Safety and Feasibility of Autologous Myoblast Transplantation in Patients With Ischemic Cardiomyopathy
http://www.100md.com
《循环学杂志》
the Arizona Heart Institute (N.D., S.R.O., A.C., M.G., E.D.), Phoenix
the Cleveland Clinic Foundation (M.Y.), Cleveland, Ohio
the University of Michigan (F.D.P., S.W.), Ann Arbor
the University of California at Los Angeles (W.R.M., G.F.)
Temple University (H.J.E.), Philadelphia, Pa
Ohio State University (R.E.M., P.B., D.B.), Columbus
the Lindner Clinical Trial Center (D.K., R.L., R.K.), Cincinnati, Ohio
the University of Maryland Medical Center (V.D., B.P.G.), Baltimore
the University of Minnesota (I.A., C.S.), Minneapolis
Brigham and Women’s Hospital (M.F.D.C.), Division of Nuclear Medicine, Boston, Mass
Scottsdale Medical Imaging Ltd (R.K.), Scottsdale, Ariz
GenVec, Inc (J.D.), Charlestown, Mass
Northwestern University (P.M.), Chicago, Ill
the Cleveland Clinic Foundation (J.T., W.J.), Section of Cardiovascular Imaging, Cleveland, Ohio
the Bryan LGH Heart Institute (S.K.K.), Lincoln, Neb.
Abstract
Background— Successful autologous skeletal myoblast transplantation into infarcted myocardium in a variety of animal models has demonstrated improvement in cardiac function. We evaluated the safety and feasibility of transplanting autologous myoblasts into infarcted myocardium of patients undergoing concurrent coronary artery bypass grafting (CABG) or left ventricular assist device (LVAD) implantation. In addition, we sought to gain preliminary information on graft survival and any associated changes in cardiac function.
Methods and Results— Thirty patients with a history of ischemic cardiomyopathy participated in a phase I, nonrandomized, multicenter pilot study of autologous skeletal myoblast transplantation concurrent with CABG or LVAD implantation. Twenty-four patients with a history of previous myocardial infarction and a left ventricular ejection fraction <40% were enrolled in the CABG arm. In a second arm, 6 patients underwent LVAD implantation as a bridge to heart transplantation, and patients donated their explanted native hearts for testing at the time of heart transplantation. Myoblasts were successfully transplanted in all patients without any acute injection-related complications or significant long-term, unexpected adverse events. Follow-up positron emission tomography scans showed new areas of glucose uptake within the infarct scar in CABG patients. Echocardiography measured an average change in left ventricular ejection fraction from 28% to 35% at 1 year and of 36% at 2 years. Histological evaluation in 4 of 6 patients who underwent heart transplantation documented survival and engraftment of the skeletal myoblasts within the infarcted myocardium.
Conclusions— These results demonstrate the survival, feasibility, and safety of autologous myoblast transplantation and suggest that this modality offers a potential therapeutic treatment for end-stage heart disease.
Key Words: myocardial infarction cells transplantation trials heart failure
Introduction
Heart disease remains a leading cause of morbidity and mortality despite continuing advances in various treatment options. With best medical therapy, there is still a significant subset of patients who become refractory or respond suboptimally. The impact of heart disease spans all ethnic groups and has profound economic consequences.1,2 Acute myocardial infarction (AMI) results in an immediate loss of heart muscle, but there is further deterioration in left ventricular (LV) function, and in 20% of patients, significant dilatation of the LV (GUSTO I) continues to occur long after the initial event. The decline in heart function and dilatation of the ventricle after MI result in heart failure that is an inexorable process, leading eventually to congestive heart failure and death. Cardiac muscle lacks any significant capacity to regenerate once injured3–5; however, recent results have shown that there may be regenerative cells in the heart that someday could be manipulated to effect repair.3–6 Unfortunately, the potential for cardiac self-repair is still theoretical. In contrast with cardiac muscle, skeletal muscle has the capacity for self-repair because of a resident population of proliferative muscle cells, or myoblasts.7 Skeletal myoblasts, once activated, divide and then fuse to form new muscle fibers that may restore lost functionality.8
Preclinical data from a variety of animal studies have demonstrated the capacity for skeletal myoblasts to engraft, form myotubules, and enhance cardiac function after transplantation into infarcted myocardium.9–12 More recently, preliminary human studies focusing on patients with ischemic heart disease have demonstrated successful myoblast transplantation into the postinfarction scar.13–17
Described herein are studies undertaken to test the survival, safety, and feasibility of transplanting autologous myoblasts derived from skeletal muscle into and around a scarred area of the myocardium. This was done in subjects after MI with LV dysfunction. The transplantation of autologous myoblasts was performed during coronary artery bypass graft surgery (CABG) or LV-assist device (LVAD) implantation as a bridge to heart transplantation.
Methods
Study Design and Patient Eligibility
Several phase 1 studies were conducted, and all were prospective, nonblinded, multicenter clinical trials that enrolled a total of 30 patients. Feasibility and safety of myoblast transplantation were assessed in 24 patients who were candidates for elective CABG. Feasibility, safety, and engraftment were evaluated in 6 patients who were candidates for LVAD as a bridge to heart transplantation. Eligibility criteria for the CABG group included previous MI and LV ejection fraction (LVEF) <30% (CABG00-2, 12 patients) or <40% (IIAM02-3, 12 patients). Patients who required LV aneurysmectomy were included only in CABG00-2. For the IIAM02-3 study, all patients had irreversible scar as demonstrated by positron emission tomography (PET) and magnetic resonance imaging (MRI) as entrance criteria. Eligibility criteria for the LVAD arm included patients undergoing LVAD as a bridge to heart transplantation and who were willing to donate their heart after explantation for histological examination. Excluded were patients with skeletal muscle disease, active malignancy, recent history of alcohol or drug abuse, pregnancy, or active infection. All studies were conducted in accordance with Good Clinical Practices, were regulated by the US Food and Drug Administration under an investigational new drug application, and were approved by each site’s institutional review board.
Baseline Testing
Suitable patients for all studies underwent screening and baseline evaluations, which consisted of a physical examination, ECG, 24-hour Holter monitoring, and routine blood testing. For CABG00-2, transthoracic echocardiography and single-photon-emission computed tomography (SPECT) were used to assess global LV function, and ischemia and viability, respectively. Optional MRI and [18F]fluorodeoxyglucose (FDG) PET were used to assess scar segment thickness and myocardial viability, respectively. IIAM02-3 differed from CABG00-2 in that MRI and [18F]FDG were required measures at baseline and follow-up. Procedures for SPECT and 24-hour viability imaging have been previously established.18
Echocardiography
Global and regional ventricular wall contractility for CABG00-2 and the LVAD study were measured by 2-dimensional (2D) echocardiography according to standard techniques. LVEF was calculated by the Simpson rule.
IIAM02-3 echocardiography was performed according to a standardized protocol provided by an echocardiography core laboratory (Dr J. Thomas, Cleveland Clinic Foundation, Cleveland, Ohio). LVEF%, regional wall-motion scoring according to the recently published American College of Cardiology/American Heart Association 17-segment model, and wall thickening were assessed. Complete 2D echocardiographic studies (parasternal long- and short-axis, apical 4- and 5-chamber, apical 2-chamber, and apical long-axis views) were performed. Investigators used modern ultrasound equipment with multifrequency harmonic imaging capability. Images were stored in a DICOM-compliant format and sent to the core laboratory, where all measurements were performed offline from digitally acquired data. Two-dimensional images were used to assess ventricular dimensions, systolic and diastolic function of the LV and right ventricle, segmental contractility, and valvular function.
On arrival at the Cleveland Clinic Foundation Echo Core laboratory, videotapes were checked for subject identification and echocardiogram date. Once identification and date had been verified, echocardiograms were digitized. One research sonographer made quantitative measurements. Three staff cardiologist specializing in echocardiography interpreted the echocardiograms on the basis of quantitative measurements. Readers were blinded as to the area of the ventricle transplanted with cells. Finally, the echocardiogram data analysis was digitally stored in an Access database.
