Study of the impact of intramyocardial implantation of stem cells on myocardial perfusion and contractility in patients with coronary heart disease concurrent with postinfarct cardiosclerosis and chronic heart failure

OBJECTIVE: to study of intramyocardial implantation of cultured bone marrow stem cells on myocardial perfusion and contractility in the surgical treatment of patients with coronary heart disease (CHD) and chronic heart failure (CHF), by synchronized single-photon emission computed tomography (SSPECT) of the myocardium. SUBJECTS AND METHODS: The study included 11 patients. Intramyocardial injection of cell injections into the myocardial periscarring areas was made at coronary bypass surgery. All the patients underwent 99mTc myocardial SSPECT MIBI before and 3, 6, 12 months after surgery. RESULTS AND CONCLUSIONS: Implantation of bone marrow stem cells into the left ventricular myocardium favorably affects left ventricular remodeling and contributes to the improvement of myocardial perfusion and contractility, as evidenced by 99mTc.     

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.     

Intramyocardial Injections to De-Stiffen the Heart: A Subject-Specific in Silico Approach

We hypothesized that minimally invasive injections of a softening agent at strategic locations in stiff myocardium could de-stiffen the left ventricle (LV) globally. Physics-based finite element models of the LV were created from LV echocardiography images and pressures recorded during experiments in four swine. Results confirmed animal models of LV softening by systemic agents. Regional de-stiffening of myocardium led to global de-stiffening of LV. The mathematical set up was used to design LV global de-stiffening by regional softening of myocardium. At an end diastolic pressure of 23 mmHg, when 8 ml of the free wall was covered by intramyocardial injections, end diastolic volume (EDV) increased by 15.0%, whereas an increase up to 11 ml due to intramyocardial injections in the septum and free wall led to a 26.0% increase in EDV. Although the endocardial intramyocardial injections occupied a lower LV wall volume, they led to an EDV (44 ml) that was equal compared to intramyocardial injections in the mid-wall (44 ml) and larger compared to intramyocardial injections in the epicardium (41 ml). Using an in silico set up, sites of regional myocardium de-stiffening could be planned in order to globally soften overly stiff LV in heart failure with preserved ejection fraction. This novel treatment is built on subject-specific data. Hypothesis-testing of these simulation findings in animal models is warranted.     

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.     

Mouse bladder wall injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.     

Passive diastolic modelling of human ventricles: Effects of base movement and geometrical heterogeneity

Left-ventricular (LV) remodelling, associated with diastolic heart failure, is driven by an increase in myocardial stress. Therefore, normalisation of LV wall stress is the cornerstone of many therapeutic treatments. However, information regarding such regional stress–strain for human LV is still limited. Thus, the objectives of our study were to determine local diastolic stress–strain field in healthy LVs, and consequently, to identify the regional variations amongst them due to geometric heterogeneity. Effects of LV base movement on diastolic model predictions, which were ignored in the literature, were further explored. Personalised finite-element modelling of five normal human bi-ventricles was carried out using subject-specific myocardium properties. Model prediction was validated individually through comparison with end-diastolic volume and a new shape-volume based measurement of LV cavity, extracted from magnetic resonance imaging. Results indicated that incorporation of LV base movement improved the model predictions (shape-volume relevancy of LV cavity), and therefore, it should be considered in future studies. The LV endocardium always experienced higher fibre stress compared to the epicardium for all five subjects. The LV wall near base experienced higher stress compared to equatorial and apical locations. The lateral LV wall underwent greater stress distribution (fibre and sheet stress) compared to other three regions. In addition, normal ranges of different stress–strain components in different regions of LV wall were reported for five healthy ventricles. This information could be used as targets for future computational studies to optimise diastolic heart failure treatments or design new therapeutic interventions/devices.     

