Abstract
Objectives
The aim was to create a model of myocardial infarction with a borderline myocardial impairment which would enable evaluation of the retrograde cellular cardiomyoplasty through the venous coronary sinus in a large animal model.
Materials and methods
Fifteen (study group) and 10 juvenile farm pigs (control group) underwent distal left anterior descending artery ligation. One month later the study group animals underwent sternotomy and a murine myoblastic line C2-C12 was injected at a constant pressure of 30 mmHg, into the coronary sinus. Thirty days later all animals that survived from both groups underwent transthoracic echocardiography and 99Tc scintigraphy and were later euthanized and specimens were taken for microscopic evaluation.
Results
Cardiac output decreased significantly after ligation (p < 0.001) and increased significantly after cardiomyoplasty (p < 0.001). In all animals, the surgical induction of myocardial infarction caused a marked decline in the echocardiographic values of cardiac function; however, the cardiac function and dimensions were significantly improved in the study group after cardiomyoplasty versus the control group. All animals undergoing cardiomyoplasty demonstrated a significant reduction of the perfusion deficit in the left anterior descending artery territory, instead such data remained unchanged in the control group. The histological examination demonstrated the engrafted myoblasts could be distinguished from the activated fibroblasts in the scar tissue because they never showed any signs of collagen secretion and fiber buildup.
Conclusions
In conclusion, the venous retrograde delivery route through the coronary sinus is safe and effective, providing a significant improvement in function and viability.
1
Introduction
Cellular cardiomyoplasty (CCM) as a possible substitute of damaged myocardium has becoming a clinical reality and several reports are today available about the results of direct implant of myoblasts on the scar tissue of the heart . However, many questions are still unresolved such as the best cellular substitute, the correct time to engraft the new cells, the best way to administer cells itself etc. As previously signaled, clinical studies have evaluated local cells administration as a possible way, but there are experimental studies turned to evaluation of direct coronary injection and to retrograde venous injection . Several other models have been reported in the literature: microspheres injection, intracoronary coils deployment leading to an elevated degree of myocardial impairment with a subsequent high mortality rate. Our experimental work deals with the possibilities offered by a retrograde venous administration through the coronary sinus (CS) using pigs with moderate left ventricular (LV) dysfunction realized by ligature of the left anterior descending coronary artery (LAD) on its middle third.
Aim of our experimental study is to create a model of simple myocardial infarction (AMI) with a borderline myocardial impairment which would enable to evaluate the retrograde CCM through the CS in a large animal model with low mortality and complications’ rate and a high reproducibility.
2
Materials and methods
Experience has been carried out on 25 juvenile farm pigs, of both sexes, weighing 35–40 kg. Fifteen pigs were subjected to the procedure, 10 were used as control. Animals were handled in accordance with the position of the American Heart Association and the European Community Rules on Animal Use for researches.
2.1
Study protocol
2.1.1
Phase 1: Induction of AMI in all animals from both groups
Few days before surgery animals on light sedation with i.m. Ketamine (0.2 mg/kg) were subjected to transthoracic echocardiography and myocardial 99Tc MIBI scintigraphy for basal evaluation of myocardial function and perfusion. The day of surgery, animals, fasted from the evening before, were sedated with Azaperone (2.5–3 mg/kg), ketamine (4 mg/kg) and diazepam (0.5 mg/kg) given i.m. Intravenous access was established through an ear vein and then animals after administration of 1 mg/kg of ketamine, were orotracheally intubated and ventilated with a mixture of O 2 and isoflurane 1–1.5% and N2O 40%. Peripheral EKG leads were placed, femoral artery was percutaneously cannulated and signals were recorded on a Marquette Multi channels recorder. Through a small cut-down the right jugular vein was exposed and a Swan-Ganz catheter was advanced into the pulmonary artery for pressures and cardiac output (CO) measurements.
Under sterile condition a right antero-lateral thoracotomy on the fifth intercostal space was made, pleura was entered, lungs gently retracted and pericardium opened longitudinally. The apex of the heart was exposed easy in its inferolateral aspect. LAD exposure requires a gentle displacement of the heart toward the surgeon achieved by a sponge introduced into the pericardial sac. LAD was identified in its middle portion, freed from the satellite vein and ligated after the emission of the second diagonal branch on a teflon felt with a 6–0 non adsorbable suture ( Fig. 1 A ). The extension of the AMI was evaluated with methylene blue infusion in the ascending aorta with a short period of aortic clamping ( Fig. 1 B). Pericardium was then approximated and the chest closed in layers. A second evaluation of CO was made after chest closure, then the Swann-Ganz was pulled out and neck incision closed. During the procedure animals received Mg infusion started at the opening of the chest and continued during all the procedure as well lidocaine (2 mg/min/kg). Lidocaine infusion rate is increased in case of severe ventricular arrhythmias and or ventricular fibrillation.