Magnetic Resonance Imaging
Ventricular mass and volume were obtained from cine-MRI images with an MRI scanner operating at a field strength of 1.5 T. Before acquisition of the cine-MRI images, localizers (ie, scout images) were acquired to prescribe the image planes for the cine studies. Scout images were acquired with a pulse sequence that collected an image in a fraction of an RR interval, eg, with a fast gradient-echo sequence with steady-state free precession (true FISP on Siemens or FIESTA on GE scanners). Cine image sets were acquired for a horizontal long-axis section, a vertical long-axis section of the heart, and adjacent short-axis sections from base to apex. The cine set for each slice was acquired during a breath-hold. The cine-MRI studies were ECG gated.
For cine images, the short-axis scout image was used as a localizer to define or prescribe the horizontal long-axis image plane. The slice for the horizontal long-axis cine study intersected the center of the LV and was perpendicular to the midseptum. For the vertical long axis, the operator prescribed a slice parallel to the septum, centered in the LV cavity, and positioned from the LV apex, through the midportion of the mitral valve.
Images were analyzed at the cardiac MRI core laboratory at the University of Minnesota, Minneapolis, with a dedicated software package with automatic edge detection. Segmentation accuracy was verified and edited when necessary by hand, and standard structural and functional parameters were assessed by using accepted formulas. The following MRI measurements were made: LV end-diastolic and end-systolic volumes and diameters; LVEF; and delayed gadolinium hyperenhancement (viability imaging). Imaging of delayed hyperenhancement was performed with a pulse sequence for 3D, rapid, T1-weighted imaging. A contrast agent dosage of 0.2 mmol/kg was used, with an inversion time of 200 ms as a starting value.
[18F]FDG PET Scanning
A 15- to 20-minute transmission scan was acquired for correction of photon attenuation. Regional myocardial glucose utilization was evaluated with FDG and PET. Studies were acquired in the glucose-loaded state. All measurements of glucose levels and insulin doses were recorded in the PET imaging transmittal form and in the patient’s chart. Ten to 15 mCi of FDG was injected intravenously, and after 45 to 60 minutes (to allow for metabolic trapping of FDG in the myocardium), images were acquired for 25 minutes. The PET images were reconstructed at each participating site with use of a Hanning filter with a 0.30 cycles/pixel cutoff frequency. Reconstructed data were transferred to the Nuclear Core Laboratory for analysis.
Myoblast Procurement and Culturing
Depending on the cell dose to be administered, a skeletal muscle biopsy sample of 2 to 5 g was obtained from each patient 3 to 5 weeks before the scheduled surgery. The muscle specimens were immediately placed in a resealable container filled with transport medium, packed in an insulated shipping container with sufficient gel ice packs to maintain temperature between –2°C and 7°C, and shipped to the cell processing facility (GenVec, Inc, Charlestown, Mass). During processing, connective tissue was removed from each specimen, and the rest of the muscle tissue was minced into a slurry. The slurry underwent several cycles of enzymatic digestion at 37°C with trypsin/EDTA (0.5 mg/mL trypsin, 0.53 mmol/L EDTA; Gibco-BRL) and collagenase (0.5 mg/mL; Gibco-BRL) to release satellite cells. Skeletal myoblasts were cultured according to a modified Ham’s method.8 The satellite cells were plated and grown in myoblast basal growth medium (SkBM, Clonetics), which contained 15% to 20% fetal bovine serum (Hyclone), recombinant human epidermal growth factor (10 ng/mL), and dexamethasone (3.9 μg/mL). To prevent myotube formation during the culturing process, cell densities were maintained throughout the process at <80% confluence. Myoblasts were expanded for 11 to 13 doublings, harvested, and cryopreserved before transplantation. Myoblasts were thawed, washed, and resuspended in transplantation medium at 1 to 1.60x108 cells/mL, loaded into 1-mL tuberculin syringes, chilled to 4°C, and shipped on ice to the clinical center for transplantation. At the time of transplantation, cells were warmed to room temperature and were ready for injection.
Myoblast Transplantation
At the time of surgery, the cultured myoblasts were injected into the epicardial surface of the infarcted area according to an escalating-dose regimen. All patients underwent CABG or LVAD implantation concurrent with myoblast transplantation. In the CABG group, 12 patients were divided into 4 escalating-dose groups (3 patients per group) of 1, 3, 10, and 30x107 cells, and 12 patients received a fixed dose of 3x108 cells. The cell dose for LVAD patients was 3x108 cells, except for 1 patient, who received only 2.2x106 cells. Autologous myoblasts were injected into and around the area of infarction from the epicardial surface over a period of 15 seconds, in 3 to 30 injections of 0.1 mL at a concentration of 1 to 1.6x108 cells/mL, with the exception of group 1 (107) patients, who received 3 injections of 3.3x107 cells/mL. Cell injections did not overlap and were performed on-pump or on a beating heart that was off-pump.
Follow-Up Evaluations
The safety of myoblast transplantation was assessed by clinical observations, ECG, echocardiography, Holter monitoring, blood tests, and urinalysis for the length of the study (24 months). Twenty-four-hour Holter monitoring was performed at follow up weeks 1, 3, 6, 12, and 24 for each patient. SPECT, PET, and MRI were also assessed at various follow-up intervals. Engraftment was assessed by histology in the LVAD group. During the cell transplantation procedure and hospitalization, patients were monitored for evidence of serious complications, including arrhythmias, cerebrovascular events, and bleeding.
Statistical Analysis
The primary outcome measure was the incidence of adverse events. In addition, we assessed changes in echocardiographic, MRI, and PET measures among the patients with CABG. Mean values at 6 to 24 months were compared with those at baseline, and the statistical significance was calculated by using the paired t test or ANOVA, when appropriate. Missing data were attributed to failures to record observations or to missed appointments and were assumed to be missing at random.
Results
Study Group
A total of 30 patients were enrolled in the study and underwent myoblast cell transplantation concurrent with CABG or LVAD. Baseline demographics of CABG patients are presented in Table 1. Twenty-four patients with a mean age of 55.2 years (range, 34 to 76) were enrolled in the CABG arm. Patients received an average of 2.7 bypass grafts, 22 of 24 had left internal mammary grafts, and 3 patients had an LV aneurysmectomy procedure. Six patients (mean age, 56 years; range, 43 to 65) underwent LVAD implantation as a bridge to heart transplantation. Fourteen patients in the CABG group showed nonsustained ventricular tachycardia on baseline Holter monitoring, which persisted in only 7 patients on follow-up Holter monitoring after CABG and cell transplantation.
Cell Culturing
There were no serious complications related to biopsy sample procurement; however, all patients experienced mild discomfort and 1 patient had a small hematoma. The myoblast cultures were maintained for 11 to 13 population doublings, with an average doubling time of 24 hours. Analysis of the cultures before transplantation by phase-contrast microscopy showed only single cells and no evidence of fused, multinucleated myotubes. There was no bacterial or fungal contamination as determined by USP sterility and Mycoplasma testing. In all but 1 case that required early surgery, growth of the number of target cells was achieved. Purity of the myoblast preparation, based on anti-CD56 monoclonal antibody staining and fluorescence-activated cell-sorting analysis, ranged from 42% to 98% (mean, 79%). Trypan blue viability testing of the injected cells at the time of transplantation ranged from 85% to 98% (mean, 92%).
Adverse Events
A summary of adverse events is presented in Table 2. For both the CABG and LVAD groups, the transplantation procedure was clinically well tolerated, and the myoblasts were delivered successfully. No deaths or arrhythmias occurred during surgery or injection of the cells. Minimal bleeding from the injection sites was seen on occasion. Four deaths occurred during the follow-up period: 3 in the LVAD group and 1 in the CABG group. None of them were deemed related to the myoblasts or cell transplantation procedure.