Myocardium at the base of the aorta and pulmonary trunk is prefigured in the outflow tract of the heart and in subdomains of the second heart field

Outflow tract myocardium in the mouse heart is derived from the anterior heart field, a subdomain of the second heart field. We have recently characterized a transgene (y96-Myf5-nlacZ-16), which is expressed in the inferior wall of the outflow tract and then predominantly in myocardium at the base of the pulmonary trunk. Transgene A17-Myf5-nlacZ-T55 is expressed in the developing heart in a complementary pattern to y96-Myf5-nlacZ-16, in the superior wall of the outflow tract at E10.5 and in myocardium at the base of the aorta at E14.5. At E9.5, the two transgenes are transcribed in different subdomains of the anterior heart field. A clonal analysis of cardiomyocytes in the outflow tract, at E10.5 and E14.5, provides insight into the behaviour of myocardial cells and their progenitors. At E14.5, most clones are located at the base of either the pulmonary trunk or the aorta, indicating that these derive from distinct myocardial domains. At E10.5, clones are observed in subdomains of the outflow tract. The distribution of small clones indicates proliferative differences, whereas regionalisation of large clones, that derive from an early myocardial progenitor cell, reflect coherent cell growth in the heart field as well as in the myocardium. Our results suggest that myocardial differences at the base of the great arteries are prefigured in distinct progenitor cell populations in the anterior heart field, with important implications for understanding the etiology of congenital heart defects affecting the arterial pole of the heart.     

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.     

Improvement in the severity of mitral regurgitation after direct intramyocardial injection of angiogenic cell precursor

We reported a case of dilated cardiomyopathy and moderate-severe mitral regurgitation (MR) who we treated by surgical direct intramyocardial angiogenic cell precursors injection. The patient was a New York Heart Association functional class III-IV, 56 year old man, who presented with end-stage congestive heart failure, moderate/severe mitral regurgitation, and myocardial fibrosis with the left ventricular ejection fraction of 13%. After he underwent direct surgical intramyocardial cell implantation, the myocardial fibrosis was resolved at 3 months follow-up. The severity of MR reduced to moderate and mild at 3 and 9 months, respectively. The left ventricular function gradually improved up to 53% at 19 months. To our knowledge, this is one of the only reports of successful direct surgical intramyocardial peripheral blood stem cell implantation to treat MR in dilated cardiomyopathy patient.     

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.     

Refinement of intrathymic injection in mice

Direct intrathymic injection is a common procedure used in several types of experimental protocols in the mouse. Currently available approaches involve major surgical procedures that expose the thoracic cavity, resulting in an increased risk of poor recovery and postsurgical complications. The authors sought to refine this surgery to reduce animal pain and distress without compromising overall efficiency of the technique. Using a minimally invasive method that does not expose the thoracic cavity, the authors gave accurately placed intrathymic injections, as confirmed by analyses with a reporter dye. They describe this new approach for intrathymic injection in mice that reduces complications associated with lengthy periods of anesthesia and thoracic cavity exposure.     

Perfusion studies of steady flow in poroelastic myocardium tissue

The behaviour of the heart has always elicited interest and particularly the study of its myocardium, as 5–10% of the blood pumped by the heart is passed through the coronary arteries to the myocardium itself. An in-depth investigation of the myocardium behaviour is useful. The present work aims to investigate how myocardium perfusion is influenced by myocardial stress and diseased states, and in general by LV pumping abnormalities. LV myocardial perfusion can then serve as a possible index of the capacity of the LV to respond to its work demand, and thus of the risk of heart failure. The poroelastic analysis of the myocardium based on finite element method (FEM) for regional perfusion through a rectangular element with various physiological ranges of loading conditions was studied.     

