From the Cardiology Division

Background-Cell therapy for myocardial infarction (MI) may be limited by poor cell survival and lack of transdifferentiation. We report a novel technique of implanting whole autologous myocardial tissue from preserved myocardial regions into infarcted regions. Methods and Results-Fourteen rats were used to optimize cardiomyotissue size with peritoneal wall implantation (300 m identified as optimal size). Thirty-nine pigs were used to investigate cardiomyotissue implantation in MI induced by left anterior descending balloon occlusion (10 animals died; male-to-female transplantation for tracking with in situ hybridization for Y chromosome, nϭ4 [2 donors and 2 MI animals]; acute MI implantation cohort at 1 hour, nϭ13; and healed MI implantation at 2 weeks, nϭ12 Key Words: myocardial infarction Ⅲ remodeling Ⅲ stem cells M yocardial infarction (MI) and its resultant left ventricular (LV) dysfunction remain a leading cause of mortality and morbidity. Different therapies for myocardial regeneration have been investigated with varying results and no definitive beneficial effects. [1][2] We have developed a novel approach to adult cardiomyocyte transplantation. In a porcine model of MI, core biopsies of myocardial tissue are obtained from the preserved basal ventricular septum and used for implantation into the myocardial scar Received January 28, 2006; accepted October 8, 2010 Methods All experiments were performed in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center. Myotissue Sizing Experiments Fourteen inbred rats (50 to 60 g) were used. Donor rats (nϭ3) were euthanized with CO 2 inhalation, and the heart was excised and placed in normal saline. Strips of varying diameters (100 to 500 mol/L) were obtained under a dissecting microscope. Eleven rats were then anesthetized with intraperitoneal ketamine/xylaxine (0.1 mL /100 g). The abdominal skin was incised and rectus muscle was exposed. Strips were implanted, and rectus fascia was closed with 10 -0 Prolene suture. Animals were divided into 5 groups (nϭ2 per group) with implantation of 100-, 200-, 300-, 400-, and 500-m strips (1 strip per animal). The animals were allowed to recover and were euthanized after 1 week, and the abdominal wall was formalin fixed, paraffin embedded, and sectioned for hematoxylin and eosin staining. Porcine Balloon Occlusion Catheter Model of Anterior MI Thirty-nine 30-to 40-kg Yorkshire pigs were anesthetized with intramuscular ketamine (10 mg/kg) and isoflurane (2 normal animals as donors for male-to-female transplantation and 37 infarcted animals: 13 animals for acute infarct study, 12 animals for healed infarct study, 2 recipient animals for tracking experiments, and 10 animals died during balloon occlusion of intractable ventricular fibrillation before being randomized, leading to more aggressive pretreatment with Mg, KCl, metoprolol, and lidocaine). A 2.75ϫ20-mm angioplasty balloon (Maverick, Boston Scientific, MA) was advanced over the wire and positioned in the mid left anterior descending artery after the takeoff of the first diagonal branch. The balloon was inflated to 6 atm for 60 minutes to produce an anterior MI. Balloon occlusion and Thrombolysis in Myocardial Infarction grade 0 flow were confirmed with contrast injection. The balloon was deflated after a minimum of 60 minutes of uninterrupted occlusion, and the surviving animals were allowed to recover. LV angiography was performed in all animals to confirm the presence of anterior wall motion abnormality. Septal Biopsy and Myotissue Implantation Into Myocardial Scar Tissue Implantations were performed acutely in acute MI cohort and 2 weeks after the initial infarction (ie, after the period of acute inflammation when scar formation and remodeling process have ensued in the healed infarct cohort). A right anterior thoracotomy through the fourth intercostal space was performed. The pericardium was opened and the lung retracted. The right ventricular free wall was incised, and a short 8F sheath (Cordis) was inserted and secured with a purse string suture. A liver bioptome (Cook Inc, Bloomington, IN) was inserted via an 8F sheath into the right ventricle and aimed at the basal septum under fluoroscopic guidance. Between 6 and 10 core biopsies (average, 9) were obtained with the bioptome from the basal septum. The anterior wall was exposed by rotating the heart slightly with a sponge stick. Six biopsies were then implanted into the anterior wall of the LV 0.5 cm distal to the left anterior descending artery and D1 bifurcation (visually identified). This was done by unloading