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In the past few years there has been an explosive growth in the development of animal models of cardiovascular disease based on transgenic and gene-targeted strategies (1). Almost exclusively, the mouse has been the animal model employed in these studies owing to the relative ease with which its genome may be manipulated, short reproductive cycle, and low cost. Further advances now provide the ability to control gene expression in a temporal manner in the postnatal mouse heart (2) and, as described in other chapters of this monograph, the potential for assessing the feasibility and effectiveness of treating cardiovascular disease at the molecular genetic level through the use of gene therapy. Phenotypic analysis in the mouse though, has generally lagged behind the rapid advances in molecular biology owing to the unique challenges posed by the small size of the mouse heart (<6 mm length) and rapid heart rate (550-650 beats/min). [ABSTRACT FROM AUTHOR]

Alterations in β-adrenergic receptor (βAR) signaling typically occur in cardiomyocytes from hearts progressing toward failure (1). Regulation of G-protein-coupled βARs mostly involves phosphorylation of agonist-occupied receptors by a βAR kinase (βARK1), leading to desensitization. This process also requires a second protein, β-arrestin, which binds to phosphorylated receptors and sterically interdicts further coupling. βARK1, also known as GRK2, is primarily a cytosolic enzyme that must translocate to the membrane in order to phosphorylate its receptor substrate. This is accomplished through a direct physical interaction between the GRK and the βγ-subunits of G-proteins (Gβγ) (2,3). The Gβγ-binding site on βARK1 has been mapped to a region located toward the carboxyl terminus of the enzyme, and a peptide derived from this region (βARKct) can act as an effective in vivo βARK1 inhibitor. The βARKct represents the last 194 amino acids of βARK1 containing the Gβγ-binding domain (2) and has been utilized in transgenic mice to inhibit the in vivo activity of βARK1 (4). Moreover, these mice present with enhanced cardiac contractility that has led to the rescue of several mouse models of cardiomyopathy (4,5). [ABSTRACT FROM AUTHOR]

Gene therapy has tremendous potential as a treatment option for heart disease. Before successful implementation of gene transfer strategies, however, several problems need to be addressed. These include: increased efficiency and homogeneity of delivery to cardiac myocytes; dosage minimization to limit untoward effects; delivery localization to avoid nonspecific gene transfer; and simplification of delivery techniques to broaden applicability of the intervention. To address the problem of efficient and homogeneous delivery, we studied adenovirus behavior in a variety of in vivo, ex vivo, and in vitro models (1-3). We found that the adenovirus behavior was characterized by a diffusional component and a receptor-ligand interaction. The percentage of cells infected by the vector varied with coronary flow rate, virus contact time, and virus concentration. A significant improvement in gene transfer efficiency was noted with modulation of microvascular permeability. The focus of this chapter is on improving in vivo delivery by increasing microvascular permeability. [ABSTRACT FROM AUTHOR]

It is now expected that gene transfer to somatic cells will offer, in the future, a new therapeutic approach to correct various pathogenic processes, including those responsible for common acquired or degenerative diseases such as heart failure. Indeed, a number of recent studies have shown that adenovirus-mediated gene transfer to cardiac myocytes can improve cardiac contractility (1-4). However, a number of methodologic obstacles still need to be overcome regarding, for instance, vector safety and infection efficiency, cardiac restriction and regulation of transgene expression, and, especially in terms of myocardial gene therapy, vector delivery procedures. Indeed, gene transfer to the myocardium should be as diffuse and homogeneous as possible, especially in heart failure therapy. [ABSTRACT FROM AUTHOR]

The objective of in vivo gene transfer is the expression of a specific gene product into a target cell population with the general intention of not altering other cell populations. Because of their ability to transduce nonreplicating cells, as well as their fairly large packaging capabilities, recombinant adenoviral vectors have frequently been utilized as a vector system. However, the immune response to recombinant adenoviral vectors utilized for gene transfer limits their clinical efficacy for certain applications. Adenovirus receptors are present on multiple cell populations, limiting the ability of systemic vector administration to target myocardial cells. Nonspecific gene transfer affects gene product expression in cell populations other than the population of interest. This alteration of nontargeted cell populations may interfere with cell homeostasis in multiple organs and may expand the immune response consequences. Thus, vector delivery techniques that optimize tissue specificity of delivery are advantageous. [ABSTRACT FROM AUTHOR]

Targeting genes to the heart through somatic gene transfer is allowing investigators to manipulate specific molecules and signal transduction pathways in both in vitro and in vivo models of cardiovascular disease (1,2). This approach permits us to test the role of these molecules and pathways of disease pathogenesis and progression (1). [ABSTRACT FROM AUTHOR]

