Nature Medicine8, 872 - 877 (2002)
Published online: 15 July 2002; | doi:10.1038/nm737
Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: A genetic predisposition to viral heart disease
Dingding Xiong1, Gil-Hwan Lee1, Cornel Badorff1, Andrea Dorner1, Sang Lee2, Paul Wolf3
& Kirk U. Knowlton1
1 Department of Medicine, Institute of Molecular Medicine and The Cardiovascular Center, University of California, San Diego, California, USA
2 Department of Surgery, University of California, San Diego, California, USA
3 Department of Pathology, University of California, San Diego, California, USA
Correspondence should be addressed to Kirk U. Knowlton kknowlton@ucsd.edu
Both enteroviral infection of the heart and mutations in the dystrophin gene can cause cardiomyopathy. Little is known, however, about the interaction between genetic and acquired forms of cardiomyopathy. We previously demonstrated that the enteroviral protease 2A cleaves dystrophin; therefore, we hypothesized that dystrophin deficiency would predispose to enterovirus-induced cardiomyopathy. We observed more severe cardiomyopathy, worsening over time, and greater viral replication in dystrophin-deficient mice infected with enterovirus than in infected wild-type mice. This difference appears to be a result of more efficient release of the virus from dystrophin-deficient myocytes. In addition, we found that expression of wild-type dystrophin in cultured cells decreased the cytopathic effect of enteroviral infection and the release of virus from the cell. We also found that expression of a cleavage-resistant mutant dystrophin further inhibited the virally mediated cytopathic effect and viral release. These results indicate that viral infection can influence the severity and penetrance of the cardiomyopathy that occurs in the hearts of dystrophin-deficient individuals.
Dilated cardiomyopathy is a multifactorial disease that results in heart failure and premature death. It has not yet been possible either to consistently identify its underlying mechanisms or to design etiologically specific therapies. Viral infections and hereditary disorders are among the best-defined and most commonly identified causes, each accounting for approximately 20−35% of cases1,
2,
3. Infection with enteroviruses, such as coxsackie B viruses, is among the most frequently recognized viral etiologies2,
4,
5.
Hereditary disorders that cause dilated cardiomyopathy have been linked to genes for cytoskeletal proteins, such as dystrophin, in individuals with Duchenne muscular dystrophy and X-linked dilated cardiomyopathy6,
7,
8,
9,
10,
11,
12. The severity and penetrance of the cardiomyopathy phenotype are variable, however9. Although it is clear that both viral infection of the heart and genetic mutations in the dystrophin gene can cause dilated cardiomyopathy, it is not known whether there are mechanistically important interactions between these causes. Determining the environmental factors that affect the severity and penetrance of the phenotype arising from a particular genetic abnormality would contribute substantially to our understanding of the etiology of dilated cardiomyopathy.
Recently, we identified a potential link between viral and hereditary causes of cardiomyopathy when we discovered that enteroviral protease 2A can directly cleave dystrophin in the heart, leading to a disruption of the dystrophin−glycoprotein complex similar to that resulting from hereditary abnormalities in dystrophin expression13,
14,
15.
We hypothesized that cleavage of dystrophin and disruption of the dystrophin−glycoprotein complex were crucial for enteroviral replication in the heart and for enteroviral cardiomyopathy; and that the absence of dystrophin would facilitate viral replication and increase the severity of virus-induced cardiomyopathy. To test this hypothesis, we infected dystrophin-deficient mdx mice, bearing a frameshift mutation in the dystrophin gene similar to the mutation in humans with Duchenne muscular dystrophy, with coxsackievirus B3 (CVB3). We saw greater viral replication, and markedly greater cardiomyopathy, in the hearts of infected dystrophin-deficient mice as compared with infected wild-type mice with normal dystrophin. In cell culture, expression of wild-type dystrophin and a cleavage-resistant mutant of dystrophin protected the cells from the virus-induced cytopathic effect and viral release. These results support the possibility that disruption of the dystrophin−glycoprotein complex in virally infected myocytes is important to viral propagation in the heart, and identify an interaction between an acquired and a hereditary etiology of dilated cardiomyopathy.
