary antibody against skeletal myosin (mouse monoclonal, MY-32, 1:400, 1 hour, room temperature) in phosphate-buffered saline solution containing 2% normal sheep serum, washed, and then incubated with tetramethyl rhodamine isothiocyanate conjugated sheep anti mouse immunoglobulin G (75 g/ml, 1 hour, room temperature). Sections were then incubated with primary antibody against connexin 43 (rabbit polyclonal, C6219, 1:400, Sigma, 1 hour, room temperature) in phosphatebuffered saline solution with 2% normal goat serum, washed, and then incubated with fluorescein isothiocyanate conjugated goat anti-rabbit immunoglobulin G (75 g/ml, 1 hour, room temperature). Sections were evaluated using a computerized image analysis system (Neurolucida, MicroBrightField Inc, Williston, VT), allowing the total infarct area (absence of connexin 43 labeling) and the area occupied by engrafted SKMBs (positive for skeletal myosin) to be quantified for the entirety of each section. The volume of the infarct and the volume of

3 Ann Thorac Surg O BLENES ET AL 2010;89: HGF AND TIMING OF MYOBLAST TRANSPLANT engrafted SKMBs were calculated according to the following formula: n 1 Volume (S i T)[(A i A i 1 ) 2] i 1 where n is the number of serial sections per heart, S is the spacing to next analyzed section, T is the thickness of sections, and A is the area measured on each section. The overall area occupied by the infarct was measured and the average thickness calculated. Immunohistochemical Staining and Quantification of Hepatocyte Growth Factor Hepatocyte growth factor expression was evaluated by immunohistochemical staining in MIs of various ages. Endogenous peroxidase activity was quenched with 3% H 2 O 2 for 30 minutes at 70 C. Sections were incubated overnight with rabbit anti-human HGF antibody (1:125, Immuno-Biological Laboratories Co Ltd, Gunma, Japan) in 0.05 mol/l Tris-buffered saline solution containing 2% normal donkey serum and sodium azide (0.01%), washed, and then incubated with biotinylated donkey anti-rabbit immunoglobulin G (90 minutes, room temperature, 1:1000) and Vectastain ABC reagent (90 minutes, room temperature, Vector Laboratories, Burlingame, CA). Immunolabeling was visualized using 0.02% diaminobenzidine, 0.65% nickel ammonium sulfate, and 0.006% H 2 O 2, which produces a dark blue reaction. Control sections were processed with the omission of primary antibody. High-resolution composite images of each tissue section were generated under constant lighting and exposure conditions. Chromogen intensity in the infarct was quantified using a previously described technique that 831 measures both the number of positively labeled pixels and the intensity of labeling for each positive pixel [18]. Skeletal Myoblast In Vitro Studies Rat SKMBs cultured as described above were replated in laminin-coated 96-well plates at a concentration of 20,000 cells per well in media containing 20% FBS. After the interventions described below, cell number in each well was determined using a 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide (MTT) based assay involving spectrophotometric detection of MTT converted to a purple formazan by the mitochondria of viable cells [19]. Proliferation was assessed using a commercial 5-bromo- 2=-deoxyuridine incorporation assay (Calbiochem, La Jolla, CA). IMPACT OF HEPATOCYTE GROWTH FACTOR ON MYOBLAST PROLIFERATION. Nonconfluent SKMB cultures were serumrestricted (FBS in growth media reduced to 0.5%) and recombinant human HGF (0, 0.3, 3, or 30 ng/ml, R&D Systems Inc, Minneapolis, MN) was added. Cell number and proliferation was assayed 24 hours later. IMPACT OF HEPATOCYTE GROWTH FACTOR ON MYOBLAST NUMBER WITH EXPOSURE TO OXIDATIVE STRESS. Confluent SKMB cultures were serum-restricted and recombinant human HGF (0, 0.3, 3, or 30 ng/ml) was added. After 18 hours, H 2 O 2 (200 mol/l) was added to each of the wells (excluding controls) and cell number was assayed 3 hours later. IMPACT OF HEPATOCYTE GROWTH FACTOR ON MYOBLAST NUMBER WITH EXPOSURE TO HYPOXIA. Nonconfluent SKMB cultures Fig 2. Delaying skeletal myoblast (SKMB) transplantation reduces engraftment. Graphic representation of typical planimetry results from sections cut parallel to the endocardium in which myocardial infarction (MI; green) and engrafted SKMBs (red) were quantified 6 weeks after SKMB transplantation either immediately (A) or delayed by 5 weeks (B) after coronary artery ligation. Ticks on the y and z axes represent 2-mm intervals; the spacing between the sections is 200 m. (C) Graph of total infarct volume and volume of engrafted SKMBs. The total infarct volume is not different when SKMBs are transplanted immediately (n 12) or delayed by 5 weeks (n 11) after coronary artery ligation. However, the volume of SKMBs engrafted in the MI was reduced by 95% if transplantation was delayed 5 weeks. (D) Graph showing average thickness of MI. The Infarct scar was 54% thinner 6 weeks after delayed SKMB transplantation compared with 6 weeks after SKMB transplantation immediately after coronary artery ligation. (E) Graph showing MI area. Average infarct area was larger in the delayed transplant. (*p )

