1University School of Medical Sciences, Department of Cardiology, District Hospital, ul. Juraszow 7/19, PL 60-479, Pozna, Poland
2Institute of Human Genetics, Polish Academy of Sciences, Pozna, Poland
Received 2 August 2004; revised 30 December 2004; accepted 6 January 2005; online publish-ahead-of-print 10 March 2005.
* Corresponding author. Tel: +48 602 217 202; fax: +48 61 8212319. E-mail address: tomasz.siminiak{at}usoms.poznan.pl
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Abstract |
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Methods and results Ten patients with heart failure and presence of an akinetic or a dyskinetic post-infarction injury with no viable myocardium were included in the study. Skeletal myoblasts were obtained from a biopsy specimen and grown in cell culture. Patients were treated with prophylactic amiodarone infusion before and during the procedure, except one patient. Skeletal myoblast transplantations were performed uneventfully in nine patients using the TransAccess® catheter system under fluoroscopic and intravascular ultrasound guidance. In one patient, the procedure was not performed because of the inability of appropriate coronary sinus guiding wire positioning across venous valve. In five patients, the anterior interventricular vein and in four patients, the middle cardiac vein were used to access the myocardium. Two to four intramyocardial injections 1.54.5 cm deep were performed in each patient, delivering up to 100 million cells in 0.42.5 mL of saline. During 6 months follow-up, New York Heart Association class improved in all patients and ejection fraction increased 38% in six out of nine cases.
Conclusion These data suggest the feasibility and procedural safety of myoblast transplantation performed via the trans-coronary-venous approach using the TransAccess catheter system.
Key Words: Myocytes Heart failure Cells Catheters Myocardial infarction
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Introduction |
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Both pre-clinical data and initial clinical experience obtained in the recent years indicate that restoration or improvement of the left ventricular contractile performance can be observed as a result of supplementing the area of infarcted myocardium with precursor cells that can differentiate into myocytes.16 Several cell types have been investigated as possible substrates for myocardial replacement therapy. Potential use of embryonic stem cells or foetal cardiomyocytes raises serious issues of political, ethical, and even religious nature and, therefore, is far from consideration for clinical applications. Experimental data suggest that autologous bone marrow stem cells when injected into healthy or recently infarcted myocardium differentiate into myocytes and/or fuse with existing cardiomyocytes, but their implantation within post-infarction scar may result in their differentiation into fibroblasts.3,4 Therefore, direct myocyte precursors, myoblasts, are under investigation for the enhancement of contractile function within the area of fibrous post-infarction scar. Data7 obtained on an experimental model of post-infarction injury have shown that foetal cardiomyocytes and skeletal myoblasts (satellite cells) are similarly effective in improving left ventricular haemodynamic parameters including contractile function. Implantation of both cell types resulted in a significant increase in ejection fraction, whereas injection of cell culture medium alone resulted in a decrease in left ventricular ejection fraction (LVEF). In addition, histological signs of differentiation of both cell types into mature myotubes have been noted.7
Data from experimental studies have encouraged Menasché et al.8 to perform the first autologous skeletal myoblast transplantation in a patient, a survivor from MI, during coronary artery bypass grafting (CABG). This was shortly followed by an independent phase I clinical study initiated at our centre.9,10 Phase II clinical studies evaluating the efficacy of autologous myoblast transplantation performed at the time of CABG are ongoing, but despite careful patient selection, it may be problematic to assess the effect on contractility of transplanted cells independently of the positive effects of revascularization. In addition, single case reports of percutaneous myoblast transplantations were published by our group11 and others.12
We report the procedural feasibility and safety results of the first phase I clinical trial on percutaneous transvenous autologous myoblast transplantation into an area of post-infarction injury. The procedures were performed as a sole therapy, that is, without concomitant revascularization, using a catheter system (TransAccess®, TransVascular Inc., Menlo Park, CA, USA), allowing intramyocardial injections performed via the cardiac venous approach under intravascular ultrasound (IVUS) guidance.