Induction of myocardial infarcts of a predictable size and location by branch pattern probability-assisted coronary ligation in C57BL/6 mice

The ability to create experimental myocardial infarctions of reproducible size and location is tantamount to progress in multiple facets of ischemic heart disease research. Branches of the mouse left main descending coronary artery penetrate the myocardium close to their origin and require "blind" ligation. Our objective was to develop a technique for ligation of nonvisible coronary artery branches to permit the reliable creation of infarcts of uniformly small size and location. From latex castings of the left coronary artery of C57BL/6J mice (n = 53), we calculated the highest probability for the location of branch points of two of three left ventricular (LV) branches distal to the origin of the left main descending artery. On the basis of these anatomic probabilities, we blindly ligated two areas that were likely to be locations of these nonvisible LV branches. We were successful in producing two types of small transmural myocardial infarctions (16.04 +/- 3.64 and 4.68 +/- 1.47% of the LV) in 57% of attempts. Thus our branch pattern probability-assisted method permits routine creation of small infarcts of uniform size in the mouse.     

Perfusion studies of steady flow in poroelastic myocardium tissue

The behaviour of the heart has always elicited interest and particularly the study of its myocardium, as 5-10% of the blood pumped by the heart is passed through the coronary arteries to the myocardium itself. An in-depth investigation of the myocardium behaviour is useful. The present work aims to investigate how myocardium perfusion is influenced by myocardial stress and diseased states, and in general by LV pumping abnormalities. LV myocardial perfusion can then serve as a possible index of the capacity of the LV to respond to its work demand, and thus of the risk of heart failure. The poroelastic analysis of the myocardium based on finite element method (FEM) for regional perfusion through a rectangular element with various physiological ranges of loading conditions was studied.     

Micro-structurally detailed model of a therapeutic hydrogel injectate in a rat biventricular cardiac geometry for computational simulations

Biomaterial injection-based therapies have showed cautious success in restoration of cardiac function and prevention of adverse remodelling into heart failure after myocardial infarction (MI). However, the underlying mechanisms are not well understood. Computational studies utilised simplified representations of the therapeutic myocardial injectates. Wistar rats underwent experimental infarction followed by immediate injection of polyethylene glycol hydrogel in the infarct region. Hearts were explanted, cryo-sectioned and the region with the injectate histologically analysed. Histological micrographs were used to reconstruct the dispersed hydrogel injectate. Cardiac magnetic resonance imaging data from a healthy rat were used to obtain an end-diastolic biventricular geometry which was subsequently adjusted and combined with the injectate model. The computational geometry of the injectate exhibited microscopic structural details found the in situ. The combination of injectate and cardiac geometry provides realistic geometries for multiscale computational studies of intra-myocardial injectate therapies for the rat model that has been widely used for MI research.     

L-selectin and SDF-1 enhance the migration of mouse and human cardiac mesoangioblasts

Efficient delivery of stem cells to heart regions is still a major problem for cell therapy. Here, we report experiments aimed to improve migration of mouse and human cardiac mesoangioblasts to the damaged heart. Cardiac mesoangioblasts were induced to transmigrate through the endothelium by factors released by cardiomyocytes or cytokines, among which stromal-derived factor 1 (SDF-1) was the most potent. Cardiac mesoangioblasts were also delivered into the left ventricular (LV) chamber of mice after coronary artery ligation (CAL), and their in vivo homing to the damaged heart was found to be quite modest. Pretreatment of cardiac mesoangioblasts with SDF-1 or transient expression of L-selectin induced a two- to three-fold increase in their transmigration and homing to the damaged heart. Therefore, combined pretreatment with SDF-1 and L-selectin generated modified cardiac mesoangioblasts, 50% of which, after injection into the LV chamber of mice early after CAL, home directly to the damaged free wall of the heart. Finally, modified mouse cardiac mesoangioblasts, injected into the LV chamber regenerate a larger surface of the ventricle in long-term experiments in comparison with their control counterparts. This study defines the requirements for efficient homing of cardiac mesoangioblasts to the damaged heart and offers a new potent tool to optimize efficiency of future cell therapy protocols for cardiovascular diseases.     