In the CABG group, 3 patients in each escalating-dose group received 1, 3, 10, and 30x107 cells/mL, and an additional 12 patients received 3x108 cells; in the LVAD group, 5 patients received 3x108 and 1 patient received 2.2x106 cells. As of March 4, 2005, postprocedure follow-up for safety on all CABG patients extends to 45 months (minimum, 11; mean, 27; median, 24) and in the LVAD group extends to 33 months (minimum 5 days; mean, 9 months; median, 4 months). Importantly, there was no mortality and no evidence of infection related to cells at any time after transplantation, as determined by fever or elevated leukocyte counts.
One patient who received a dose of 108 cells experienced multiple episodes of symptomatic, nonsustained ventricular tachycardia 7 days after surgery. The patient was hospitalized and angiography was performed. Two areas of stenosis, 70% and 60%, were found in the left internal mammary artery graft. Coronary flow reserve as assessed by Doppler FloWire was reduced to 1.5, indicating ischemia. Electrophysiology studies demonstrated inducible, sustained, monomorphic ventricular tachycardia. Treatment included an increased dose of -blockers and placement of an internal cardioverter-defibrillator (ICD). No further arrhythmias were observed up to 37 months’ follow-up. PET scans revealed improvement in the uptake of [18F]FDG in the scar area (data not shown).
One patient was scheduled to receive and did receive cardiac resynchronization therapy with an ICD at the time of cell transplantation. This patient experienced 2 episodes of ICD firing 9 months after transplantation. This was likely due to sustained ventricular arrhythmias but could not be confirmed by device interrogation. Follow-up evaluation revealed proper functioning of the device. Although we cannot exclude the possibility that ICD firing was related to myoblast engraftment, alterations in his medical management were made, and no further events were reported. These events were considered by an independent safety monitor to be unrelated to cell transplantation.
An additional serious adverse event occurred when a patient who received 3x108 cells experienced nonsustained ventricular tachycardia during week-1 Holter monitoring. A review of Holter monitoring records described multiple episodes of arrhythmia, including wide-complex tachycardia consistent with ventricular tachycardia, intraventricular junctional rhythm, and bigeminy. All episodes were asymptomatic and similar to baseline Holter monitor reports. The patient was hospitalized for observation and electrophysiology consultation. Mildly decreased left systolic function was noted, and an automatic ICD was recommended if the patient was found to be inducible. The patient underwent electrophysiology study that revealed normal sinoatrial node function, mildly abnormal atrioventricular node function, and no inducible, sustained ventricular tachycardia. Digoxin was discontinued, and carvedilol was increased gradually to a final dose of 12.5 mg BID with no further episodes of chest pain, shortness of breath, palpitations, or edema.
For the IIAM02-3, 4 patients received implantation of automatic ICDs and there was 1 patient death: Two received an ICD before the 1-month time point, and 2 received an ICD before the 6-month time point. Because of automatic ICD placement, these patients are no longer evaluable by MRI but are being followed up with all other protocol-specified measures. For 1 patient, the automatic ICD implantation was not deemed a serious adverse event because the device was implanted prophylactically, based on a positive T-wave alternans test result. The implantation did not prolong the patient’s hospitalization, and there have been no firings to date. For the remaining 3 patients, 1 received an ICD for asymptomatic, nonsustained ventricular tachycardia recorded from a Holter monitor performed 15 days after treatment (part of the standard study follow-up), and the other 2 received ICDs not for arrhythmias but because of a continued LVEF <30%. There have been no firings of the ICDs in any of those patients.
One death occurred in the IIAM02-3 study. Twelve days after CABG, subject C.E.E. experienced a suspected AMI, with complaints of severe indigestion, chest pain, and shortness of breath. Autopsy report indicated a large, organizing thrombus in the vein graft to the distal right coronary artery system. All other recently placed grafts were fully patent.
Three deaths occurred in the LVAD implantation group. One death, due to LVAD infection and sepsis, occurred 68 days after the procedure. Autopsy revealed obvious purulence in the driveshaft tract and around the LVAD itself. The other death occurred on the fifth postoperative day due to complications of acute right cerebral infarct and thrombus of the LVAD device, causing cardiac and respiratory failure. The third death occurred at 33 months while the patient was waiting for a heart transplant. The data safety monitor reviewed all available information on these deaths and considered them both unrelated to the myoblasts or implantation procedure. Although arrhythmias were noted in the LVAD group (Table 2), they are common and expected for this procedure.
Feasibility
Feasibility was determined by 3 criteria: first, the ability to culture sufficient numbers of myoblasts for transplantation; second, the ability to deliver myoblasts into the region of myocardial infarct; and third, the ability to demonstrate engraftment. Five patients underwent LVAD explantation. Histological evaluation was performed on 6 patients in total, 3 patients after heart transplantation and 3 patients after death, at 5 days, 2 months, 3 months, 5 months, 6 months, and 33 months after injection, respectively. Trichrome staining of cross sections of damaged myocardium that had received transplant injections clearly demonstrated engraftment of striated myotubes containing multiple nuclei developing within the fibrotic tissue, with no evidence of lymphocyte infiltration (see Pagani et al12). There was no evidence of myoblast migration into normal myocardium. Immunostaining with the MY-32 antibody (a skeletal muscle–reactive anti-myosin that does not stain cardiac muscle) confirmed myoblast engraftment. Verification of engrafted myofibers was visualized in all LVAD recipients, except the first patient who received only 2.2x106 cells and a patient who remained alive for 33 months and received a second LVAD.
Functional Assessment
PET scanning, which was optional in CABG00-2, was performed in 7 of 12 patients enrolled in CABG00-2 at baseline and at the 6-month follow-up. Evidence of cell viability in the region of myocardial scar after myoblast transplantation was seen in 3 of those patients. For 2 patients who received 3x107cells, the PET scan showed no improvement. One of 3 scanned patients who received 108 cells and the 2 of 2 patient scanned who received 3x108 cells showed an increase in FDG uptake in the transplanted zone (data not shown). All 12 patients in the IIAM02-3 group who received 3x108 cells had baseline PETs performed, and 11 of those patients underwent follow-up scans at 6 months: The first patient in this study died 12 days after CABG because of an apparent AMI resulting from an occluded bypass graft. Two of 11 patients showed significant improvement from baseline to 6 months, with the remainder showing no change. A PET scan from 1 of the 2 patients showing PET improvement is shown in Figure 1.
In addition to PET scanning, MRI delayed-hyperenhancement scans were performed on patients in study IIAM02-3 who received 3x108 cells. Scans were performed at baseline, 6 months’, and 12 months’ follow-up. Three of 12 patients were ineligible for follow-up scans because of either an automatic ICD (n=2) or death (n=1). A significant change in hyperenhancement was seen in 1 of the 9 patients at follow-up. A 17-segment polar map for hyperenhancement is shown in Figure 2. This shows a significant increase in the area of tissue viability, and the area corresponds to the same area shown in Figure 1 that shows increased glucose metabolism by PET.
Individual LVEFs for each patient and their corresponding cell doses are shown in Table 3. The New York Heart Association classification at baseline was 2.1; 1.4 at 12 months (change, –0.7, P=0.004 by ANOVA); 1.5 at 18 months (change, –0.6, P=0.03, by ANOVA); and 1.7 at 24 months (change, –0.5, P=0.18, by ANOVA), with too few patients to reach statistical significance (Table 3). The mean baseline LVEF, measured by echocardiography, for the CABG patients was 28% at baseline; 35% at 12 months’ follow up (change, +7%, P=0.02, by ANOVA); 37% at 18 months (change, +9%, P=0.008 by ANOVA); and 36% at 24 months (change, +8%, P=0.01 by ANOVA). The average LV systolic volumes decreased from an average of 129 mL at baseline to 104 mL at 12 months (P=0.01 by paired t test), to 98 mL at 18 months (P=0.02 by paired t test), and to 96 mL at 24 months (P=0.04 by paired t test). The average end-diastolic volumes decreased from an average 187 to 156 mL at 12 months (P=0.02 by paired t test), to 166 mL at 18 months (P=0.08 by paired t test), and to 146 mL at 24 months (P=0.006 by paired t test). A comparison of ventricular dimensions measured by MRI in 9 of the 12 patients in IIAM02-3 with that observed in all patients by echocardiography revealed similar dimensions.
