Cardiac and skeletal muscle are categorized as striated muscle, having actin and myosin filaments aligned in orderly arrays to form a series of contractile units which give the cells a striated appearance. Numerous types of muscular disorders affecting striated muscle share an underlying pathology characterized by loss of muscle mass and function. This would include cardiac disorders such as myocardial infarction, certain forms of idiopathic non-ischemic cardiomyopathies, degenerative muscular diseases (e.g., muscular dystrophies and myasthenia gravis) and even traumatic physical injuries to striated muscle. Therapeutic approaches which contribute to restoration of muscle mass and function will be useful in the treatment of all disorders characterized by this common pathology.
Myocardial infarction (MI) is characterized by the death of myocytes, by coagulative necrosis, myocytolysis, contraction band necrosis, or apoptosis, resulting from a critical imbalance between the oxygen supply and demand of the myocardium. The most common cause of MI is coronary artery thrombosis following the rupture of atheromatous plaques in epicardial blood vessels resulting in regional myocardial ischemia. Though once strictly defined as a lack of blood flow, the modern definition of ischemia emphasizes both the imbalance between oxygen supply and demand while also emphasizing the inadequate removal of metabolic waste products. Impaired oxygen delivery results in a reduction in oxidative-phosphorylation resulting in dependence on anaerobic glycolysis for the production of high-energy phosphates. This shift in metabolism produces excess lactate, which accumulates in the myocardium. Impaired ATP production and acidosis prevails with a resultant decline in myocardial contractility, which may further reduce blood delivery past the obstruction in the coronary vessel. Similarly, ischemia reperfusion injury without total occlusion can also cause cardiac damage
The exposure of the contents of the plaque to the basement membrane following plaque rupture ultimately results in vessel blockage culminating from a series of events including platelet aggregation, thrombus formation, fibrin accumulation, and vasospasm. Total occlusion of the vessel for more than 4-6 hours results in irreversible myocardial necrosis. Ultimately, death and morbidity due to myocardial infarction is the result of fatal dysrhythmia or progressive heart failure. Progressive heart failure is chiefly the result of insufficient muscle mass or due to improper function of the heart muscle, which can be caused by various conditions including but not limited to hypertension, and is therefore the focus of cellular based therapy.
All current strategies for the treatment of myocardial infarction focus on limiting myocyte death. Annually in the United States, 500,000 patients undergo angioplasty with stent placement, 400,000 will undergo coronary artery bypass, while an unknown additional number of patients will be treated by thrombolytic therapy. Overall prognosis is highly variable and depends on a number of factors related to the timing and nature of intervention, success of the intervention in limiting infarct size, and post-MI management. Better prognosis is associated with early reperfusion, inferior wall infarct, preserved LV function, short-term and long-term treatment with beta-blockers, aspirin, and ACE inhibitors. In contrast, poor prognosis is associated with delay in reperfusion or unsuccessful reperfusion.
One approach that has received recent attention focuses on repopulation and engraftment of the injured myocardium by transplantation of healthy cells, which is otherwise known as cellular cardiomyoplasty (Reffelmann, T., and Kloner, R. A. (2003) Cardiovasc Res. 58(2): 358-68). Many cell types that might replace necrotic tissue and minimize regional scarring have been considered. Cells that have already committed to a specific lineage, such as satellite cells, cardiomyocytes, primary myocardial cell cultures, fibroblasts, and skeletal myoblasts have been readily used in cellular cardiomyoplasty with limited success in restoring damaged tissue and improving cardiac function (Menasche, P. (2003) Cardiovasc Res. 58(2): 351-7; Etzion, S., et al, (2001) J Mol Cell Cardiol. 33(7): 1321-30; Sakai, T. et al, (1999) J. Thorac. Cardiovasc. Surg. 118(4): 715-24).