The effect of hydrogel injection on cardiac function and myocardial mechanics in a computational post-infarction model

An emerging therapy to limit adverse heart remodelling following myocardial infarction (MI) is the injection of polymers into the infarcted left ventricle (LV). In the few numerical studies carried out in this field, the definition and distribution of the hydrogel in the infarcted myocardium were simplified. In this computational study, a more realistic biomaterial distribution was simulated after which the effect on cardiac function and mechanics was studied. A validated finite element heart model was used in which an antero-apical infarct was defined. Four infarct models were created representing different temporal phases in the progression of a MI. Hydrogel layers were simulated in the infarcted myocardium in each model. Biomechanical and functional improvement of the LV was found after hydrogel inclusion in the ischaemic models representing the early phases of MI. In contrast, only functional but no mechanical restitution was shown in the scar model due to hydrogel presence.     

大鼠胸主动脉缩窄诱导心肌肥厚模型的建立

目的建立大鼠胸主动脉部分缩窄诱导心肌肥厚动物模型。方法雄性SD大鼠30只,随机分为两组：胸主动脉缩窄组20只和同期假手术组10只。在右无名动脉和左颈总动脉之间将主动脉结扎于8G针头上,随后将针头退出即可。术后10周,采用超声心动图检测心脏、观察心脏的大体剖面以及HE染色、测量心肌肥厚指数评价心肌肥厚的效果。结果术后10周,肉眼观：模型组心脏体积明显大于对照组。M型超声示：模型组较假手术组缩短分数下降,左室内径和室壁厚度明显增加。超声测量结果示：模型组与假手术组比较：室间隔厚度增加明显（2.527±0.269 vs.1.943±0.1）mm,（P〈0.01）;后壁厚度增加明显（2.492±0.242 vs.1.902±0.076）mm,（P〈0.01）;缩短分数略减小（49±7.681 vs.55.7±9.828）（P〉0.05）;左室舒张末期内径、左室收缩末期内径及射血分数均无明显变化。心脏肥厚指数明显增大（3.196±0.11 vs.1.785±0.099）,P〈0.01。结论胸主动脉缩窄可以导致大鼠心肌肥厚,为研究心室肥厚、心肌功能障碍以及心肌重构提供了一个很好的模型。     

Granulocyte colony-stimulating factor treatment enhances the efficacy of cellular cardiomyoplasty with transplantation of embryonic stem cell-derived cardiomyocytes in infarcted myocardium

We tested the hypothesis that granulocyte colony-stimulating factor (G-CSF) administration would enhance the efficacy of cellular cardiomyoplasty with embryonic stem (ES) cell-derived cardiomyocytes in infarcted myocardium. Three weeks after myocardial infarction by cryoinjury, Sprague-Dawley rats were randomized to receive either an injection of medium, ES cell-derived cardiomyocyte transplantation, G-CSF administration, or a combination of G-CSF administration and ES cell-derived cardiomyocyte transplantation. Eight weeks after treatment, the cardiac tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. The left ventricular (LV) dimensions and function of the treated heart were evaluated by echocardiography. Transplanted ES cell-derived cardiomyocytes survived and participated in the myocardial regeneration in the infarcted heart. A combination of G-CSF treatment and ES cell-derived cardiomyocyte transplantation significantly promoted angiogenesis and reduced the infarct area and cell apoptosis in the infarcted myocardium compared with ES cell-derived cardiomyocyte transplantation alone. The combination therapy also attenuated LV dilation, as compared with ES cell-derived cardiomyocyte transplantation alone. G-CSF treatment can enhance the efficacy of cellular cardiomyoplasty by ES cell-derived cardiomyocyte transplantation to treat myocardial infarction.     

Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts

The concept of regenerating diseased myocardium by implantation of tissue-engineered heart muscle is intriguing, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts. Here we created large (thickness/diameter, 1-4 mm/15 mm), force-generating engineered heart tissue from neonatal rat heart cells. Engineered heart tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered heart tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted hearts.