Gene transfer into the murine myocardium represents a powerful strategy for studying the mechanisms and potential treatments of cardiac disease. The mouse can be genetically engineered precisely, and cardiac function can be examined at both the organ and the cellular level (1). Several mouse models have been developed that mimic features of acquired heart disease, such as hypertrophy, contractile dysfunction, or arrhythmias. Also, models of cardiac developmental defects as well as dilated and hypertrophic cardiomyopathy have been created by overexpression or deletion of specific genes. Gene transfer into the myocardium of such models of cardiac disease has already given insight into molecular mechanisms of cardiac dysfunction at the cellular level (2). Furthermore, high-efficiency, long-term expression of foreign genes in the neonatal and embryonic heart is particularly valuable as a strategy for rescue of the various murine cardiac disease models. Finally, gene transfer to the myocardium may also allow genetic targeting of the heart prenatally and postnatally by virus-mediated delivery of Cre recombinase to mice with floxed alleles (3). [ABSTRACT FROM AUTHOR]

Virus-based gene transfer results in the rapid and efficient expression of individual contractile proteins within the myofilaments of adult cardiac myocytes (1-3). This ability to express genetically modified contractile proteins in myocytes now makes it possible to investigate the relative phosphorylation of a given contractile protein as it resides within the intact myofilament. Contractile proteins, including troponin I (TnI), troponin T (TnT), myosin light chain 2 (MLC2), and myosin binding protein C (MyBPC) are in vitro targets for protein kinase C (PKC) phosphorylation (4). Several protocols have been developed to detect contractile protein phosphorylation using 32P-ATP and PKC preparations (5-7). However, questions remain about whether these proteins are phosphorylated by agonist-activated PKC in intact adult cardiac myocytes (5,8,9). Thus, there is a need to identify the relative phosphorylation of myofilament proteins in response to PKC activation in adult cardiac myocytes. This goal is accomplished by studying myofilament protein phosphorylation in genetically modified myocytes expressing contractile proteins with and without modified phosphorylation sites. [ABSTRACT FROM AUTHOR]

The ability to isolate individual canine cardiac myocytes that maintain normal rod-shaped morphology and contractility with stimulation is integral to the study of cardiac myocyte function and response to intervention in vitro. The literature is replete with the use of isolated murine and rat myocytes for the investigation of cardiac physiology. Methods include morselization or perfusion of the myocardium with various enzymatic solutions to disperse cells. Similar methods have been translated to larger species with variable yield and viability. [ABSTRACT FROM AUTHOR]

The transfer of genes encoding immunomodulatory agents into allografts holds promise as an inductive therapy in transplantation (reviewed in refs. 1-3). This approach is clinically applicable, since vascularized transplants are routinely perfused at the time of organ harvest and therefore may be transfected by perfusion. However, many fundamental aspects of this technology must be addressed before it may be optimally applied to clinical transplantation. For example, it has been suggested that immunosuppressive gene therapy may provide advantages over conventional immunosuppression (1-3). Notably, gene transfer should allow for the persistent, local release of the agent within the microenvironment of the graft, thereby negating the deleterious side effects of systemic immunosuppression. Although this feature of immunosuppressive gene transfer is attractive, it has not been validated. Indeed, adenovirus mediated transfer of CTLA4Ig in liver allografts results in readily detectable levels of the transgene product in the sera (4). Hence, local secretion of the transgene product may result in systemic immunosuppression and increased susceptibility to infections and neoplasia. This fundamental aspect of immunosuppressive gene therapy has not been fully addressed and should be rigorously investigated. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotide technology has emerged as an important technique for manipulating gene expression. The theory behind this technique derives from an understanding of transcription and translation. Synthetic oligodeoxyribonucleotide chains are engineered complementary to a given messenger RNA (mRNA). By binding to the mRNA, the oligonucleotide prevents protein translation. This blockade may be mediated by RNAase-H degradation of the RNA-oligonucleotide complex. In addition to negative effects on protein translation, oligonucleotides may inhibit mRNA transcription through triple helix formation with complementary DNA regions (1-3). [ABSTRACT FROM AUTHOR]

Although tRNA-mediated suppression has been mainly used to study chainterminating mutations in bacteria and yeast (1,2), suppressor tRNAs have also been employed for a variety of other purposes. For example, by introducing a nonsense mutation into the diphtheria toxin-coding sequence, its expression in vivo can be regulated by the presence or absence of a suppressor tRNA. This technique has provided new approaches to cancer therapy (3) as well as to the study of visual system development in Drosophila melanogaster (4). [ABSTRACT FROM AUTHOR]

Despite impressive advances in the last decade in treating coronary atherosclerosis, myocardial infarction remains the number one cause of death and disability in industrialized countries. A great deal of effort has been expended over the last 25 years toward identifying strategies to limit the amount of myocardium lost to infarction (1,2). Although infarct size limitation remains a highly desirable goal, it has been extremely difficult to achieve clinically. A fundamental problem is the fact that ischemic myocardium dies quite rapidly, whereas most patients wait for more than 3 hours before seeking medical attention. Over the last 5 years, our group has become increasingly interested in working on strategies to enhance the repair phase of myocardial infarction (3-7). Our long term goals are to induce muscular regeneration of the infarcted region. This chapter focuses on our work using skeletal muscle cell transplantation for cardiac regeneration. [ABSTRACT FROM AUTHOR]