the liver bioptome (Cook Inc) and rotating it in situ so as not to remove the implanted biopsy material (see the online-only Data Supplement for a detailed description). In each cohort, animals were randomized to cardiomyotissue implantation or sham injections. Sham animals also underwent the septal biopsy. The empty liver bioptome without the implant tissue was then introduced into the anterior wall. In Situ Hybridization of the Y Chromosome for Implant Viability Assessment Four sibling pairs of male and female pigs were used for this experiment. Two animals died during MI induction, leaving 2 animals for implantation. Two weeks later, the female recipients were brought back with their male siblings. Male hearts were harvested via median sternotomy, and basal septal biopsies were immediately taken after opening of the right ventricular cavity. Cardiomyotissue from the basal septum of the male sibling was implanted into the anterior wall myocardial infarct area of the females under direct vision. The area of implantation (9 to 11 implants per animal) was demarcated with 6 -0 Prolene sutures. The female recipients received 3 days of pulse dose steroids (40 mg or 1 mg/kg) and 10 mg thereafter to prevent rejection (not HLA matched). The hearts of the recipients were harvested 2 weeks after implantation and 4 weeks after the initial MI. In situ hybridization for Y chromosome was performed on the harvested female recipient hearts to quantify the number of viable implants. Hybridization with Starfish biotinylated pig Y chromosome DNA probe (Cambio, Guildford, Surrey, UK) was performed overnight at 37°C in a humidified chamber. A streptavidin-biotin system with diaminobenzidine development (Vector, Burlingame, CA) followed by hematoxylin counterstaining was used to visualize male cells. Infarct Volume, Myocardial Perfusion, and Functional Assessment by Cardiac Magnetic Resonance Animals in the acute MI cohort underwent cardiovascular magnetic resonance on a 1.5-T General Electric TwinSpeed Scanner (GE Healthcare Technologies, Milwaukee, WI) 4 weeks after infarction as previously described. Functional Assessment of LV Function by Echocardiography (Transthoracic and Epicardial) In the healed infarct cohort, 2 weeks after MI, baseline 2-dimensional and 2-dimensional-directed M-mode epicardial echocardiography was performed in multiple views (standard short-axis and long-axis views, as well as epicardial views) 24 to assess LV ejection fraction (EF) and LV end-diastolic dimension. 25 Transthoracic echocardiography was performed before implantation with the animal chest closed. Epicardial echocardiography windows were obtained before implantation after the chest was open. Magnetic resonance imaging (MRI) was not performed because of multiple procedures in this cohort to reduce time under anesthesia. End-systolic and end-diastolic LV cavity dimensions at the level of midpapillary muscles were determined in the M mode. EF was calculated from the M-mode-derived cavity dimensions in the Teicholz formula: (enddiastolic dimensionϪend-systolic dimension)/end-diastolic dimensionϫ100. Measurements were repeated at 4 weeks after infarction at the time of tissue harvest. In the acute cohort, echocardiography was performed at the time of death (at 4 weeks). Echocardiographic analysis was performed quantitatively and qualitatively by 2 experienced echocardiographers in a blinded fashion. Hemodynamic Assessment LV pressure was measured with a high-fidelity micromanometer catheter placed in the LV cavity in a retrograde fashion. The rate of change of LV pressure was measured and averaged over 10 beats (dP/dt). Left atrial pressure was measured with a 3.5-JL 5F catheter advanced (retrograde) to left atrium. All data were recorded digitally and stored for offline analysis (Sonosoft, Sonometrics Corp, London, ON, Canada). Wykrzykowska et al Myotissue Implantation Regenerates Myocardium 63 at Harvard University on February 3, 2011 circ.ahajournals.org Downloaded from Histology, Morphometric Analysis, and Immunohistochemistry Four weeks after the initial infarction, animals were euthanized under general anesthesia, and the hearts were harvested and cut into 5 transverse slices. The apical and middle slices were used for myocardial viability with 1% triphenyltetrazolium chloride (TTC) in phosphate buffer (Sigma Chemical) and incubated for 20 minutes at 38°C as previously described. Molecular Studies Myocardial tissue samples were lysed in radioimmunoprecipitation assay solution (Boston Bioproducts, Ashland, MA). Protein concentrations were determined by Bradford assay. Equal