Only 9 patients could be followed up after CABG by MRI because of 1 patient death and ICD placement in 2 patients. Data for changes in ventricular dimensions from MRI are shown in Table 3. Statistically significant changes were observed as early as 3 months after CABG and were maintained at 6 months.
Discussion
The main findings of this study can be summarized as follows: (1) Epicardial transplantation of autologous skeletal myoblasts is feasible and safe, in that there were no deaths directly related to transplantation and no infections or allergic reactions resulting from the procedure or the myoblast preparation. (2) Myoblast transplantation in combination with bypass surgery was accompanied by an increase in LVEF, an increase in tissue viability (PET scanning and gadolinium MRI delayed hyperenhancement), and a reduction in ventricular systolic and diastolic volumes (echocardiography and MRI), further verifying the safety of the procedure. However, changes may be related only to benefits of the CABG, and further studies are needed to assess any improvements related to the cells per se. The most serious adverse event that the safety monitoring board determined could have been possibly related to cell transplantation was nonsustained ventricular tachycardia. This occurred in only 3 of 24 CABG patients and was effectively treated with medications and an ICD. Previous experience with autologous myoblast transplantation noted the occurrence of ventricular arrhythmias in 4 of 10 patients between postoperative days 9 and 22.13,14 However, the data presented herein and recent data from a multicenter myoblast transplantation study being performed in France and other countries indicate that the 4 of 10 patients with arrhythmia represented an anomalously high incidence because of small sample size. The current multicenter European trial has shown 6 of 63 patients with post-CABG arrhythmias (Dr Menasche, personal communication, March 8, 2005). Coupled with the 3 of 24 patients shown here, the cumulative experience indicates that the post-CABG arrhythmia rate ranges from 10% to 15%, the expected rate for arrhythmias in patients an LVEF <40% undergoing CABG alone.
Histological analysis in tissues from patients in the LVAD group confirmed graft survival in scarred myocardial tissue, minimal localized migration of myoblasts, and no lymphocyte invasion of normal or grafted tissue.17 Multiple CABG-group patients have shown evidence for renewed glucose metabolism and decreased gadolinium hyperenhancement in the area of transplant, consistent with cell survival. However, because of concomitant bypass grafting, these changes cannot be conclusively ascribed to the myoblast transplant. There were no patients in whom worsening of PET or MRI measures of tissue viability were observed. These data support the conclusion that the myoblast procedure is safe.
PET scanning demonstrated increased FDG uptake in some but not all patients, and there were no patients who received <108 cells who showed any increase. Changes in PET and MRI measures of viability in areas of transmural scar were detected (Figures 1 and 2 ). The significance of these changes cannot be determined because of the small number of patients in this study, but data do indicate that PET and MRI may be useful modalities to include in a larger study designed to measure improvement.
Because this procedure was combined with bypass surgery in the absence of a control arm, the improvement in LVEF, LV dimensions, and LV volumes, as related to cell transplantation, are unknown. However, positive changes after transplantation further confirm the safety of the procedure. In a similar group of 83 patients from Yale University with severe coronary artery disease and LVEFs <30%, the average LVEF improved from 24.6% to 33.2%.18,19
In conclusion, autologous myoblast transplantation at cell doses up to 3x108 into infarcted myocardium in patients undergoing CABG or LVAD appears to be safe and technically feasible. Most important, no increased risk for arrhythmia was detected. Histology confirmed myoblast survival, myofiber formation, and engraftment. Furthermore, we conclude that additional clinical trials are warranted to explore the impact of this and other cell-dosage regimens on regional as well as global ventricular function. Consideration should be given to clinical trials in the absence of CABG as well as with increasing dosage regimens and with a control group.
Acknowledgments
The authors thank the entire GenVec staff in the Charlestown location for the production and quality release of patients’ myoblasts and to the clinical staff for all their efforts in the successful initiation and completion of these clinical trials. We thank Dr Neal Salomon for his assistance in patient qualification and data safety monitoring. We also thank Sheila Ulrich for administrative support.
Disclosures
This study was sponsored by GenVec, Inc, Charlestown, Mass, which is developing myoblasts as a commercial product. Dr Anand has received a research grant from and has served as a consultant to GenVec. Dr Dinsmore is employed by GenVec. Dr Di Carli has received grants from Bracco Diagnostics and BMS Medical Imaging and served on the speakers’ bureaus of and/or received honoraria from GE Healthcare, Fujisawa USA, and BMS Medical Imaging. Dr MacLellan has received research grants from the American Heart Association and National Institutes of Health.
References H1439–H1445.
Demirovic J, Prineas R, Rudolph M. Epidemiology of congestive heart failure in three ethnic groups. Congest Heart Fail. 2001; 7: 93–96.
Szucs TD. The growing healthcare burden of CHF. J Renin Angiotens Aldost Syst. 2000; 1 (suppl 1): 2–6.
Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol. 1997; 272: H220–H226.
Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis during hypertrophy in adult mice. Am J Physiol. 1994; 266:
Soonpaa MH, Field LJ. Survey of studies examining mammalian cardiomyocyte DNA synthesis. Circ Res. 1998; 83: 15–26.
Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961; 9: 493–495.
Bischoff R. Regeneration of single skeletal muscle fibers in vitro. Anat Rec. 1975; 182: 215–235.
Jain M, DerSimonian H, Brenner DA, Ngoy S, Teller P, Edge AS, Zawadzka A, Wetzel K, Sawyer DB, Colucci WS, Apstein CS, Liao R. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation. 2001; 103: 1920–1927.
Atkins BZ, Hueman MT, Meuchel JM, Cottman MJ, Hutcheson KA, Taylor DA. Myogenic cell transplantation improves in vivo regional performance in infarcted rabbit myocardium. J Heart Lung Transplant. 1999; 18: 1173–1180.
Taylor DA, Atkins BZ, Hungspreugs P, Jones TR, Reedy MC, Hutcheson KA, Glower DD, Kraus WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med. 1998; 4: 929–933.
Pouzet B, Vilquin JT, Hagege AA, Scorsin M, Messas E, Fiszman M, Schwartz K, Menasche P. Factors affecting functional outcome after autologous skeletal myoblast transplantation. Ann Thorac Surg. 2001; 71: 844–50;discussion 850–851.
Pagani FD, DerSimonian H, Zawadzka A, Wetzel K, Edge AS, Jacoby DB, Dinsmore JH, Wright S, Aretz TH, Eisen HJ, Aaronson KD. 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.
Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Bruneval P, Benbunan M, Marolleau JP, Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol. 2003; 41: 1078–1083.
Menasche P, Vilquin JT, Desnos M. Early results of autologous skeletal myoblast transplantation in patients with severe ischemic heart failure. Circulation. 2001; 104 (suppl II): II-598.
Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M, Duboc D, Schwartz K, Vilquin JT, Marolleau JP. Myoblast transplantation for heart failure. Lancet. 2001; 357: 279–280.
Herreros J, Prosper F, Perez A, Gavira JJ, Garcia-Velloso MJ, Barba J, Sanchez PL, Canizo C, Rabago G, Marti-Climent JM, Hernandez M, Lopez-Holgado N, Gonzalez-Santos JM, Martin-Luengo C, Alegria E. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J. 2003; 24: 2012–2020.
Hassink RJ, Dowell JD, Brutel de la Riviere A, Doevendans PA, Field LJ. Stem cell therapy for ischemic heart disease. Trends Mol Med. 2003; 9: 436–441.
Auerbach MA, Schoder H, Hoh C, Gambhir SS, Yaghoubi S, Sayre JW, Silverman D, Phelps ME, Schelbert HR, Czernin J. Prevalence of myocardial viability as detected by positron emission tomography in patients with ischemic cardiomyopathy. Circulation. 1999; 99: 2921–2926.