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Methods |
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Patients studied
The inclusion criteria for the study were as follows: documented MI occurring a minimum of 3 months before the procedure; age <65 years; ejection fraction >25% and <40%; one to three akinetic or dyskinetic segments seen on echocardiography and lack of myocardial viability on dobutamine echocardiography; heart failure NYHA class 2 or 3; and good coronary artery blood flow into the area of post-infarction injury as a result of previous revascularization or development of collateral flow. The main exclusion criteria consisted of episodes of unstable ischaemia after the infarction onset or other causes requiring urgent revascularization, known inflammatory disease, renal insufficiency, history of stroke, peripheral muscular dystrophy, and inability to fully understand the protocol and the experimental nature of the procedure.
Transthoracic two-dimensional echocardiography was performed to screen patients, survivors from acute MI presenting with heart failure NYHA class 2 or 3. Global left ventricular ejection fraction (LVEF) (%) was calculated from LV end-diastolic volume (mL) and LV end-systolic volume (mL) using the Simpson's rule. Regional contractile function was assessed semiquantitatively after division of the left ventricle into 16 segments, according to the recommendations of the American Society of Echocardiography.10 After optimization of endocardial visualization on three parasternal short-axis views at three levels of the left ventricle, as well as apical two- and four-chamber views, segmental thickening was blindly graded by two experienced echocardiographers. Patients having a documented history of MI and with an akinetic or a dyskinetic area within the left ventricle, were screened by means of dobutamine stress echocardiography and included in the study when no viable myocardium was detected. Absence of viability in the akinetic/dyskinetic area was assumed if no change in segmental contractility after administration of 5, 10, and 15 µg/kg per min of dobutamine with no increase in the heart rate >10 beats/min has been observed. Patients were enrolled into the study group when both blinded examiners clearly agreed upon the lack of signs of viable myocardium.
As typical phase I study requires inclusion of 10 patients, 10 consecutive patients fulfilling the inclusion criteria, who received extensive information on the procedure and subsequently gave their written informed consent, were enrolled. Eight patients from the study group underwent previous revascularization, and in two cases the infarct area was supplied by well-developed collateral circulation. The minimum thickness of the left ventricular wall at the site of post-infarction myocardial akinesia/dyskinesia was 3 mm. Two patients from the study group had previously had an implantable cardioverter defibrillator (ICD) implanted. All patients received beta-blockers, ACE-inhibitors, and aspirin.
The investigation was approved by the Bioethical Committee of the University School of Medical Sciences in Poznan.
Cell preparation
A biopsy specimen of skeletal muscle was obtained from each patient, and myoblasts were isolated and grown in cell in vitro culture, as previously described.8,9,13 Briefly, a biopsy sample 1 cm3 was obtained from the vastus lateralis by an incision performed under local anaesthesia. The biopsy specimen was cleared of remains of fatty and connective tissue and minced into
1 mm3 pieces in Hanks' solution. After centrifugation (1200 r.p.m., 10 min at 20°C), the supernatant was suspended in culture medium. The tissue was digested with collagenase I (0.01%, 15 min at 33°C) and then filtered in sterile conditions through a nylon filter of 80 µm. Digestion and filteration were repeated, and cells resuspended in culture medium (Dulbecco MEM) supplemented with 20% FCS. The cells were taken in a logarithmic phase of expansion, washed, and resuspended in physiological saline in order to avoid provoking foreign antigens contained in culture medium. Cell viability as assessed by 1% Trypan Blue exclusion always exceeded >95%. Myoblasts were assessed by histochemistry using anti-CD 56 antibody and anti-desmin antibody, which indicated altogether >65% of myoblasts contained in the cell suspension. After in vitro expansion, serum was withdrawn and albumin added for proof of myotube formation. Myosin heavy chain staining provided evidence for almost 100% of cells positive for MHC.13 As a result of 3 weeks cell culture, up to 108 myoblasts were obtained. Two microbiological controls of the cell culture were performed and were both negative. No effect on viability as assessed by Trypan Blue staining was observed after passaging of the cells through the 27-gauge microinfusion catheter.
