Although the dystrophin cDNA was cloned more than a decade ago,67 the exact physiologic role of the dystrophin protein is poorly defined. The current model assumes a structural role for dystrophin51 because its N-terminus and an area in the rod domain bind F-actin,70 whereas the cysteine-rich region near the carboxy-terminus binds the transmembrane P-dystroglycan.71 Dystrophin, therefore, connects the internal cytoskeleton to the sarco-
lemma via the transmembrane glycoprotein complex (Fig. 10-5). Genetic dystrophin deficiency leads to a loss of the dystrophin-glycoprotein complex, with increased myocyte permeability.51'72 However, the downstream effectors that ultimately lead to myocyte death in Duchenne muscular dystrophy are not clear. Several potential mechanisms, including activation of reactive oxygen species73 and lymphocyte-mediated myocyte damage,28 have been proposed.
Our findings in enteroviral myocarditis and cardiomyopathy suggest a pathogenic link between virally induced dystrophin abnormalities and genetic dystrophin deficiency. Protease 2A cleaves dystrophin during CVB3 infection, adversely affecting myocytes by impairing transmission of mechanical force and increasing cell permeability.65 At the organ level, myocyte dropout or dysfunction and loss of contractile force may ultimately contribute to the induction of dilated cardiomyopathy.
In addition, it is likely that cleavage of dystrophin may significantly affect viral replication in the myocyte. Previous studies with other viruses have demonstrated that viral-mediated cleavage of cytoskeletal proteins may be important for disruption of the cell membrane and release of virus from the cell. For example, the adenoviral L3 protease is able to cleave cytokeratin-18 at the junction of the globular head domain and the alpha-helical rod domain.74 In addition, the human immunodeficiency virus (HIV) type I protease cleaves intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein.75 It was shown that the rhinoviral protease 2A is able to cleave cytokeratin-8 late in the infection cycle in HeLa cells.48 These studies indicate that cleavage of cytoskeletal proteins may be an important component of viral replication, perhaps facilitating efficient release of virus from infected cells, and highlight the significance of dystrophin cleavage by enteroviral protease 2A.
Important distinctions exist between virus-induced cardiomyopathy and the hereditary absence of dystrophin. In contrast to a genetic defect, enteroviral infection is focal in viral cardiomyopathy. The percentage of virally infected myocytes differs in the various stages of the disease. In the acute stage, up to 7 days after infection with CVB3, more than 10% of myocytes are infected in C3H mice (unpublished observation). This percentage may be high enough that acute cleavage of dystrophin could significantly affect overall cardiac function. A similar mechanism could occur in acute, fulminant myocarditis as is observed in children and young adults. In the chronic stage of murine infection, the percentage of cells with viral persistence appears to be much lower.76 A similarly low percentage has been reported in human heart samples from patients with dilated cardiomyopathy.76
How then can enteroviruses induce dilated cardiomyopathy if the percentage of cells that express viral proteins, such as the protease 2A, is small? First, the number of infected cells in any sample indicates only the number of cells infected at the time when the tissue was harvested for analysis. Even if few cells are infected at any given time, a replicating virus can infect many cells over a prolonged time and thus cause substantial myocyte loss. The same concept has been applied to myocyte apoptosis where the percentage of apoptotic nuclei is several-fold increased in human heart failure but the absolute numbers of apoptotic cells are low.77 Second, increased sarcolemmal permeability that occurs with cleavage of dystrophin can expose myocyte contents to immunoregulatory cells. For instance, low-level expression of coxsackieviral genomes in cultured cardiomyocytes results in release of cardiac enzymes from the cells.20
"Leaking" myocyte proteins from even a few infected cells could act as autoantigens and may initiate an autoreactive response. T and B lymphocytes activated in such a way may recognize diverse myocardial antigens such as myosin, the adenine-nucleotide-translocator, or the sarcolemma, all of which have been reported as autoantigens in human dilated cardiomyopathy.78-81 This inappropriate immune response may attack and destroy many uninfected myocytes and thus cause substantial myocyte loss or dysfunction.82 In this regard, increased lymphocytic infiltrates and enhanced expression of cytolytic mediators such as perforin and TIA-1 have been reported in endomyocardial biopsy specimens from patients with dilated cardiomyopathy.79
Another difference between hereditary dystrophin deficiency and cleavage of dystrophin by protease 2A is the chronic versus acute loss of dystrophin. In hereditary dystrophin deficiency, dystrophin is absent in embryonic muscle and throughout the animal's life. This allows for compensatory mechanisms such as up-regulation of the related utrophin, as has been observed in dystrophin-deficient mice.83 However, in virally infected cells there is acute cleavage of dystrophin that is likely to occur in a cell that cannot promptly compensate for the loss of dystrophin because host cell translation mechanisms are impaired in the virally infected cells. How this would affect the cell is not known, but it may contribute to the more dramatic loss of sarcolemmal integrity that is observed in virally infected cells.65'69 An alternative mechanism by which disruption of the dystrophin-glycoprotein complex could cause cardiomyopathy has been proposed. In mice with genetic disruption of a-sarcoglycan, there is disruption of the typical sarcolemmal organization in the cardiac myocyte but not in the vascular smooth muscle cells; however, genetic knockout of 8- or P-sarcoglycan leads to disruption of the sarcoglycans in cardiac and vascular smooth muscle cells. Significant cardiomyopathy occurs in the mice with genetic disruption of 8- and P-sarcoglycan and disruption of the sarcoglycan complex in vascular smooth muscle cells, but the cardiomyopathy is significantly less in mice that have disruption of a-sarcoglycan with loss of the sarcoglycan complex in cardiac muscle alone. The cardiomyopathy associated with disruption of P- and 8-sarcoglycan is associated with arteriolar constriction leading to areas of apparent myocardial ischemia and cell death that are increased with exercise.84,85 Because coxsackievirus can infect smooth muscle cells,86 it is conceivable that enteroviral infection of smooth muscle cells can lead to disruption of the dystrophin-glycoprotein complex with focal areas of arteriolar vasospasm and cell loss through oxygen supply-demand mismatch. Arteriolar vasoconstriction has also been reported in mice that have been infected with CVB3.87 Such a paradigm would not require that a large number of myocytes be simultaneously infected to affect a significant amount of myocardium.
The mechanisms by which disruption of the dystrophin-glycoprotein complex during coxsackieviral infection induces cardiomyopathy are still unknown. Nevertheless, cleavage of dystrophin by protease 2A is the first potential molecular mechanism that relates the pathogenesis of hereditary cardiomyopathy to events occurring in acquired cardiomyopathy. Ultimately, a complete understanding of the mechanisms by which cleavage of dystrophin by protease 2A can contribute to viral replication and cardiomyopathy depends on an improved understanding of how genetic loss of the dystrophin-glycoprotein complex causes muscular dystrophy and dilated cardiomyopathy.
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