Elefteriades JA, Tolis G Jr, Levi E, Mills LK, Zaret BL. Coronary artery bypass grafting in severe left ventricular dysfunction: excellent survival with improved ejection fraction and functional state. J Am Coll Cardiol. 1993; 22: 1411–1417.(Nabil Dib, MD, MSc; Rober)
the Cleveland Clinic Foundation (M.Y.), Cleveland, Ohio
the University of Michigan (F.D.P., S.W.), Ann Arbor
the University of California at Los Angeles (W.R.M., G.F.)
Temple University (H.J.E.), Philadelphia, Pa
Ohio State University (R.E.M., P.B., D.B.), Columbus
the Lindner Clinical Trial Center (D.K., R.L., R.K.), Cincinnati, Ohio
the University of Maryland Medical Center (V.D., B.P.G.), Baltimore
the University of Minnesota (I.A., C.S.), Minneapolis
Brigham and Women’s Hospital (M.F.D.C.), Division of Nuclear Medicine, Boston, Mass
Scottsdale Medical Imaging Ltd (R.K.), Scottsdale, Ariz
GenVec, Inc (J.D.), Charlestown, Mass
Northwestern University (P.M.), Chicago, Ill
the Cleveland Clinic Foundation (J.T., W.J.), Section of Cardiovascular Imaging, Cleveland, Ohio
the Bryan LGH Heart Institute (S.K.K.), Lincoln, Neb.
Abstract
Background— Successful autologous skeletal myoblast transplantation into infarcted myocardium in a variety of animal models has demonstrated improvement in cardiac function. We evaluated the safety and feasibility of transplanting autologous myoblasts into infarcted myocardium of patients undergoing concurrent coronary artery bypass grafting (CABG) or left ventricular assist device (LVAD) implantation. In addition, we sought to gain preliminary information on graft survival and any associated changes in cardiac function.
Methods and Results— Thirty patients with a history of ischemic cardiomyopathy participated in a phase I, nonrandomized, multicenter pilot study of autologous skeletal myoblast transplantation concurrent with CABG or LVAD implantation. Twenty-four patients with a history of previous myocardial infarction and a left ventricular ejection fraction <40% were enrolled in the CABG arm. In a second arm, 6 patients underwent LVAD implantation as a bridge to heart transplantation, and patients donated their explanted native hearts for testing at the time of heart transplantation. Myoblasts were successfully transplanted in all patients without any acute injection-related complications or significant long-term, unexpected adverse events. Follow-up positron emission tomography scans showed new areas of glucose uptake within the infarct scar in CABG patients. Echocardiography measured an average change in left ventricular ejection fraction from 28% to 35% at 1 year and of 36% at 2 years. Histological evaluation in 4 of 6 patients who underwent heart transplantation documented survival and engraftment of the skeletal myoblasts within the infarcted myocardium.
Conclusions— These results demonstrate the survival, feasibility, and safety of autologous myoblast transplantation and suggest that this modality offers a potential therapeutic treatment for end-stage heart disease.
Key Words: myocardial infarction cells transplantation trials heart failure
Introduction
Heart disease remains a leading cause of morbidity and mortality despite continuing advances in various treatment options. With best medical therapy, there is still a significant subset of patients who become refractory or respond suboptimally. The impact of heart disease spans all ethnic groups and has profound economic consequences.1,2 Acute myocardial infarction (AMI) results in an immediate loss of heart muscle, but there is further deterioration in left ventricular (LV) function, and in 20% of patients, significant dilatation of the LV (GUSTO I) continues to occur long after the initial event. The decline in heart function and dilatation of the ventricle after MI result in heart failure that is an inexorable process, leading eventually to congestive heart failure and death. Cardiac muscle lacks any significant capacity to regenerate once injured3–5; however, recent results have shown that there may be regenerative cells in the heart that someday could be manipulated to effect repair.3–6 Unfortunately, the potential for cardiac self-repair is still theoretical. In contrast with cardiac muscle, skeletal muscle has the capacity for self-repair because of a resident population of proliferative muscle cells, or myoblasts.7 Skeletal myoblasts, once activated, divide and then fuse to form new muscle fibers that may restore lost functionality.8
Preclinical data from a variety of animal studies have demonstrated the capacity for skeletal myoblasts to engraft, form myotubules, and enhance cardiac function after transplantation into infarcted myocardium.9–12 More recently, preliminary human studies focusing on patients with ischemic heart disease have demonstrated successful myoblast transplantation into the postinfarction scar.13–17
Described herein are studies undertaken to test the survival, safety, and feasibility of transplanting autologous myoblasts derived from skeletal muscle into and around a scarred area of the myocardium. This was done in subjects after MI with LV dysfunction. The transplantation of autologous myoblasts was performed during coronary artery bypass graft surgery (CABG) or LV-assist device (LVAD) implantation as a bridge to heart transplantation.
Methods
Study Design and Patient Eligibility
Several phase 1 studies were conducted, and all were prospective, nonblinded, multicenter clinical trials that enrolled a total of 30 patients. Feasibility and safety of myoblast transplantation were assessed in 24 patients who were candidates for elective CABG. Feasibility, safety, and engraftment were evaluated in 6 patients who were candidates for LVAD as a bridge to heart transplantation. Eligibility criteria for the CABG group included previous MI and LV ejection fraction (LVEF) <30% (CABG00-2, 12 patients) or <40% (IIAM02-3, 12 patients). Patients who required LV aneurysmectomy were included only in CABG00-2. For the IIAM02-3 study, all patients had irreversible scar as demonstrated by positron emission tomography (PET) and magnetic resonance imaging (MRI) as entrance criteria. Eligibility criteria for the LVAD arm included patients undergoing LVAD as a bridge to heart transplantation and who were willing to donate their heart after explantation for histological examination. Excluded were patients with skeletal muscle disease, active malignancy, recent history of alcohol or drug abuse, pregnancy, or active infection. All studies were conducted in accordance with Good Clinical Practices, were regulated by the US Food and Drug Administration under an investigational new drug application, and were approved by each site’s institutional review board.
Baseline Testing
Suitable patients for all studies underwent screening and baseline evaluations, which consisted of a physical examination, ECG, 24-hour Holter monitoring, and routine blood testing. For CABG00-2, transthoracic echocardiography and single-photon-emission computed tomography (SPECT) were used to assess global LV function, and ischemia and viability, respectively. Optional MRI and [18F]fluorodeoxyglucose (FDG) PET were used to assess scar segment thickness and myocardial viability, respectively. IIAM02-3 differed from CABG00-2 in that MRI and [18F]FDG were required measures at baseline and follow-up. Procedures for SPECT and 24-hour viability imaging have been previously established.18
Echocardiography
Global and regional ventricular wall contractility for CABG00-2 and the LVAD study were measured by 2-dimensional (2D) echocardiography according to standard techniques. LVEF was calculated by the Simpson rule.
IIAM02-3 echocardiography was performed according to a standardized protocol provided by an echocardiography core laboratory (Dr J. Thomas, Cleveland Clinic Foundation, Cleveland, Ohio). LVEF%, regional wall-motion scoring according to the recently published American College of Cardiology/American Heart Association 17-segment model, and wall thickening were assessed. Complete 2D echocardiographic studies (parasternal long- and short-axis, apical 4- and 5-chamber, apical 2-chamber, and apical long-axis views) were performed. Investigators used modern ultrasound equipment with multifrequency harmonic imaging capability. Images were stored in a DICOM-compliant format and sent to the core laboratory, where all measurements were performed offline from digitally acquired data. Two-dimensional images were used to assess ventricular dimensions, systolic and diastolic function of the LV and right ventricle, segmental contractility, and valvular function.
On arrival at the Cleveland Clinic Foundation Echo Core laboratory, videotapes were checked for subject identification and echocardiogram date. Once identification and date had been verified, echocardiograms were digitized. One research sonographer made quantitative measurements. Three staff cardiologist specializing in echocardiography interpreted the echocardiograms on the basis of quantitative measurements. Readers were blinded as to the area of the ventricle transplanted with cells. Finally, the echocardiogram data analysis was digitally stored in an Access database.