Technique of cell delivery
The procedures of percutaneous trans-coronary-venous cell transplantation were performed as previously described.11,15 After introducing 6-French (F) arterial and 11F venous femoral sheaths, coronary angiography was performed, allowing prolonged recording (Axiom Artist, Siemens, Germany) of the venous flow to determine coronary venous anatomy, anomalies, and coronary sinus location. The coronary sinus was engaged with a 7F SIM1 catheter (Cook, USA), and over it a 10F CS guiding catheter (TransVascular) was introduced into the coronary sinus. A balloon located at the tip of the guiding catheter was briefly inflated under low pressure to slow down the coronary sinus blood outflow, and a retrograde dye injection was performed via the guiding catheter in order to visualize the coronary venous system. The SIM1 catheter was replaced by an exchange length, 0.014 in. Hi Torque Floppy guide wire (Guidant, Temecula, CA, USA), and the wire was advanced into the anterior interventricular coronary vein via the great cardiac vein or into the middle cardiac vein. If the retrograde dye injection through the guiding catheter was not able to opacify the distal part of the target coronary vein because of the presence of venous valves, a sub-selective catheterization of the target vein over the guide wire was performed. After appropriate positioning of the guiding catheter and the guide wire, the TransAccess catheter (TransVascular) was advanced over the guide wire into the target coronary vein. The TransAccess catheter (Figure 1) is a 6.2F, monorail, composite catheter system combining a phased-array IVUS (compatible with Volcano IVUS system, Volcano Therapeutics, Rancho Cordova, CA, USA) and a pre-shaped 24-gauge nitinol needle. As the TransAccess system was placed in the target coronary vein, the intravascular orientation was performed using the corresponding artery, pericardium, and ventricular myocardium as landmarks with IVUS imaging. After confirmation of the appropriate TransAccess catheter position within the coronary vein by IVUS imaging and after verification of the depth of the catheter system in the vein by fluoroscopy, the nitinol needle was extended 37 mm into the myocardium. A 27-gauge microinfusion catheter (IntraLumeTM, TransVascular Inc.) was then advanced through the needle, centimetres into the myocardium with simultaneous injection of cell suspension into the target area.
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Safety issues
Muscle biopsy and myoblast culture were successfully completed in 12 patients. Two patients were not admitted to the study for the following reasons: one patient experienced ventricular tachycardia (VT) accompanied by chest pain just prior to attempting the procedure, despite prior amiodarone infusion; and the cells of the very first patient expired (time post-harvest from culture) prior to the procedure being attemptedcells were resuspended in saline prior to injection.
Successful intramyocardial injections of the cell suspensions into the target area of the left ventricle were performed in 9 out of 10 patients; procedural details are given in Table 1. In one patient, we were unable to advance the guide wire past a valve in the great cardiac vein and, therefore, the procedure was abandoned. In five patients, the anterior interventricular vein (Figure 2) and in four patients, the middle cardiac vein (Figure 3) was used to access to myocardium with the TransAccess catheter system. In each of the nine patients, two to four intramyocardial injections were performed delivering cell suspensions in 0.62.5 mL of saline. Each of intramyocardial injections resulted in formation of channels 1.54.5 cm deep within the myocardial injured area, as the IntraLumeTM catheter was advanced into the myocardium during slow injection of cell suspension. No injection-related chest pain was observed. The target region has been reached in all nine successful cases.
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Nine patients from the study group were treated with prophylactic amiodarone infusions started the day before the procedure (1200 mg/24 h i.v. the first day; 600 mg/24 h i.v. during the procedure) and kept on oral amiodarone (initiated day 1 after the procedure) during the next 23 weeks. One patient from the study group, reporting allergic reaction to amiodarone in the past and having ICD implanted, was not treated with prophylactic amiodarone.
Only single ventricular extrasystoles were seen during intramyocardial cell injections in all cases, and no important ventricular arrhythmia was observed on computer-assisted Holter monitoring throughout 1016 days of continuous ECG monitoring after the procedure in eight of the nine cases. One patient, not receiving prophylactic amiodarone, had episodes of VT and experienced two shots from his internal defibrillator at day 8 post-procedure.