amounts of protein were subjected to fractionation on 10% SDS-polyacrylamide under reducing conditions. Protein extracts were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). MMP-2 and TIMP-2 (Chemicon) were detected with specific antibodies. Immunoblots were visualized with appropriate secondary antibodies conjugated to horseradish peroxidase and chemiluminescence detection reagents (Amersham, Life Science, Arlington Heights, IL). Values of image densitometry were obtained with ImageJ software and adjusted by the ratio of sample loading as determined by Ponceau Red staining. Statistical Analysis Data analysis and graphing were performed with the Statview software package (SAS Institute Inc, Cary, NC). Groups were compared through the use of 2-tailed Student t test with a cutoff for statistical significance of Pϭ0.05. For comparisons of data at 2 and 4 weeks, paired t tests were used to compare means within groups, whereas unpaired tests were used to compare mean changes between groups. Normal distribution of the data was verified before parametric analysis was performed. Correction was made for multiple comparisons. Data are expressed as meanϮSD. Results Sizing Experiments Abdominal wall sections were obtained from implanted rats. Tissues Յ300 m remained viable at 1 week after implantation in all animals. Tissues 400 and 500 m in diameter showed necrosis, indicating that the maximal tissue size for implantation would be 300 m, probably related to revascularization of the implant Feasibility and Safety of Cardiomyotissue Implantation The initial creation of the MI model with balloon occlusion was associated with 31% mortality secondary to ventricular fibrillation during balloon occlusion. Twenty-seven of 37 animals survived: 2 for male-female transplantation, 13 in the acute MI cohort (6 sham and 7 treated animals), and 12 in the healed infarct cohort (6 sham and 6 treated animals). There was no additional mortality associated with cardiomyotissue implantation. The animals tolerated both the biopsy of the basal septum and the anterior wall implantation without hypotension or sustained arrhythmias. Histological Analysis and Male-Into-Female Transplantation Model Histological analysis by hematoxylin and eosin staining confirmed the presence of extensive areas of infarction and fibrosis in the anteroseptal area. In the treated animals, all implants were identified and were viable (marked with 6 -0 suture) in multiple tissue sections Acute MI Cohort MRI and Echocardiography Results The perfusion ratio of anterior to septal wall was greater in the treated animals than in controls (1.2Ϯ0.01 versus 0.86Ϯ0.05; PϽ0.01; Hemodynamics (dP/dt) Contractility as measured by positive maximal dP/dt was greater in the treated animals compared with controls (1235Ϯ215 versus 817Ϯ817; PϽ0.05), indicating that the overall systolic myocardial function was improved in treated animals. TTC Staining The percent infarct size of the anterior wall area in the treated animals was 3-fold smaller than in controls (10.3Ϯ4.6 versus 28.9Ϯ5.8; PϽ0.03; Healed MI Cohort Echocardiographic Assessment of LV Function Treated animals had the same EF at 2 and 4 week time points (49Ϯ6.5% versus 46Ϯ7.4%; Pϭ0.5; Hemodynamics Both systolic (positive dP/dt) and diastolic (negative dP/dt) function and LA pressures did not change in the treated animals between weeks 2 and 4 after infarction ( Morphometric Analysis Percent infarct size in the anterior wall of treated animals was significantly smaller than control animals (21Ϯ11 versus 38Ϯ8; Pϭ0.006). There was also a significant difference in Wykrzykowska et al Myotissue Implantation Regenerates Myocardium 65 at Harvard University on February 3, 2011 circ.ahajournals.org Downloaded from the infarct size in the untreated septum, suggesting a global effect of cardiomyotissue on myocardial salvage (16Ϯ11 versus 27Ϯ10; Pϭ0.02), an effect not seen in acute MI cohort. TTC staining assessment was consistent between the 2 independent observers (rϭ0.82, Pϭ0.0005). Adjacent to the implants and within the infarct region, a 2-fold greater number of cells positive for mdr-1 were observed Molecular Analysis We explored the expression of MMP-2 and TIMP-2, known to be involved in remodeling after infarction. Discussion Cell-based therapies for myocardial regeneration have demonstrated variable initial results. 