4 832 O BLENES ET AL Ann Thorac Surg HGF AND TIMING OF MYOBLAST TRANSPLANT 2010;89: Fig 3. Hepatocyte growth factor (HGF) declines with time in myocardial infarction (MI). Representative low-power composite photomicrographs of sections cut parallel to the endocardium through rat MIs 48 hours (A), 1 week (B), 2 weeks (C), and 5 weeks (D) after coronary artery ligation stained using antibodies against HGF. Hepatocyte growth factor labeling within the MI (outlined in red) declines with time. (E) Graph showing signal intensity for HGF labeling within the MIs quantified from immunohistochemically labeled sections. There is a progressive decline in HGF immunolabeling in MI with time. (*p versus 48 hours; **p versus 48 hours and p 0.05 versus 1 week; #p versus 48 hours and p 0.05 versus 1 week. Scale bar 5 mm.) were serum-restricted and recombinant human HGF was added to each well (0, 1, 3, 30 ng/ml). The SKMB cultures were maintained under normoxic conditions or exposed to a hypoxic environment (partial pressure of oxygen 1%, BD GasPak EZ, Beckton, Dickson, and Company, Shannon, Ireland) for 64 hours, after which they were returned to a normoxic environment and cell number was assayed. Statistical Analysis All data are expressed as mean standard error of the mean. Differences between means were evaluated using either a two-tailed, unpaired Student s t test or a oneway analysis of variance with post-hoc testing by Tukey s. All analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA). Results Coronary Artery Ligation and Skeletal Myoblast Transplantation Thirty-six rats underwent coronary artery ligation. Nine (25%) died in the perioperative period because of endotracheal tube displacement (n 4), ventricular arrhythmias (n 3), or unknown causes (n 2). Fourteen rats received SKMB transplantation immediately after coronary artery ligation. Thirteen received SKMB transplantation 5 weeks after coronary artery ligation (delayed group). One animal in the delayed group died of hemorrhage during the cell transplant procedure; there were otherwise no deaths associated directly with injection of the cells. Animals were sacrificed 6 weeks after SKMB transplantation. Two animals in the immediate transplant group and 1 in the delayed group showed no evidence of MI and were not analyzed. All other animals had large anterior MIs evident on gross examination. In all, 12 rats receiving immediate SKMB transplantation and 11 receiving delayed SKMB transplantation were analyzed. Skeletal Myoblast Engraftment Is Improved When Transplantation Performed Early After Myocardial Infarction Six weeks after SKMBs were transplanted into MIs immediately after coronary artery ligation, engrafted SKMBs occupied 53% of the total infarct volume (Figs 2A, 2C). However, if SKMB transplantation was delayed for 5 weeks after coronary artery ligation, engrafted SKMBs occupied only 2.5% of the total infarct volume (Figs 2B, 2C). This represents a 95% reduction in the volume of engrafted SKMBs when transplantation is performed into a chronic infarct scar as opposed to a fresh MI ( versus mm 3 ; p 0.001). Although the total volume of the infarct scar 6 weeks after SKMB transplantation was similar in each group (Fig 2C), the delayed group had 55% thinner infarcts ( versus mm; p ) and occupied a larger area ( versus mm 2 ; p 0.005), suggesting more advanced adverse remodeling (Figs 2D, 2E). Hepatocyte Growth Factor Declines With Time in Myocardial Infarction Twenty-four rats underwent left anterior descending coronary artery ligation for evaluation of HGF at various time points after MI; 3 died intraoperatively. Myocardial infarctions from the surviving animals were examined 48 hours (n 4), 1 week (n 6), 2 weeks (n 6), and 5 weeks (n 5) after coronary artery ligation and compared with control myocardium (area remote from MI in 48-hours group, n 6). Hepatocyte growth factor evaluated by quantitative analysis of immunohistochemical staining in early MI (48 hours after coronary artery ligation) was similar to that found in control myocardium ( versus units; Figs 3A, 3E). However, there was a progressive decline of HGF immunolabeling (Figs 3B 3E) in MIs at 1, 2, and 5 weeks after coronary artery ligation ( , , and units, respectively; p for 1, 2, and 5 weeks versus 48 hours; p 0.05 for 2 and 5 weeks versus 1 week).