Magnetic Resonance Imaging
Ventricular mass and volume were obtained from cine-MRI images with an MRI scanner operating at a field strength of 1.5 T. Before acquisition of the cine-MRI images, localizers (ie, scout images) were acquired to prescribe the image planes for the cine studies. Scout images were acquired with a pulse sequence that collected an image in a fraction of an RR interval, eg, with a fast gradient-echo sequence with steady-state free precession (true FISP on Siemens or FIESTA on GE scanners). Cine image sets were acquired for a horizontal long-axis section, a vertical long-axis section of the heart, and adjacent short-axis sections from base to apex. The cine set for each slice was acquired during a breath-hold. The cine-MRI studies were ECG gated.
For cine images, the short-axis scout image was used as a localizer to define or prescribe the horizontal long-axis image plane. The slice for the horizontal long-axis cine study intersected the center of the LV and was perpendicular to the midseptum. For the vertical long axis, the operator prescribed a slice parallel to the septum, centered in the LV cavity, and positioned from the LV apex, through the midportion of the mitral valve.
Images were analyzed at the cardiac MRI core laboratory at the University of Minnesota, Minneapolis, with a dedicated software package with automatic edge detection. Segmentation accuracy was verified and edited when necessary by hand, and standard structural and functional parameters were assessed by using accepted formulas. The following MRI measurements were made: LV end-diastolic and end-systolic volumes and diameters; LVEF; and delayed gadolinium hyperenhancement (viability imaging). Imaging of delayed hyperenhancement was performed with a pulse sequence for 3D, rapid, T1-weighted imaging. A contrast agent dosage of 0.2 mmol/kg was used, with an inversion time of 200 ms as a starting value.
[18F]FDG PET Scanning
A 15- to 20-minute transmission scan was acquired for correction of photon attenuation. Regional myocardial glucose utilization was evaluated with FDG and PET. Studies were acquired in the glucose-loaded state. All measurements of glucose levels and insulin doses were recorded in the PET imaging transmittal form and in the patient’s chart. Ten to 15 mCi of FDG was injected intravenously, and after 45 to 60 minutes (to allow for metabolic trapping of FDG in the myocardium), images were acquired for 25 minutes. The PET images were reconstructed at each participating site with use of a Hanning filter with a 0.30 cycles/pixel cutoff frequency. Reconstructed data were transferred to the Nuclear Core Laboratory for analysis.
Myoblast Procurement and Culturing
Depending on the cell dose to be administered, a skeletal muscle biopsy sample of 2 to 5 g was obtained from each patient 3 to 5 weeks before the scheduled surgery. The muscle specimens were immediately placed in a resealable container filled with transport medium, packed in an insulated shipping container with sufficient gel ice packs to maintain temperature between –2°C and 7°C, and shipped to the cell processing facility (GenVec, Inc, Charlestown, Mass). During processing, connective tissue was removed from each specimen, and the rest of the muscle tissue was minced into a slurry. The slurry underwent several cycles of enzymatic digestion at 37°C with trypsin/EDTA (0.5 mg/mL trypsin, 0.53 mmol/L EDTA; Gibco-BRL) and collagenase (0.5 mg/mL; Gibco-BRL) to release satellite cells. Skeletal myoblasts were cultured according to a modified Ham’s method.8 The satellite cells were plated and grown in myoblast basal growth medium (SkBM, Clonetics), which contained 15% to 20% fetal bovine serum (Hyclone), recombinant human epidermal growth factor (10 ng/mL), and dexamethasone (3.9 μg/mL). To prevent myotube formation during the culturing process, cell densities were maintained throughout the process at <80% confluence. Myoblasts were expanded for 11 to 13 doublings, harvested, and cryopreserved before transplantation. Myoblasts were thawed, washed, and resuspended in transplantation medium at 1 to 1.60x108 cells/mL, loaded into 1-mL tuberculin syringes, chilled to 4°C, and shipped on ice to the clinical center for transplantation. At the time of transplantation, cells were warmed to room temperature and were ready for injection.
Myoblast Transplantation
At the time of surgery, the cultured myoblasts were injected into the epicardial surface of the infarcted area according to an escalating-dose regimen. All patients underwent CABG or LVAD implantation concurrent with myoblast transplantation. In the CABG group, 12 patients were divided into 4 escalating-dose groups (3 patients per group) of 1, 3, 10, and 30x107 cells, and 12 patients received a fixed dose of 3x108 cells. The cell dose for LVAD patients was 3x108 cells, except for 1 patient, who received only 2.2x106 cells. Autologous myoblasts were injected into and around the area of infarction from the epicardial surface over a period of 15 seconds, in 3 to 30 injections of 0.1 mL at a concentration of 1 to 1.6x108 cells/mL, with the exception of group 1 (107) patients, who received 3 injections of 3.3x107 cells/mL. Cell injections did not overlap and were performed on-pump or on a beating heart that was off-pump.
Follow-Up Evaluations
The safety of myoblast transplantation was assessed by clinical observations, ECG, echocardiography, Holter monitoring, blood tests, and urinalysis for the length of the study (24 months). Twenty-four-hour Holter monitoring was performed at follow up weeks 1, 3, 6, 12, and 24 for each patient. SPECT, PET, and MRI were also assessed at various follow-up intervals. Engraftment was assessed by histology in the LVAD group. During the cell transplantation procedure and hospitalization, patients were monitored for evidence of serious complications, including arrhythmias, cerebrovascular events, and bleeding.
Statistical Analysis
The primary outcome measure was the incidence of adverse events. In addition, we assessed changes in echocardiographic, MRI, and PET measures among the patients with CABG. Mean values at 6 to 24 months were compared with those at baseline, and the statistical significance was calculated by using the paired t test or ANOVA, when appropriate. Missing data were attributed to failures to record observations or to missed appointments and were assumed to be missing at random.
Results
Study Group
A total of 30 patients were enrolled in the study and underwent myoblast cell transplantation concurrent with CABG or LVAD. Baseline demographics of CABG patients are presented in Table 1. Twenty-four patients with a mean age of 55.2 years (range, 34 to 76) were enrolled in the CABG arm. Patients received an average of 2.7 bypass grafts, 22 of 24 had left internal mammary grafts, and 3 patients had an LV aneurysmectomy procedure. Six patients (mean age, 56 years; range, 43 to 65) underwent LVAD implantation as a bridge to heart transplantation. Fourteen patients in the CABG group showed nonsustained ventricular tachycardia on baseline Holter monitoring, which persisted in only 7 patients on follow-up Holter monitoring after CABG and cell transplantation.
Cell Culturing
There were no serious complications related to biopsy sample procurement; however, all patients experienced mild discomfort and 1 patient had a small hematoma. The myoblast cultures were maintained for 11 to 13 population doublings, with an average doubling time of 24 hours. Analysis of the cultures before transplantation by phase-contrast microscopy showed only single cells and no evidence of fused, multinucleated myotubes. There was no bacterial or fungal contamination as determined by USP sterility and Mycoplasma testing. In all but 1 case that required early surgery, growth of the number of target cells was achieved. Purity of the myoblast preparation, based on anti-CD56 monoclonal antibody staining and fluorescence-activated cell-sorting analysis, ranged from 42% to 98% (mean, 79%). Trypan blue viability testing of the injected cells at the time of transplantation ranged from 85% to 98% (mean, 92%).
Adverse Events
A summary of adverse events is presented in Table 2. For both the CABG and LVAD groups, the transplantation procedure was clinically well tolerated, and the myoblasts were delivered successfully. No deaths or arrhythmias occurred during surgery or injection of the cells. Minimal bleeding from the injection sites was seen on occasion. Four deaths occurred during the follow-up period: 3 in the LVAD group and 1 in the CABG group. None of them were deemed related to the myoblasts or cell transplantation procedure.
In the CABG group, 3 patients in each escalating-dose group received 1, 3, 10, and 30x107 cells/mL, and an additional 12 patients received 3x108 cells; in the LVAD group, 5 patients received 3x108 and 1 patient received 2.2x106 cells. As of March 4, 2005, postprocedure follow-up for safety on all CABG patients extends to 45 months (minimum, 11; mean, 27; median, 24) and in the LVAD group extends to 33 months (minimum 5 days; mean, 9 months; median, 4 months). Importantly, there was no mortality and no evidence of infection related to cells at any time after transplantation, as determined by fever or elevated leukocyte counts.