4 -7,29 We have developed a new method of myocardial autotransplantation that obviates the need for cell culture and could be implemented during planned revascularization procedures. Implantation of cardiomyotissue appears to reduce infarct size and to prevent the decline in myocardial function after extensive anterior MI. This was evident in the preservation of EF, LV dimensions, and hemodynamic parameters and the decrease in infarct size in the treated animals compared with controls. In addition, implants remain viable and appear to express connexin 43 66 Circulation January 4/11, 2011 at Harvard University on February 3, 2011 circ.ahajournals.org Downloaded from and N-cadherin (gap junctions and desmosomes between the implant and surrounding myocardium). The effects in acute MI cohort were more robust than in the healed MI cohort, but this finding may be related in part to shorter treatment duration in this cohort. Our study was controlled for nonspecific effects of implantation by use of sham implants. We propose that several mechanisms and intrinsic properties of whole-tissue implantation may be responsible for preventing the decline in myocardial function. It may avoid cell shearing and preserve tissue architecture and growth factor milieu within the extracellular matrix scaffold. In addition, we hypothesize that our biopsies may contain resident stem cells that may contribute to myocardial repair. Wykrzykowska et al Myotissue Implantation Regenerates Myocardium 67 at Harvard University on February 3, 2011 circ.ahajournals.org Downloaded from definition of a stem cell and its recognition based on cell surface markers. Furthermore, the differentiation potential of the adult cardiac stem cells may be limited by the trophic factor-impoverished milieu of the infarct. We observed that treated animals tended to have lower levels of MMP-2 and higher levels of TIMP-2, with more favorable hemodynamic parameters in treated animals. As demonstrated in murine models of MI, MMP-2 expression increases and is maintained for several weeks after infarction. MMP-2 knockout mice appear to have decreased dilation response after infarction. 32 TIMP knockout mice, on the other hand, have an exaggerated unfavorable remodeling with higher incidence of LV rupture and mortality. Finally, the effects of cardiomyotissue implantation can be contrasted with the effects of skin microorgan transplantation. Limitations This preliminary "proof of concept" study suffers from the limitation of lack of long-term follow-up and safety data. A major limitation was that randomization was not based on baseline infarct size and LV function. Furthermore, the worsening LV function in the acute MI control cohort conflicts with previous studies that surprisingly showed preserved LV function. Conclusions We presented here a novel approach to cellular therapy for MI, which involves septal biopsy and implantation of the intact tissue into the infarcted area. This novel implantation technique has low mortality in our swine model of MI and is technically simple to perform. These whole-biopsy implants were efficacious in preventing myocardial dysfunction as measured by MRI, echocardiography, and hemodynamic parameters and in decreasing infarct size. They obviate the need for tissue manipulation and culture. The implants are viable at 2 weeks after implantation and may be electromechanically coupled with the host myocardium. Further studies are needed to explore the beneficial mechanism of this novel technology

Original Studies Outcomes of a Dedicated Stent in Coronary Bifurcations with Large Side Branches: A Subanalysis of the Randomized TRYTON Bifurcation Study

Objectives: To examine the benefit of the Tryton dedicated side branch (SB) stent compared with provisional stenting in the treatment of complex bifurcation lesions involving large SBs. Background: The TRYTON Trial was designed to evaluate the utility of a dedicated SB stent to treat true bifurcation lesions involving large (!2.5 mm by visual estimation) SBs. Patient enrolled in the trial had smaller SB diameters than intended (59% SB 2.25 mm by Core Lab QCA). The TRYTON Trial did not meet its primary endpoint due to an increased rate of peri-procedural myocardial infarctions (MIs). Methods: The TRYTON Trial randomized 704 patients to the Tryton SB stent with main vessel DES versus provisional SB treatment with main vessel DES. The rates of the primary end point of target vessel failure and the secondary powered end point of angiographic percent diameter stenosis in the SB at 9 months were assessed and compared between the two treatment strategies among patients with a SB !2.25 mm diameter at Additional Supporting Information may be found in the online version of this article. Catheterization and Cardiovascular Interventions 00:00-00 V C 2015 Wiley Periodicals, Inc