5 Ann Thorac Surg O BLENES ET AL 2010;89: HGF AND TIMING OF MYOBLAST TRANSPLANT 833 associated with an increase in cell proliferation as measured by 5-bromo-2=-deoxyuridine incorporation (Fig 4B). Hepatocyte growth factor has no significant effect on SKMB number in confluent cultures or if serum concentration is kept high (20% FBS, data not shown). Hepatocyte Growth Factor Protects Skeletal Myoblasts Against Oxidative Stress and Hypoxia In Vitro In confluent, serum-restricted cultures of rat SKMBs, exposure to oxidative stress caused a 77% loss of viable cells (Fig 4C; versus ; p 0.001). However, pretreatment with HGF protected, in a dose-dependent manner, against loss of viable cells on exposure to oxidative stress ( , , for HGF 0.3, 3, and 30 ng/ml, respectively; p 0.01 for H 2 O 2 plus HGF 30 mg/ml versus H 2 O 2 alone). In these confluent SKMB cultures, pretreatment with HGF does not influence cell number in the absence of H 2 O 2 (data not shown). Exposure of serum-restricted rat SKMB cultures to a hypoxic environment caused a 90% reduction in cell number relative to control cultures maintained in a normoxic environment (Fig 4D; versus ; p 0.001). However, when HGF was added to the SKMB cultures immediately before exposure to hypoxia, a dosedependent increase in cell number toward normoxic control values was seen ( , , cells for HGF 1, 10, and 30 ng/ml, respectively; p 0.01 for hypoxia plus HGF 10 and 30 ng/ml versus hypoxia alone). Figure 4. Hepatocyte growth factor (HGF) promotes skeletal myoblast (SKMB) proliferation and protects against oxidative stress and hypoxia in vitro. (A) Graph representing SKMB number after incubation with increasing doses of recombinant HGF. Hepatocyte growth factor causes a dose-dependent increase in SKMB number (n 4 per group; *p 0.05 versus control, **p 0.01 versus control). (B) Graph representing SKMB proliferation (5-bromo-2=-deoxyuridine incorporation) after incubation with increasing doses of recombinant HGF. Skeletal myoblast proliferation increases with HGF administration (n 4 per group; *p 0.05 versus control, **p 0.01 versus control). (C) Graph representing effect of increasing doses of HGF on SKMB number during exposure to oxidative stress (H 2 O 2 ). Hepatocyte growth factor reduces SKMB loss in response to oxidative stress in a dose-dependent manner (n 4 per group; *p 0.01 versus H 2 O 2 alone). (D) Graph representing effect of increasing doses of HGF on SKMB number with exposure to hypoxia. Hepatocyte growth factor minimizes the impact of hypoxia on SKMB number in a dose-dependent manner (n 4 per group; *p 0.01 versus hypoxia alone, **p versus hypoxia alone). Hepatocyte Growth Factor Enhances Skeletal Myoblast Proliferation In Vitro In nonconfluent, serum-restricted cultures of rat SKMBs, HGF caused a dose-dependent increase in viable cell number (Fig 4A; , , , and cells for control; 0.3, 3, and 30 ng/ml for HGF, respectively; p 0.05 for 3 and 30 ng/ml versus control). This increase in cell number is Comment In the rat model of MI, acute inflammation has resolved and a chronic infarct scar developed by 4 weeks after coronary artery ligation [20, 21]. We found that SKMB engraftment was minimal when transplants were performed into chronic infarcts 5 weeks after coronary artery ligation in rats. This may be one reason why patients in the Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial who had autologous SKMBs transplanted into their chronic MI scars did not enjoy as much recovery of their ventricular function as was anticipated [9]. Our observations are consistent with those of McCue and colleagues [22] who could detect only a small percentage of SKMBs transplanted 1 month after MI in rabbits. Only about 1% of transplanted SKMBs are detectable on histologic examination of hearts from humans who had cell transplantation into chronic infarct scars [23]. Li and associates [24] found that transplantation of fetal cardiomyocytes was most effective if performed early (2 weeks) rather than late (4 weeks) after myocardial cryoinjury in rats, but failed to see any engraftment or functional recovery when fetal cardiomyocytes were transplanted immediately after cryoinjury. This is in contrast to our observation of a 20-fold increase in the volume of engrafted SKMBs when transplantation was performed immediately after coronary artery ligation rather than 5 weeks later. These differences could be