One patient who received a dose of 108 cells experienced multiple episodes of symptomatic, nonsustained ventricular tachycardia 7 days after surgery. The patient was hospitalized and angiography was performed. Two areas of stenosis, 70% and 60%, were found in the left internal mammary artery graft. Coronary flow reserve as assessed by Doppler FloWire was reduced to 1.5, indicating ischemia. Electrophysiology studies demonstrated inducible, sustained, monomorphic ventricular tachycardia. Treatment included an increased dose of -blockers and placement of an internal cardioverter-defibrillator (ICD). No further arrhythmias were observed up to 37 months’ follow-up. PET scans revealed improvement in the uptake of [18F]FDG in the scar area (data not shown).
One patient was scheduled to receive and did receive cardiac resynchronization therapy with an ICD at the time of cell transplantation. This patient experienced 2 episodes of ICD firing 9 months after transplantation. This was likely due to sustained ventricular arrhythmias but could not be confirmed by device interrogation. Follow-up evaluation revealed proper functioning of the device. Although we cannot exclude the possibility that ICD firing was related to myoblast engraftment, alterations in his medical management were made, and no further events were reported. These events were considered by an independent safety monitor to be unrelated to cell transplantation.
An additional serious adverse event occurred when a patient who received 3x108 cells experienced nonsustained ventricular tachycardia during week-1 Holter monitoring. A review of Holter monitoring records described multiple episodes of arrhythmia, including wide-complex tachycardia consistent with ventricular tachycardia, intraventricular junctional rhythm, and bigeminy. All episodes were asymptomatic and similar to baseline Holter monitor reports. The patient was hospitalized for observation and electrophysiology consultation. Mildly decreased left systolic function was noted, and an automatic ICD was recommended if the patient was found to be inducible. The patient underwent electrophysiology study that revealed normal sinoatrial node function, mildly abnormal atrioventricular node function, and no inducible, sustained ventricular tachycardia. Digoxin was discontinued, and carvedilol was increased gradually to a final dose of 12.5 mg BID with no further episodes of chest pain, shortness of breath, palpitations, or edema.
For the IIAM02-3, 4 patients received implantation of automatic ICDs and there was 1 patient death: Two received an ICD before the 1-month time point, and 2 received an ICD before the 6-month time point. Because of automatic ICD placement, these patients are no longer evaluable by MRI but are being followed up with all other protocol-specified measures. For 1 patient, the automatic ICD implantation was not deemed a serious adverse event because the device was implanted prophylactically, based on a positive T-wave alternans test result. The implantation did not prolong the patient’s hospitalization, and there have been no firings to date. For the remaining 3 patients, 1 received an ICD for asymptomatic, nonsustained ventricular tachycardia recorded from a Holter monitor performed 15 days after treatment (part of the standard study follow-up), and the other 2 received ICDs not for arrhythmias but because of a continued LVEF <30%. There have been no firings of the ICDs in any of those patients.
One death occurred in the IIAM02-3 study. Twelve days after CABG, subject C.E.E. experienced a suspected AMI, with complaints of severe indigestion, chest pain, and shortness of breath. Autopsy report indicated a large, organizing thrombus in the vein graft to the distal right coronary artery system. All other recently placed grafts were fully patent.
Three deaths occurred in the LVAD implantation group. One death, due to LVAD infection and sepsis, occurred 68 days after the procedure. Autopsy revealed obvious purulence in the driveshaft tract and around the LVAD itself. The other death occurred on the fifth postoperative day due to complications of acute right cerebral infarct and thrombus of the LVAD device, causing cardiac and respiratory failure. The third death occurred at 33 months while the patient was waiting for a heart transplant. The data safety monitor reviewed all available information on these deaths and considered them both unrelated to the myoblasts or implantation procedure. Although arrhythmias were noted in the LVAD group (Table 2), they are common and expected for this procedure.
Feasibility
Feasibility was determined by 3 criteria: first, the ability to culture sufficient numbers of myoblasts for transplantation; second, the ability to deliver myoblasts into the region of myocardial infarct; and third, the ability to demonstrate engraftment. Five patients underwent LVAD explantation. Histological evaluation was performed on 6 patients in total, 3 patients after heart transplantation and 3 patients after death, at 5 days, 2 months, 3 months, 5 months, 6 months, and 33 months after injection, respectively. Trichrome staining of cross sections of damaged myocardium that had received transplant injections clearly demonstrated engraftment of striated myotubes containing multiple nuclei developing within the fibrotic tissue, with no evidence of lymphocyte infiltration (see Pagani et al12). There was no evidence of myoblast migration into normal myocardium. Immunostaining with the MY-32 antibody (a skeletal muscle–reactive anti-myosin that does not stain cardiac muscle) confirmed myoblast engraftment. Verification of engrafted myofibers was visualized in all LVAD recipients, except the first patient who received only 2.2x106 cells and a patient who remained alive for 33 months and received a second LVAD.
Functional Assessment
PET scanning, which was optional in CABG00-2, was performed in 7 of 12 patients enrolled in CABG00-2 at baseline and at the 6-month follow-up. Evidence of cell viability in the region of myocardial scar after myoblast transplantation was seen in 3 of those patients. For 2 patients who received 3x107cells, the PET scan showed no improvement. One of 3 scanned patients who received 108 cells and the 2 of 2 patient scanned who received 3x108 cells showed an increase in FDG uptake in the transplanted zone (data not shown). All 12 patients in the IIAM02-3 group who received 3x108 cells had baseline PETs performed, and 11 of those patients underwent follow-up scans at 6 months: The first patient in this study died 12 days after CABG because of an apparent AMI resulting from an occluded bypass graft. Two of 11 patients showed significant improvement from baseline to 6 months, with the remainder showing no change. A PET scan from 1 of the 2 patients showing PET improvement is shown in Figure 1.
In addition to PET scanning, MRI delayed-hyperenhancement scans were performed on patients in study IIAM02-3 who received 3x108 cells. Scans were performed at baseline, 6 months’, and 12 months’ follow-up. Three of 12 patients were ineligible for follow-up scans because of either an automatic ICD (n=2) or death (n=1). A significant change in hyperenhancement was seen in 1 of the 9 patients at follow-up. A 17-segment polar map for hyperenhancement is shown in Figure 2. This shows a significant increase in the area of tissue viability, and the area corresponds to the same area shown in Figure 1 that shows increased glucose metabolism by PET.
Individual LVEFs for each patient and their corresponding cell doses are shown in Table 3. The New York Heart Association classification at baseline was 2.1; 1.4 at 12 months (change, –0.7, P=0.004 by ANOVA); 1.5 at 18 months (change, –0.6, P=0.03, by ANOVA); and 1.7 at 24 months (change, –0.5, P=0.18, by ANOVA), with too few patients to reach statistical significance (Table 3). The mean baseline LVEF, measured by echocardiography, for the CABG patients was 28% at baseline; 35% at 12 months’ follow up (change, +7%, P=0.02, by ANOVA); 37% at 18 months (change, +9%, P=0.008 by ANOVA); and 36% at 24 months (change, +8%, P=0.01 by ANOVA). The average LV systolic volumes decreased from an average of 129 mL at baseline to 104 mL at 12 months (P=0.01 by paired t test), to 98 mL at 18 months (P=0.02 by paired t test), and to 96 mL at 24 months (P=0.04 by paired t test). The average end-diastolic volumes decreased from an average 187 to 156 mL at 12 months (P=0.02 by paired t test), to 166 mL at 18 months (P=0.08 by paired t test), and to 146 mL at 24 months (P=0.006 by paired t test). A comparison of ventricular dimensions measured by MRI in 9 of the 12 patients in IIAM02-3 with that observed in all patients by echocardiography revealed similar dimensions.