Patients were discharged at days 1016 post-transplantation and were well after 10 weeks of follow-up. Ambulatory Holter monitoring repeated every week during the follow-up period showed no episodes of sustained VT despite discontinuation of oral amiodarone 23 weeks after the procedure.
Secondary endpoint
Although efficacy evaluation was not the main purpose of the study, regular follow-up visits during 6 months follow-up were completed. After the procedure, the NYHA class improved in all nine patients and all subjects were in class I after 6 months follow-up. Ejection fraction evaluated independently by two blinded experienced investigators increased 38% in six out of nine cases, and no change in the ejection fraction, despite improvement in the NYHA class, in the remaining three patients was observed. Segmental contractility analysis was not statistically evaluated at this number of patients, but at least seven previously akinetic segments in the study population were hypokinetic 6 months after the procedure. None of the segments analysed deteriorated during 6 months follow-up.
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Discussion |
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The use of the TransAccess catheter system allows direct myocardial access with IVUS guided needle punctures via the coronary venous system and cell infusion through a microcatheter into remote areas of injured myocardium. The system has been tested extensively on animal models15 and may have potential advantages over the currently available approaches in the precise delivery of therapeutic agents into remote areas of myocardium. Although coronary sinus catheterization requires additional training even for an experienced interventionalist, it seems that IVUS guided needle injection (Figure 4) enables appropriate accuracy for intramyocardial advancement of microlumen injection catheter.
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The results of our phase I trial show that the intramyocardial injections were possible to perform in nine out of 10 cases. The visibility of the coronary arteries parallel to the target coronary veins was good in all nine patients, allowing the safe intramyocardial needle injections with limited risk of artery puncture. Precise advancement of the microlumen catheter in the remote target area was evaluated under fluoroscopy using the coronary angiogram as a landmark for positioning the catheter tip within the injured myocardium (Figures 2 and 3). We believe that injections during the advancement of the microlumen catheter form a channel produced by the liquid pressure and, therefore, limit the myocardial trauma. As a result, no significant troponin release was observed, despite long, up to 4 cm deep injections, to deliver the cells to the target area. It should be noted that the use of the middle cardiac vein, parallel to the posterior descending coronary artery, in four cases resulted in the advancement of the TransAccess system closer to the apical segments of the left ventricle, when compared with the anterior interventricular vein. Lack of procedural success in one patient, related to the inability of appropriate positioning of the guiding catheter across the venous valve present at the bifurcation of the great cardiac vein, suggests the need for better coronary sinus guiding catheter design.
Experimental data as well as initial clinical experience have led to the initiation of clinical studies evaluating the use of totipotent stem cell or autologous skeletal myoblast transplantations as a possible means of myocardial replacement therapy1 in patients with post-infarction myocardial injury. Both cell types have different properties, especially with respect to their different abilities to differentiate after transplantation under influence of surrounding tissue. Current data indicate that, for clinical application, bone marrow cells may be the best option when transplanted early after infarction, whereas autologous skeletal myoblast transplantation may be capable of restoring contractile performance within a fibrous post-infarction scar.1 Because bone marrow derived stem cells do have the potential of extravasation into the injured tissue, clinical protocols evaluating the use of intracoronary administration of bone marrow stem cells have been applied16 and are undergoing evaluation in several phase II/III trials. Skeletal myoblasts (satellite cells) do not extravasate under normal circulation pressure,17 and therefore their potential application in myocardial regeneration requires cell injection into the area of myocardial injury.