Cardiogenic progenitors are precursor cells that have committed to the cardiac lineage, but have not differentiated into cardiac muscle. Cardiomyocytes are the cells that comprise the heart, also known as cardiac muscle cells. Use of cardiomyocytes in the repair of cardiac tissue has been proposed, however, this approach is hindered by an inability to obtain sufficient quantities of cardiomyocytes for the repair of large areas of infarcted myocardium. Doubt has also been cast over the incorporation and tissue-specific function of intra-cardiac grafts derived from cardiomyocytes, even when harvested from embryonic sources (Etzion, S., et al, (2001) J. Mol. Cell. Cardiol. 33(7): 1321-30). Intra-cardiac grafts using this cell type can be successfully grafted and are able to survive in the myocardium after permanent coronary artery occlusion and extensive infarction. However, rat-engrafted embryonic cardiomyocytes attenuate, but do not fully reverse left ventricular dilatation and prevention of wall thinning. While survival is improved during 8 weeks of follow-up, the implanted cells did not develop into fully differentiated myocardium. Surprisingly, they remained isolated from the host myocardium by scar tissue and did not result in an improvement in systolic function over time. (Etzion, S. et al, (2001) J. Mol. Cell. Cardiol. 33(7): 1321-30).
The term muscular dystrophy describes a group of diseases characterized by hereditary progressive muscle weakness and degeneration. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic, lethal degenerative muscle diseases with an incidence of 1:3500 male births (Clemens et al, In: Current Neurology, Appel, Ed., Mosby-Year Book, Chicago, Ill., Vol. 14, pp. 29-54 (1994)). In DMD, mutations in the dystrophin gene usually result in the absence of dystrophin, a cytoskeletal protein in skeletal and cardiac muscle. In BMD, dystrophin is usually expressed in muscle, but at a reduced level and/or as a shorter, internally deleted form, resulting in a milder phenotype. No effective treatment is available for DMD or BMD at this time.
Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of autosomal recessive neuromuscular disorders of early onset. In the classic form of CMD, clinical manifestations are limited to skeletal muscle with no clinical involvement of the central nervous system (CNS) although changes in the white matter have been detected by MRI. The histological changes in muscle biopsies consist of connective tissue proliferation, large variation in the size of the muscle fibers as well as some necrotic and regenerating fibers.
The myotonic muscular dystrophy (DM) disease is the most common adult muscular dystrophy in man with a prevalence of 1 in 10,000. The disorder is inherited in an autosomal dominant manner with variable expression of symptoms from individual to individual within a given family. Furthermore, the phenomenon of anticipation (increasing disease severity over generations) is well documented for DM. This is particularly evident when an affected mother transmits the gene for the disease to her offspring. These offspring have a high incidence of mental retardation and profound infantile myotonia. Adult patients with DM manifest a pleiotropic set of symptoms including myotonia, cardiac arrhythmias, cataracts, frontal baldness, hypogonadism, and other endocrine dysfunctions. Other muscular dystrophies include limb-girdle muscular dystrophy, facioscapulohumeral (FSH) muscular dystrophy and the like.
Myasthenia gravis is a neuromuscular conduction defect that is responsible for progressive weakening of skeletal muscle strength, involvement of the extraocular muscles and levator palpebral and results in diplopia and ptosis. Diplopia, in particular, can be disabling. Myasthenia gravis is defined as a condition typified by a fluctuating condition of easy fatigability of voluntary muscles aggravated by exertion, emotion, menstruation or infection, and relieved, both subjectively and objectively, by rest and anti-cholinesterase drugs.
Like myocardial infarction, degenerative muscular diseases such as muscular dystrophies and myasthenia gravis affect striated muscle and share an underlying pathology characterized by loss of muscle mass or proper function. Treatments focusing on myocardial repair through autologous cellular substitution have also been proposed for various degenerative muscular diseases.