Only 9 patients could be followed up after CABG by MRI because of 1 patient death and ICD placement in 2 patients. Data for changes in ventricular dimensions from MRI are shown in Table 3. Statistically significant changes were observed as early as 3 months after CABG and were maintained at 6 months.
Discussion
The main findings of this study can be summarized as follows: (1) Epicardial transplantation of autologous skeletal myoblasts is feasible and safe, in that there were no deaths directly related to transplantation and no infections or allergic reactions resulting from the procedure or the myoblast preparation. (2) Myoblast transplantation in combination with bypass surgery was accompanied by an increase in LVEF, an increase in tissue viability (PET scanning and gadolinium MRI delayed hyperenhancement), and a reduction in ventricular systolic and diastolic volumes (echocardiography and MRI), further verifying the safety of the procedure. However, changes may be related only to benefits of the CABG, and further studies are needed to assess any improvements related to the cells per se. The most serious adverse event that the safety monitoring board determined could have been possibly related to cell transplantation was nonsustained ventricular tachycardia. This occurred in only 3 of 24 CABG patients and was effectively treated with medications and an ICD. Previous experience with autologous myoblast transplantation noted the occurrence of ventricular arrhythmias in 4 of 10 patients between postoperative days 9 and 22.13,14 However, the data presented herein and recent data from a multicenter myoblast transplantation study being performed in France and other countries indicate that the 4 of 10 patients with arrhythmia represented an anomalously high incidence because of small sample size. The current multicenter European trial has shown 6 of 63 patients with post-CABG arrhythmias (Dr Menasche, personal communication, March 8, 2005). Coupled with the 3 of 24 patients shown here, the cumulative experience indicates that the post-CABG arrhythmia rate ranges from 10% to 15%, the expected rate for arrhythmias in patients an LVEF <40% undergoing CABG alone.
Histological analysis in tissues from patients in the LVAD group confirmed graft survival in scarred myocardial tissue, minimal localized migration of myoblasts, and no lymphocyte invasion of normal or grafted tissue.17 Multiple CABG-group patients have shown evidence for renewed glucose metabolism and decreased gadolinium hyperenhancement in the area of transplant, consistent with cell survival. However, because of concomitant bypass grafting, these changes cannot be conclusively ascribed to the myoblast transplant. There were no patients in whom worsening of PET or MRI measures of tissue viability were observed. These data support the conclusion that the myoblast procedure is safe.
PET scanning demonstrated increased FDG uptake in some but not all patients, and there were no patients who received <108 cells who showed any increase. Changes in PET and MRI measures of viability in areas of transmural scar were detected (Figures 1 and 2 ). The significance of these changes cannot be determined because of the small number of patients in this study, but data do indicate that PET and MRI may be useful modalities to include in a larger study designed to measure improvement.
Because this procedure was combined with bypass surgery in the absence of a control arm, the improvement in LVEF, LV dimensions, and LV volumes, as related to cell transplantation, are unknown. However, positive changes after transplantation further confirm the safety of the procedure. In a similar group of 83 patients from Yale University with severe coronary artery disease and LVEFs <30%, the average LVEF improved from 24.6% to 33.2%.18,19
In conclusion, autologous myoblast transplantation at cell doses up to 3x108 into infarcted myocardium in patients undergoing CABG or LVAD appears to be safe and technically feasible. Most important, no increased risk for arrhythmia was detected. Histology confirmed myoblast survival, myofiber formation, and engraftment. Furthermore, we conclude that additional clinical trials are warranted to explore the impact of this and other cell-dosage regimens on regional as well as global ventricular function. Consideration should be given to clinical trials in the absence of CABG as well as with increasing dosage regimens and with a control group.
Acknowledgments
The authors thank the entire GenVec staff in the Charlestown location for the production and quality release of patients’ myoblasts and to the clinical staff for all their efforts in the successful initiation and completion of these clinical trials. We thank Dr Neal Salomon for his assistance in patient qualification and data safety monitoring. We also thank Sheila Ulrich for administrative support.
Disclosures
This study was sponsored by GenVec, Inc, Charlestown, Mass, which is developing myoblasts as a commercial product. Dr Anand has received a research grant from and has served as a consultant to GenVec. Dr Dinsmore is employed by GenVec. Dr Di Carli has received grants from Bracco Diagnostics and BMS Medical Imaging and served on the speakers’ bureaus of and/or received honoraria from GE Healthcare, Fujisawa USA, and BMS Medical Imaging. Dr MacLellan has received research grants from the American Heart Association and National Institutes of Health.
References H1439–H1445.
Demirovic J, Prineas R, Rudolph M. Epidemiology of congestive heart failure in three ethnic groups. Congest Heart Fail. 2001; 7: 93–96.
Szucs TD. The growing healthcare burden of CHF. J Renin Angiotens Aldost Syst. 2000; 1 (suppl 1): 2–6.
Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol. 1997; 272: H220–H226.
Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis during hypertrophy in adult mice. Am J Physiol. 1994; 266:
Soonpaa MH, Field LJ. Survey of studies examining mammalian cardiomyocyte DNA synthesis. Circ Res. 1998; 83: 15–26.
Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961; 9: 493–495.
Bischoff R. Regeneration of single skeletal muscle fibers in vitro. Anat Rec. 1975; 182: 215–235.
Jain M, DerSimonian H, Brenner DA, Ngoy S, Teller P, Edge AS, Zawadzka A, Wetzel K, Sawyer DB, Colucci WS, Apstein CS, Liao R. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation. 2001; 103: 1920–1927.
Atkins BZ, Hueman MT, Meuchel JM, Cottman MJ, Hutcheson KA, Taylor DA. Myogenic cell transplantation improves in vivo regional performance in infarcted rabbit myocardium. J Heart Lung Transplant. 1999; 18: 1173–1180.
Taylor DA, Atkins BZ, Hungspreugs P, Jones TR, Reedy MC, Hutcheson KA, Glower DD, Kraus WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med. 1998; 4: 929–933.
Pouzet B, Vilquin JT, Hagege AA, Scorsin M, Messas E, Fiszman M, Schwartz K, Menasche P. Factors affecting functional outcome after autologous skeletal myoblast transplantation. Ann Thorac Surg. 2001; 71: 844–50;discussion 850–851.
Pagani FD, DerSimonian H, Zawadzka A, Wetzel K, Edge AS, Jacoby DB, Dinsmore JH, Wright S, Aretz TH, Eisen HJ, Aaronson KD. 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.
Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Bruneval P, Benbunan M, Marolleau JP, Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol. 2003; 41: 1078–1083.
Menasche P, Vilquin JT, Desnos M. Early results of autologous skeletal myoblast transplantation in patients with severe ischemic heart failure. Circulation. 2001; 104 (suppl II): II-598.
Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M, Duboc D, Schwartz K, Vilquin JT, Marolleau JP. Myoblast transplantation for heart failure. Lancet. 2001; 357: 279–280.
Herreros J, Prosper F, Perez A, Gavira JJ, Garcia-Velloso MJ, Barba J, Sanchez PL, Canizo C, Rabago G, Marti-Climent JM, Hernandez M, Lopez-Holgado N, Gonzalez-Santos JM, Martin-Luengo C, Alegria E. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J. 2003; 24: 2012–2020.
Hassink RJ, Dowell JD, Brutel de la Riviere A, Doevendans PA, Field LJ. Stem cell therapy for ischemic heart disease. Trends Mol Med. 2003; 9: 436–441.
Auerbach MA, Schoder H, Hoh C, Gambhir SS, Yaghoubi S, Sayre JW, Silverman D, Phelps ME, Schelbert HR, Czernin J. Prevalence of myocardial viability as detected by positron emission tomography in patients with ischemic cardiomyopathy. Circulation. 1999; 99: 2921–2926.
Elefteriades JA, Tolis G Jr, Levi E, Mills LK, Zaret BL. Coronary artery bypass grafting in severe left ventricular dysfunction: excellent survival with improved ejection fraction and functional state. J Am Coll Cardiol. 1993; 22: 1411–1417.(Nabil Dib, MD, MSc; Rober)