At the end of surgical procedure furosemide 25 mg, antibiotics and pain medications were given, chest tube removed, and animals allowed to breathe spontaneously. Antibiotics and pain medications were continued for the next 2 days. Blood samples for myocardial specific enzymes (CK-Mb, troponin and myoglobin) were taken and measured in established time interval after AMI ( Table 1 ).
| Group I | Baseline | 4 h | 8 h | 12 h | 1 week | 24 h post cardiomyoplasty |
|---|---|---|---|---|---|---|
| Troponin I (μg/l) | 0.06 ± 0.052 | 15.1 ± 10.2 | 36.6 ± 15.6 | 44.8 ± 17.4 | 0.36 ± 0.01 | 0.442 ± 0.085 |
| CK-MB (ng/ml) | 0.03 ± 0.01 | 0.072 ± 0.012 | 0.19 ± 0.058 | 0.26 ± 0.09 | 0.08 ± 0.015 | 0.068 ± 0.013 |
| Myoglobin (ng/ml) | 33.5 ± 20 | 696 ± 345 | 580 ± 314 | 422 ± 213 | 40 ± 21 | 36 ± 15 |
| Control Group | Baseline | 4 h | 8 h | 12 h | 1 week | At time of CCM in Group I |
|---|---|---|---|---|---|---|
| Troponin I (μg/l) | 0.04 ± 0.01 | 13.4 ± 7.5 | 39 ± 17 | 47 ± 21 | 0.3 ± 0.012 | 0.12 ± 0.05 |
| CK-MB (ng/ml) | 0.026 ± 0.01 | 0.066 ± 0.01 | 0.24 ± 0.04 | 0.28 ± 0.02 | 0.073 ± 0.02 | 0.055 ± 0.017 |
| Myoglobin (ng/ml) | 31 ± 12 | 678 ± 331 | 601 ± 355 | 445 ± 254 | 38 ± 19 | 33 ± 18 |
2.1.2
Phase 2: Follow-up after AMI
After 1 month all survivors underwent on light sedation to a transthoracic echo and to a second myocardial perfusion evaluation by a 99 mTc MIBI gated scintigraphy.
2.1.3
Phase 3: CCM (only the study group)
Few days after the control exams, the animals on general anesthesia and orotracheal intubation, monitored for EKG and arterial blood pressure detected percutaneously from the femoral artery underwent, sternotomy; pericardium was opened freeing the adhesions. Pericardium was suspended and a purse string was employed on the superior vena cava: a Swann–Ganz catheter was advanced on the pulmonary artery and pressures and CO were evaluated. The same catheter was then withdraw from the pulmonary artery and advanced through the CS to the anterior descending vein, parallel to the omonimous artery ( Fig. 2 A ).
Pressure was registered with the balloon inflated and then a murine myoblastic line C2-C12 in 20 cm 3 of medium was injected at a constant pressure of 30 mmHg, with the balloon inflated through a 20 min interval. The catheter was then flushed with saline and withdraw. During the injection no arrhythmic problems were observed. Pericardial remnants were then loosely closed; sternum, muscular and subcutaneous layers were approximated with non absorbable sutures in separated layers.
Animals are nursed with pain medications, antibiotics for 3 days and cyclosporine was given orally (3 mg/kg) until the animal sacrifice.
In most studies evaluating the myoblast transplantation, immunosuppressive therapy has been used to improve cell acceptance and survival. In particular, cyclosporine prevents donor-cell rejection . Similar results have been reported for allo and xenomyoblast transplantation . Therefore, because of the nonisogenic cell origins and immunogenic eGFP expression, all animals undergoing CCM received immunosuppressive therapy: a daily dose of 15 mg/kg of cyclosporine with food, beginning on day 1 of the administration of cells or placebo 10 .
2.1.4
Phase 4: Follow-up after CCM
Thirty days later all survived animals from the study group and the control group underwent transthoracic echocardiography and 99 Tc MIBI gated scintigraphy evaluation and later were euthanized through a midline sternotomy ( Fig. 2 B) and specimens were taken from the LV for microscopic evaluation.