6 834 O BLENES ET AL Ann Thorac Surg HGF AND TIMING OF MYOBLAST TRANSPLANT 2010;89: related to specific characteristics of the cryoinjury model, but we propose that SKMBs may be better suited than fetal cardiomyocytes to survive in an acute inflammatory environment because of their normal role in the repair of injured skeletal muscle. The same group recently reported that SKMB transplantation performed 30 days after coronary artery ligation in rats was as effective at improving ventricular function as transplantation performed 5 days after coronary artery ligation [25], but did not quantify the extent of SKMB engraftment. We found that in the rat, HGF immunoreactivity within an MI declines with time after coronary artery ligation. In light of the central role that HGF plays in mediating repair of skeletal muscle through its receptor c-met found on satellite cells and SKMBs [10 13], it is possible that declining HGF in infarcted myocardium with time could influence SKMB engraftment through several mechanisms. Consistent with previous observations [26], we found that recombinant HGF stimulates rat SKMB proliferation in vitro. Hepatocyte growth factor is also known to protect against apoptotic cell death through stimulation of the caspase-8 inhibitor FLICE-like inhibiting protein, blocking Bax mitochondrial translocation and enhancing expression of the anti-apoptotic protein Bcl-X L in ischemic endothelial cells [27]. Delivery of HGF either by local slow-release gel or gene transfer in 2- to 4-week-old MIs in rats augments engraftment of neonatal SKMBs by reducing apoptotic cell death [28, 29], but this has not been confirmed with clinically relevant adult SKMBs. Hepatocyte growth factor may also protect against oxidative stress, which is elevated within infarcted myocardium [30] and during heart failure [30 32]. Hepatocyte growth factor protects cardiomyocytes against injury by H 2 O 2 [31] in vitro through a pathway involving ERK1/2 phosphorylation [16, 33]. We found that HGF also protects SKMBs against hypoxia and oxidative stress in vitro. Therefore in acute MI, HGF may promote engraftment of SKMBs by enhancing proliferation of the transplanted cells or by protecting against cell loss caused by ischemia and oxidative stress. The molecular mechanism for these protective effects remains to be elucidated. Limitations This study focused exclusively on the volume of engrafted SKMBs rather than functional end points. Therefore, we cannot conclude that earlier SKMB transplantation would necessarily improve recovery of ventricular function. We saw evidence of more advanced adverse remodeling in the MI scars of the delayed SKMB transplant group, but the overall time from coronary artery ligation to harvest in this group was longer so this observation is not necessarily related to the extent of myoblast engraftment. Delayed transplantation still may have a beneficial impact on the extent of remodeling, but this was not addressed in our study because we did not examine animals that had coronary artery ligation only (no SKMB transplantation). Efforts were made to ensure accurate delivery of SKMBs into the heart, but it is impossible to completely eliminate the risk of some intracavitary loss of cells injected into thin-walled chronic MIs, which could potentially exaggerate our findings. Engrafted SKMBs were identified by labeling with skeletal myosin, which is expressed when the contractile phenotype emerges. This is a specific marker (infarcts injected with media only show no labeling, data not shown), but could theoretically underestimate engraftment if undifferentiated myoblasts still existed within the infarct. Conclusions Performing SKMB transplantation immediately after MI may be optimal but is not feasible in clinical practice because it takes approximately 3 weeks to prepare the cells [9]. Therefore, strategies to enhance engraftment of SKMBs transplanted late after MI, such as the delivery of exogenous HGF, should be explored as a method to achieve successful clinical cell transplantation. This work was funded in part by the Heart and Stroke Foundation of New Brunswick. References 1. Terrovitis J, Lautamaki R, Bonios M, et al. Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery. J Am Coll Cardiol 2009; 54: Johnston PV, Sasano T, Mills K, et al. Engraftment, differentiation, and functional benefits of autologous cardiospherederived cells in porcine ischemic cardiomyopathy. Circulation 2009;120: , Moretti A, Bellin M, Jung CB, et al. Mouse and human induced pluripotent stem cells as a source for multipotent Is11 cardiovascular progenitors. FASEB J 2009 Oct Zwi L, Caspi O, Arbel G, et al. Cardiomyocyte differentiation of human induced pluripotent stem cells. Circulation 2009; 120: Rosenzweig A. Cardiac cell therapy mixed results from mixed cells. N Engl J Med 2006;355: Assmus B, Honold J, Schachinger V, et al. Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J Med 2006;355: Ghostine S, Carrion C, Souza LC, et al. Long-term efficacy of myoblast transplantation on regional structure and function after myocardial infarction. Circulation 2002;106(12 Suppl 1):I Al Attar N, Carrion C, Ghostine S, et al. Long-term (1 year) functional and histological results of autologous skeletal muscle cells transplantation in rat. Cardiovasc Res 2003;58: Menasche P, Alfieri O, Janssens S, et al. The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized placebo-controlled study of myoblast transplantation. Circulation 2008;117: Morgan JE, Partridge TA. Muscle satellite cells. Int J Biochem Cell Biol 2003;35: Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001;91: Husmann I, Soulet L, Gautron J, Martelly I, Barritault D. Growth factors in skeletal muscle regeneration. Cytokine Growth Factor Rev 1996;7:

7 Ann Thorac Surg O BLENES ET AL 2010;89: HGF AND TIMING OF MYOBLAST TRANSPLANT Wagers AJ, Conboy IM. Cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell 2005;122: Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Enhanced expression of hepatocyte growth factor/c-met by myocardial ischemia and reperfusion in a rat model. Circulation 1997;95: Zhu Y, Hojo Y, Ikeda U, Shimada K. Production of hepatocyte growth factor during acute myocardial infarction. Heart 2000;83: Ueda H, Nakamura T, Matsumoto K, Sawa Y, Matsuda H, Nakamura T. A potential cardioprotective role of hepatocyte growth factor in myocardial infarction in rats. Cardiovasc Res 2001;51: Pouzet B, Vilquin JT, Hagege AA, et al. Intramyocardial transplantation of autologous myoblasts: can tissue processing be optimized? Circulation 2000;102(19 Suppl 3):III Tolivia J, Navarro A, del Valle E, Perez C, Ordonez C, Martinez E. Application of Photoshop and Scion Image analysis to quantification of signals in histochemistry, immunocytochemistry and hybridocytochemistry. Anal Quant Cytol Histol 2006;28: Kajio T, Kawahara K, Kato K. Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin. J Pharmacol Toxicol Methods 1992;28: Fishbein MC, Maclean D, Maroko PR. The histopathologic evolution of myocardial infarction. Chest 1978;73: Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res 1979;44: McCue JD, Swingen C, Feldberg T, et al. The real estate of myoblast cardiac transplantation: negative remodeling is associated with location. J Heart Lung Transplant 2008;27: Pagani FD, 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: Li RK, Mickle DA, Weisel RD, Rao V, Jia ZQ. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg 2001; 72: Farahmand P, Lai TY, Weisel RD, et al. Skeletal myoblasts preserve remote matrix architecture and global function when implanted early or late after coronary ligation into infarcted or remote myocardium. Circulation 2008;118(14 Suppl):S Sheehan SM, Tatsumi R, Temm-Grove CJ, Allen RE. HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro. Muscle Nerve 2000;23: Wang X, Zhou Y, Kim HP, et al. Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells. J Biol Chem 2004;279: Tambara K, Premaratne GU, Sakaguchi G, et al. Administration of control-released hepatocyte growth factor enhances the efficacy of skeletal myoblast transplantation in rat infarcted hearts by greatly increasing both quantity and quality of the graft. Circulation 2005;112(9 Suppl):I Miyagawa S, Sawa Y, Taketani S, et al. Myocardial regeneration therapy for heart failure: hepatocyte growth factor enhances the effect of cellular cardiomyoplasty. Circulation 2002;105: Inoue T, Ide T, Yamato M, et al. Time-dependent changes of myocardial and systemic oxidative stress are dissociated after myocardial infarction. Free Radic Res 2009;43: Sun Y. Myocardial repair/remodelling following infarction: roles of local factors. Cardiovasc Res 2009;81: Hill MF, Singal PK. Antioxidant and oxidative stress changes during heart failure subsequent to myocardial infarction in rats. Am J Pathol 1996;148: Kitta K, Day RM, Ikeda T, Suzuki YJ. Hepatocyte growth factor protects cardiac myocytes against oxidative stressinduced apoptosis. Free Radic Biol Med 2001;31: INVITED COMMENTARY Despite advances in treatment, ischemic cardiac injury and ventricular dysfunction that results from it, are major causes of morbidity and mortality throughout the world. The natural regenerative capacity of the heart is inadequate to completely replace injured or nonviable myocardium, leading to cumulative loss of cardiomyocytes and myocardial dysfunction during the lifetime of a patient. For this reason, experiments done in animals, suggesting that the transfer of cells derived from skeletal myoblasts or bone marrow could improve cardiac function after infarction through regeneration of the myocardium, has generated tremendous interest. The promise and hope of cellular cardiomyoplasty has been tempered by the reality of often conflicting experimental results and clinical trials. Many questions remain unanswered in this field. First, the optimal route for cell delivery (coronary vs direct myocardial injection) is not known. Second, the best cell type (skeletal myoblast vs bone marrow-derived cells or resident cardiomyocyte precursors) for injection is controversial. Third, the vast majority of cells transferred to the heart do not become engrafted, and these cells are lost within minutes of delivery. Fourth, no injected cell of any type has been shown to beat in synchronization with endogenous cardiomyocytes. Fifth, functional recovery of the myocardium has been reported in multiple studies in a time frame when no transplanted cells could be detected. This has led to the widespread belief that cell transfer to the heart may improve myocardial function through the modulation of cytokines, the stimulation of angiogenesis, or the buttressing of an infarction area by mechanical factors. The article by O Blenes and colleagues [1] addresses the question of how to achieve optimal skeletal myoblast engraftment in the heart. The authors examine the efficiency of skeletal myoblast engraftment in the left ventricle in an acute versus chronic model of myocardial infarction in the rat. They show that cell engraftment is better in early infarction compared with chronically remodeled myocardium. Studying the kinetics of hepatocyte growth factor (HGF) expression in evolving myocardial infarction tissue and the effects of HGF on survival of skeletal myoblasts in culture, the authors found that HGF may enhance the ability of skeletal myoblasts to survive after cardiac injection. Although the experiments outlined in this article [1] are suggestive of a role for this growth factor in cellular engraftment in the heart, they do not prove cause and effect. The proof of concept of the author s [1] hypothesis that HGF is important for incorporation and viability 2010 by The Society of Thoracic Surgeons /10/$36.00 Published by Elsevier Inc doi: /j.athoracsur