Satellite cells are undifferentiated skeletal myoblasts and represent a unique myogenic stem cell population with a committed fate, which is capable of regenerating injured skeletal muscle (Menasche, P. (2003) Cardiovasc. Res. 58(2): 351-7; Menasche, P. et al, (2001) Lancet 357(9252): 279-80; Campion, D. R. (1984) Int. Rev. Cytol. 87: 225-5). Autologous skeletal myoblasts are the best-characterized cell type that could be considered for myocardial repair. While successful delivery of autologous skeletal myoblasts to the heart can be achieved by direct intra-myocardial injection or via intra-arterial routes, and despite their ability to survive, adapt within the cardiac microenvironment, and improve myocardial performance in experimental animal models, fundamental differences exist between skeletal myoblasts and cardiomyocytes (Reffelmann, T., and Kloner, R. A. (2003) Cardiovasc Res. 58(2): 358-68). These differences extend to morphology, mechanism of electromechanical coupling, and response to injury, which influences the successful incorporation of skeletal myoblasts into the host myocardium (Kessler, P. D. and Byrne, B. J. (1999) Annu. Rev. Physiol. 61: 219-42). Moreover, engrafted skeletal myoblasts assume a slow-twitch phenotype in vivo, which only partially mirrors the cardiac phenotype (Reinecke, H. et al, (2002) J. Mol. Cell. Cardiol. 34(2): 241-9).
The ideal candidate for cellular renewal of the myocardium is likely to be a less committed, self-renewing stem cell that can undergo full myogenic differentiation. Such a cell population might be found in the adult bone marrow. It is now accepted that cell populations isolated from bone marrow and expanded in vitro represents a potential source of undifferentiated cells that can give rise to multiple cell types. Three such examples that have been utilized experimentally are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and endothelial progenitor cells (EPCs).
Whole bone marrow contains various populations of multipotent mesenchymal stem cells that are derived from somatic mesoderm and are involved in the self-maintenance and repair of various mesenchymal tissues. These cells can be induced in vitro and in vivo to differentiate into fat, cartilage, bone, cardiac and skeletal muscle. Bone marrow MSCs have been demonstrated to engraft and differentiate into cardiac tissue in a rat model of cardiac infarction (Wang, J. S. et al, (2001) J. Thorac. Cardiovasc. Surg. 122(4): 699-705). In some instances of bone marrow MSC engraftment, ventricular function was enhanced and bone marrow MSCs expressed a cardiac phenotype, however, the cells accumulated in the center of the scar tissue and were unlikely to have incorporated with host myocardium or to have contributed to contractile function (Tomita S., et al, (1999) Circulation 100 [suppl II]: 11-247-II-256). The MSCs induced angiogenesis in the host scar tissue and this may have contributed to improvement of cardiac function by replenishing blood flow to hibernating host myocardium (Tomita S. et al, (1999) Circulation 100 [suppl II]: II-247-II-256). Other studies also found similar results, wherein cardiac function did improve, but ultimately, engraftment of MSCs with host tissues was extremely low (Hughes, S., (2002) J. Pathol. 197(4): 468-78).
Bone marrow is also a known source of endothelial progenitor cells (EPCs). Circulating bone marrow-derived EPCs have been isolated from bone marrow and home to sites of vasculogenesis and angiogenesis, where they contribute to new vessel formation following infarction. EPCs have been used in rats, where a high level of engraftment was achieved (Asahara T. et al, (1997) Science 275(5302): 964-7). Replenishment of the vascular supply influenced the remodeling process and there was reduced scar tissue formation and improvement of cardiac function (Takahashi, T. et al, (1999) Nat Med. 5(4): 434-8). Further, compared with control animals, there was evidence that neovascularization contributed to myocardial salvage of non-infarcted tissue (Kocher, A. A. et al, (2001) Nat Med. 7(4): 430-6).