Cellular transplantation into the heart is an emerging field with numerous applications for designing new therapeutic strategies for treating various types of heart disease. The two primary applications of cellular transplantation are to generate new functional myocardium and to deliver therapeutic agents such as growth factors into diseased hearts. Cardiac cellular transplantation experiments have been performed using different cell types, including cardiomyocyte cell lines (1), primary cardiomyocytes (2,3), skeletal myocytes (4-6), and fibroblasts (7). In performing cardiac cellular transplantation experiments, various animal models have been used to assess the efficacy of autologous (8), syngeneic (9), allogeneic, and xenogeneic (1,10) cellular transplants. Many investigators are currently focusing on using embryonic stem (ES) cells (10,11) and bone marrow-derived stem cells (12,13) for in vitro differentiation into cardiomyocytes for transplantation, or as direct sources for cardiac cellular transplantation. In order for cells to be useful for transplantation into injured myocardium, they must be able to survive in the recipient tissue, have limited capacity for replication, and become physically and electrically coupled to each other and the neighboring host myocardium so that they may all contract synchronously. [ABSTRACT FROM AUTHOR]

Cardiac dysfunction resulting from various insults to the myocardium can ultimately lead to the development of heart failure. Owing to the limited regenerative capacity of adult cardiac tissue and the lack of a resident cardiac progenitor cell equivalent to the skeletal myoblast, the transplantation of myogenic cells into the myocardium has been investigated as a novel mechanism to repair damaged and dysfunctional myocardium. For maximal impact on cardiac function, the design of cellular therapies must carefully consider the identity of the transplanted cells as well as the method of cell delivery. This review details techniques for the purification of commonly investigated cell grafts, including skeletal myoblasts and mesenchymal stem cells, and the methods to deliver these grafts to the myocardium of laboratory animals. [ABSTRACT FROM AUTHOR]

Gene therapy has the potential to reverse the genetic causes and modify the pathophysiology of many innate and acquired diseases (1-4). Transduction of foreign DNA into cardiac myocytes is of potential value for therapeutic applications (5,6) and also offers an experimental approach to investigate the roles of individual genes in cardiovascular pathophysiology. Both efficient delivery and long-term expression of transduced genes is required before the full benefit of genetic manipulation strategies can be realized in the cardiovascular system. However, all the current methods of gene delivery have major limitations. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) as a vector for gene therapy of cardiovascular diseases has received much recent attention (1-6). This enthusiasm is based on the success of numerous proof of principal gene transfer experiments. Stable high-level expression has been achieved following a variety of delivery methods (direct cardiac muscle injection, coronary vascular or carotid artery infection) and in a wide range of animal models from rodents to pigs (7-12). A side-by-side comparison between viral and nonviral vectors in rabbit heart suggests that rAAV is much more efficient than nonviral vectors such as naked or liposome-complexed DNA. At the same time, rAAV also displays the least inflammatory response in comparison with other viral vectors such as adenovirus and herpes simplex virus (13). Regulated myocardial transgene expression has also been demonstrated with an AAV vector containing glucocorticoid response elements in rat heart (14). [ABSTRACT FROM AUTHOR]

Adenoviral vectors are a popular choice for gene transfer protocols because they are well characterized, have a relatively large cloning capacity (up to 36 kB), and can be grown to high titers (1013 particles/mL) (1). Despite these attributes, first-generation adenoviral vectors retain many viral genes that can elicit a strong immune response, severely limiting their utility for studies in vivo (2). Our laboratory and others have been developing "gutted" or helper-dependent adenoviruses, which lack all viral coding sequences and therefore should greatly enhance the persistence of the vector in vivo (3,4). We have used this technology to deliver to muscle full-length cDNAs of the largest known gene, dystrophin, under control of the mouse muscle creatine kinase enhancer plus promoter (4-6). [ABSTRACT FROM AUTHOR]

Current methodologies in first-generation adenoviral gene transfer, however novel their approach to vector delivery, are ultimately limited by the purity of the vector being delivered. Purity in this case is defined both from the standpoint of genetic homogeneity, and from the absence of any toxic elements that may jeopardize cellular homeostasis and/or virion-cell receptor interactions. The evolution from plasmid to recombinant adenoviral vector, therefore, necessitates the orchestration of production and purification. In vector development there is a constant need for confirmation stemming from the many vulnerabilities that may be imposed on the system in the cascade of events linking plasmid endocytosis to viral genomic encapsidation. The propensity with which wild-type virions tend to outgrow any engineered competitors is a primary concern in an effort to package and propagate a recombinant adenoviral genome. [ABSTRACT FROM AUTHOR]