2.2
Generation and culture of engineered C2C12 myoblast cell lines
Mouse skeletal C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in DMEM containing 10% fetal bovine serum (Sigma, Milan, Italy). Cells were transduced with an integrating lentiviral vector bicistronically expressing human preprorelaxin 2 cDNA and eGFP gene, or just eGFP, under a cytomegalovirus (CMV) promoter. These cell strains have been termed C2C12/RLX and C2C12/GFP, respectively. Clones were selected by cloning ring method and analyzed by fluorescent microscopy and flow cytometry for eGFP expression. Cells were grown in DMEM, containing 10% fetal bovine serum (Sigma) and 0.1% gentamycin, in a 5% CO 2 atmosphere at 37 °C. When required for transplantation, cells were detached using EDTA 0.1% in phosphate-buffered saline (PBS) and mechanical scraping, centrifuged and washed twice in PBS and finally resuspended in complete culture medium, as described below. Cell concentration was determined using a Bürker chamber and adjusted to the amount required for individual injections.
2.3
Echocardiography
A transthoracic ECHO using a Philips Envisor ECHO with a 3.5 MHz phased array transducer was used. Evaluations were done on M-mode, B-mode, continuous wave and Doppler recordings. M-mode measurements were made according to the guidelines of the American Society of Echocardiography, from right parasternal long axis view; mitral and aortic flows were recorded from the apical four and five chamber views. The following parameters were evaluated: left atrial diameter, left ventricular end diastolic and systolic diameters (LVEDD and LVESD, respectively), fractional shortening (FS), and left ventricular ejection fraction (LVEF) according to Simpson method. Myocardial performance index (MPI) as the sum of isovolumic contraction and relaxation times was also evaluated. The sum of isovolumic contraction and relaxation times was derived from the interval between the end of mitral inflow and the onset of the next mitral inflow signal minus ejection time evaluated as the interval between the beginning and the end of aortic flow.
2.4
Myocardial scintigraphy
All animals underwent evaluation of myocardial perfusion using a 99Mc Tc MIBI gated spect which enables evaluation of myocardial perfusion on three different projections: short axis, vertical long axis and horizontal long axis. Data were acquired on Picker Prism 3000 XP and Equal Cedar Emory quantitative analysis) software using a specific cardiac stress SPECT protocol at a frequency of 20 s per frame. 99 M Tc. binded with MIBI was injected through an ear vein at a dosage of 0.5 miCu/kg. Animals were kept in a separate cage for the first 3 days to allow dismission of the nuclear activity. From the data obtained a reconstruction was possible with evaluation of the volumes (end-systolic and end-diastolic) and evaluation–determination of LVEF.
2.5
Histologic analysis
To study the distribution and differentiation of myoblasts, we examined the eGFP and sarcomeric D-actin expression by immune fluorescence (10-μ-thick myocardial samples fixed in formaldehyde vapors for 10 min and then incubated with rhodamine conjugated monoclonal antibody anti-eGFP and antisarcomeric D-actin). Other sections were examined for immune reaction by means of incubation with polyclonal antibodies anti-VCAM-1 and ICAM-1. The immune reaction was detected with use of a confocal laser scanner microscope that could detect secondary antibodies. Ultrathin sections of myocardial tissue samples fixed in glutaraldehyde and osmium tetraoxide of epoxy resin were put on a slide with uranyl acetate and citrate and examined under electron microscopy. Some fragments not fixed in osmium were used for immune electron microscopy to reveal the relaxin produced in the grafted C2C12-relaxin cells by anti-H2-relaxin antibodies. Fibrosis in the cardiac tissue was studied by means of morphologic analysis of samples of tissue fixed in paraformaldehyde and paraffin. Some sections were stained in accordance with the Van Gieson method for evaluation of collagen (0.1% fuchsin in hydrogen peroxide containing 0.08% hydrochloric acid for 4 min, then washed in 95% ethanol for 5 min) and analyzed by means of optical microscopy.
2.6
Statistical analysis
Group statistics were expressed as mean ± SD. Fisher’s Exact test was used for the non continuous variables. Student t test was employed for the continuous variables. Significance between data was considered achieved when P < .05.
2
Materials and methods
Experience has been carried out on 25 juvenile farm pigs, of both sexes, weighing 35–40 kg. Fifteen pigs were subjected to the procedure, 10 were used as control. Animals were handled in accordance with the position of the American Heart Association and the European Community Rules on Animal Use for researches.