In patients with chronic heart failure, the advantage of using skeletal myoblasts for cellular transplantation is their limited plasticity (i.e. possibility of differentiation into muscle fibres independently of environmental influence), their relatively good capacity to proliferate and grow in cell culture as well as high resistance to ischaemia.1,18,19 The concept and its efficacy were evaluated in numerous studies on experimental animal models.5,7,2022
Clear evidence is missing for formation of gap-junctions between transplanted myoblasts and cardiac myocytes. Gap-junctions being direct communication channels between the cytoplasm of cardiac myocytes are involved in electrical impulse travelling across the myocardium.2,7 In addition, experimental data indicate that the beneficial effect of transplanted cells may result from strengthening the post-infarction scar and amelioration of left ventricular remodelling, independently of possible contraction effect.23,24
Initial clinical experience with autologous skeletal myoblast transplantation in a patient undergoing coronary artery bypass surgery has been reported by Menasché et al.8 Five months later, they observed significant clinical status improvement including decrease of symptoms of heart failure by one NYHA class, increase in segmental contractility and ejection fraction seen on echocardiography, and increase in tracer activity on positron emission tomography suggesting new onset metabolic activity in the previously non-viable scar area. We have recently reported our independent phase I clinical trial on autologous skeletal myoblast transplantation in patients undergoing CABG.9,10 An increase in segmental contractility was seen in all patients 6 months after the procedure, and this effect was maintained throughout a 12 month follow-up period.
Current experimental and clinical data suggest a possibility of increased risk of arrhythmogenicity in patients after autologous skeletal myoblast transplantation. In our CABG phase I experience,9,10 episodes of sustained VT were observed in first two patients during early post-operative period, and this resulted in prophylactic administration of amiodarone in the other patients. Prophylactic amiodarone prevented VT episodes, and no amiodarone treatment was continued later than 6 weeks during follow-up. Because no significant ventricular arrhythmia despite no amiodarone treatment later during follow-up period was observed, we speculate that early arrhythmogenic effect of myoblast transplantation is rather related to intramyocardial injection, and/or inflammatory reaction against transplanted cells, especially against those dying shortly after injection, and is not necessary related to inappropriate electromechanical coupling due to the lack of gap-junctions formation between transplanted cells and surrounding cardiac myocytes (25).
On the basis of our previous experience with prevention of arrhythmia related to myoblast transplantation,1,10 we decided to use prophylactic amiodarone infusion in our current series of percutaneous myoblast transplantations, with exception of patients having an ICD implanted. No episodes of VT after myoblast transplantation on prophylactic amiodarone, and VT episodes resulting in shots from his ICD in the only patient in this series not being on amiodarone suggests strongly an arrhythmogenic effect of myoblast transplantation and a preventive effect of amiodarone. However, the effect of amiodarone on electrophysiological properties of transplanted cells remains unknown. Potential relationship between the low number of cells injected and low incidence of arrhythmia could not be excluded. In contrast, at the current stage, having only small numbers of patients who have undergone autologous skeletal myoblast transplantations, it is difficult to predict whether skeletal myoblasts are really arrhythmogenic, especially when patients with ischaemic cardiomyopathy frequently do develop ventricular arrhythmia.23,24 Nevertheless, future studies on cell transplantation in patients with post-infarction heart failure will have to focus on potential arrhythmogenic effect.
During 6 months follow-up, we have observed improvement in NHYA class in all nine cases and increase ejection fraction in six out of nine patients. However, caution has to be taken, when interpreting phase I clinical trials results. Pronounced improvement in functional heart failure class and only limited increase in the ejection fraction suggest a strong placebo effect in our patients. The current paper reporting procedural phase I observations is just a description of procedural feasibility and safety of this novel technique of cell transplantation, and no final conclusions on efficacy should be drawn. We did not have minimum number of cells for this phase I trial, as it was believed that safety evaluation should be initiated with small number of cells. Definitely future phase II/III trials should have evaluated higher numbers of cells for efficacy evaluation. We believe, that our initial observations justify initiation of the phase II clinical trial to thoroughly assess safety issues and, subsequently, controlled efficacy clinical trials aiming at the evaluation of efficacy of percutaneous autologous myoblast transplantation in the treatment of post-infarction heart failure as well as to evaluate the TransAccess catheter system as a possible platform for future bioengineering therapies.
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Acknowledgements |
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Footnotes |
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References |
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