Work by Drs. Donald Orlic and Piero Anversa have demonstrated that populations of multipotent hematopoietic stem cells (HSCs) could give rise to vascular endothelium, smooth muscle cells, and cardiomyocytes. In the study by Orlic et al, large myocardial infarctions were induced by coronary ligation in mice (Orlic D., et al, (2001) Nature 410(6829): 701-5). Within hours of injury, enriched lin−, c-kit+ bone marrow cells were isolated from transgenic male donor mice and injected into the undamaged contracting tissue surrounding the infarct. These injected cells were able to migrate to the damaged myocardium, proliferate, transdifferentiate, and form a band of new myocardium and associated vasculature. The injected cells had successfully differentiated into myocardial tissue, confirming that the new cardiomyocytes represented maturing cells in the process of attaining functional competence, also expressed cardiac-specific transcription factors (Orlic D. et al, (2001) Ann. NY Acad. Sci. 938: 221-9; discussion 229-30). Repair and improved cardiac performance were obtained in 40% of the injected mice, however the associated mortality with this procedure was high. Other studies have determined that administration of cytokines such as stem cell factor and granulocyte-colony stimulating factor can mobilize and allow HSCs from adult male mice to home to and generate de novo myocardium and vascular structures in the infarcted heart and improve cardiac hemodynamics (Hughes, S. (2002) J. Pathol. 197(4): 468-78).
The most primitive cell type that has been used in cellular cardiomyoplasty is the embryonic stem cell (ES). Pluripotent ES cells can be derived from the inner cell mass of the blastocyst, while embryonic germ (EG) cells have been isolated from primordial germ cells. Undifferentiated stem cell lines have the capacity to differentiate in vitro into cells derived from all three primary germ layers, and can differentiate spontaneously into cardiomyocytes, endothelial cells, and vascular smooth muscle cells. Several groups have demonstrated the ability of ES cells to form stable intra-cardiac grafts. Klug and others engrafted genetically modified differentiated murine ES-derived cardiomyocytes into the left ventricular free wall of dystrophic mdx recipient mice (Klug, M. G. et al, (1996) J. Clin. Invest. 98(1): 216-24). These ES-derived cardiomyocytes were able to survive in damaged myocardium and improve cardiac function. A drawback of the cardiac engraftment studies to date is that pure and reproducible populations of ES-derived cardiomyocytes were not obtained. ES-derived cardiomyocyte cultures used for example, by Klug et al exhibited heterogeneous immunoreactivity to atrial natriuretic factor, which suggests that the ventricular cardiomyocyte sub-population for intra-cardiac grafting may well be a heterogeneous population (Klug, M. G. et al, (1996) J. Clin. Invest. 98(1): 216-24). Furthermore, ES-derived cardiomyocytes eventually become post-mitotic, and thus, the proliferative capacity of these cells in vivo is likely to be limited (Klug, M. G. et al, (1996) J. Clin. Invest. 98(1): 216-24). Generating sufficient numbers of these ES-derived cardiomyocytes may prove to be an obstacle, as well as the low regeneration of the myocardium by these cells. Yield was shown to be improved in vitro by addition of retinoic acid or overexpression of the GATA-4 transcription factor, however it is yet to be determined if these strategies work in vivo (Wobus, A. M. et al, (1997) J. Mol. Cell. Cardiol. 29(6): 1525-39; Grepin, C. et al, (1997) Development 124(12): 2387-95). Another potential drawback of the use of embryonic stem cells is their ability to form teratomas, which can then turn into life-threatening pathologies (Takada, T. et al, (2002) Cell Transplant. 11(7): 631-5).
While advances in the field of cardiac transplantation and cardiomyoplasty have been achieved with the advent of stem cell technology, a population of cells that are able to effectively engraft damaged myocardium and restore cardiac function without improper differentiation to other contaminating cell types is still highly desired. While pluripotent ES cells offer the promise of functional plasticity and the ability to differentiate into any cell type in vitro, extensive experimentation in vivo is still necessary to properly direct the formation of incorporated, functional cardiac tissue at the site of injury without improper differentiation to form teratomas or other non-cardiac cell types. Multipotent tissue-specific cells that have already committed to a distinct lineage, such as HSCs, MSCs, and EPCs, have also produced encouraging results. However use of these cells often results in incomplete engraftment and a failure to restore cardiac function over time. Therefore, attractive candidates would include an undifferentiated, preferably adult, stem cell population that has not committed to a specific lineage.
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