2.1
Study protocol
2.1.1
Phase 1: Induction of AMI in all animals from both groups
Few days before surgery animals on light sedation with i.m. Ketamine (0.2 mg/kg) were subjected to transthoracic echocardiography and myocardial 99Tc MIBI scintigraphy for basal evaluation of myocardial function and perfusion. The day of surgery, animals, fasted from the evening before, were sedated with Azaperone (2.5–3 mg/kg), ketamine (4 mg/kg) and diazepam (0.5 mg/kg) given i.m. Intravenous access was established through an ear vein and then animals after administration of 1 mg/kg of ketamine, were orotracheally intubated and ventilated with a mixture of O 2 and isoflurane 1–1.5% and N2O 40%. Peripheral EKG leads were placed, femoral artery was percutaneously cannulated and signals were recorded on a Marquette Multi channels recorder. Through a small cut-down the right jugular vein was exposed and a Swan-Ganz catheter was advanced into the pulmonary artery for pressures and cardiac output (CO) measurements.
Under sterile condition a right antero-lateral thoracotomy on the fifth intercostal space was made, pleura was entered, lungs gently retracted and pericardium opened longitudinally. The apex of the heart was exposed easy in its inferolateral aspect. LAD exposure requires a gentle displacement of the heart toward the surgeon achieved by a sponge introduced into the pericardial sac. LAD was identified in its middle portion, freed from the satellite vein and ligated after the emission of the second diagonal branch on a teflon felt with a 6–0 non adsorbable suture ( Fig. 1 A ). The extension of the AMI was evaluated with methylene blue infusion in the ascending aorta with a short period of aortic clamping ( Fig. 1 B). Pericardium was then approximated and the chest closed in layers. A second evaluation of CO was made after chest closure, then the Swann-Ganz was pulled out and neck incision closed. During the procedure animals received Mg infusion started at the opening of the chest and continued during all the procedure as well lidocaine (2 mg/min/kg). Lidocaine infusion rate is increased in case of severe ventricular arrhythmias and or ventricular fibrillation.
At the end of surgical procedure furosemide 25 mg, antibiotics and pain medications were given, chest tube removed, and animals allowed to breathe spontaneously. Antibiotics and pain medications were continued for the next 2 days. Blood samples for myocardial specific enzymes (CK-Mb, troponin and myoglobin) were taken and measured in established time interval after AMI ( Table 1 ).
| Group I | Baseline | 4 h | 8 h | 12 h | 1 week | 24 h post cardiomyoplasty |
|---|---|---|---|---|---|---|
| Troponin I (μg/l) | 0.06 ± 0.052 | 15.1 ± 10.2 | 36.6 ± 15.6 | 44.8 ± 17.4 | 0.36 ± 0.01 | 0.442 ± 0.085 |
| CK-MB (ng/ml) | 0.03 ± 0.01 | 0.072 ± 0.012 | 0.19 ± 0.058 | 0.26 ± 0.09 | 0.08 ± 0.015 | 0.068 ± 0.013 |
| Myoglobin (ng/ml) | 33.5 ± 20 | 696 ± 345 | 580 ± 314 | 422 ± 213 | 40 ± 21 | 36 ± 15 |
| Control Group | Baseline | 4 h | 8 h | 12 h | 1 week | At time of CCM in Group I |
|---|---|---|---|---|---|---|
| Troponin I (μg/l) | 0.04 ± 0.01 | 13.4 ± 7.5 | 39 ± 17 | 47 ± 21 | 0.3 ± 0.012 | 0.12 ± 0.05 |
| CK-MB (ng/ml) | 0.026 ± 0.01 | 0.066 ± 0.01 | 0.24 ± 0.04 | 0.28 ± 0.02 | 0.073 ± 0.02 | 0.055 ± 0.017 |
| Myoglobin (ng/ml) | 31 ± 12 | 678 ± 331 | 601 ± 355 | 445 ± 254 | 38 ± 19 | 33 ± 18 |
2.1.2
Phase 2: Follow-up after AMI
After 1 month all survivors underwent on light sedation to a transthoracic echo and to a second myocardial perfusion evaluation by a 99 mTc MIBI gated scintigraphy.
2.1.3
Phase 3: CCM (only the study group)
Few days after the control exams, the animals on general anesthesia and orotracheal intubation, monitored for EKG and arterial blood pressure detected percutaneously from the femoral artery underwent, sternotomy; pericardium was opened freeing the adhesions. Pericardium was suspended and a purse string was employed on the superior vena cava: a Swann–Ganz catheter was advanced on the pulmonary artery and pressures and CO were evaluated. The same catheter was then withdraw from the pulmonary artery and advanced through the CS to the anterior descending vein, parallel to the omonimous artery ( Fig. 2 A ).