Dystrophin deficiency increases sarcolemma disruption As dystrophin-deficient mdx mice are normally bred in an inbred mouse strain that is not susceptible to CVB3 infection, we backcrossed inbred dystrophin-deficient female mice (C57BL/10ScSn-Dmdmdx/J) with inbred dystrophin-competent C3H/HeJ male mice, which are known to be susceptible to coxsackieviral infection13. F1 male mice from this backcross are dystrophin deficient (referred to as C57mdx/C3H). We generated dystrophin-competent control mice by backcrossing female inbred dystrophin-competent mice of the same strain as the dystrophin-deficient mice (C57BL/6 10ScSn/J) with male C3H/HeJ inbred dystrophin-competent mice (referred to as C57wt/C3H) (Fig. 1a). Immunostaining of the heart with an antibody against dystrophin confirmed the absence of dystrophin in the cardiac muscle of the C57mdx/C3H mice (data not shown). F1 male C57mdx/C3H mice and F1 male C57wt/C3H mice were infected with CVB316. One-third of the C57mdx/C3H mice died within six days after infection, whereas none of the infected C57wt/C3H mice died.
Figure 1. Dystrophin deficiency markedly increases enterovirus-mediated disruption of the sarcolemma.
a, Dystrophin-deficient mice susceptible to CVB3 infection (C57mdx/C3H) were generated by backcrossing female dystrophin-deficient C57mdx mice with male C3H mice. Female dystrophin-competent C57wt mice were also bred with male C3H mice to generate control mice (C57wt/C3H). b, Staining with Evans blue dye (white on dark background), showing sarcolemmal disruption in the heart of CVB3 infected C57wt/C3H mice (left) and C57mdx/C3H (right) on day 6 after infection (scale bar, 100 m). c, Quantification of sarcolemmal disruption. Disruption of cardiomyocyte sarcolemmal membrane integrity was significantly greater in C57mdx/C3H mice () than in C57wt/C3H (l) at days 2 (n = 3), 4 (n = 3) and 6 (n = 6) after infection (*, P < 0.05). d, Serum concentration of cardiac troponin I (cTnI) after infection with CVB3. Concentrations of cTnI were significantly higher in C57mdx/C3H than C57wt/C3H mice (*, P < 0.05, n = 4). e, H&E staining of the heart from CVB3 infected C57wt/C3H mice (top) and C57mdx/C3H mice (bottom) (scale bar, 100 m). There were occasional foci of inflammation in the C57wt/C3H mice (arrow). In C57mdx/C3H, Evans blue dye staining can be seen as a bluish stain in the affected myocytes (arrow). No significant inflammation was noted near the dye−positive myocytes shown.
Because CVB3 is cardiotropic, we compared the cardiomyopathic effect of CVB3 infection in the hearts of dystrophin-deficient and wild-type mice. Viral infection of cardiomyocytes disrupts the integrity of the sarcolemmal membrane13,
15. We evaluated sarcolemmal membrane integrity in vivo by injecting the tracer dye Evans blue, a large molecule that can enter only cells with a disrupted sarcolemma13,
17. Disruption of the sarcolemmal membrane was completely absent in uninfected C57wt/C3H mice and rare in uninfected C57mdx/C3H mice. We saw markedly greater uptake of Evans blue dye, however, in infected C57mdx/C3H mice as compared with infected C57wt/C3H mice (Fig. 1b). This disruption was detectable as early as two days after infection and continued to increase up to six days after infection (Fig. 1c).
As another marker of sarcolemmal injury, we measured the release of cardiac troponin I, a cardiac-specific sarcomeric protein, into the serum18. Concentrations of troponin I were significantly higher in the serum of infected C57mdx/C3H mice than that of infected C57wt/C3H mice (Fig. 1d). Troponin I was not detectable in uninfected C57mdx/C3H or C57wt/C3H mice (data not shown). H&E staining of the infected myocardium showed only minimal cellular inflammation in regions of the myocardium that also stained with Evans blue dye (Fig. 1e). These results demonstrate that dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy in vivo.
Dystrophin deficiency increases viral propagation To assess whether the increased disruption of the sarcolemmal membrane in the C57mdx/C3H mice was associated with viral infection, we measured viral titers in the hearts of infected C57mdx/C3H and C57wt/C3H mice. We saw significantly higher viral titers in the hearts of C57mdx/C3H mice than in those of dystrophin-competent C57wt/C3H mice (Fig. 2a), indicative of greater viral replication in the absence of dystrophin.
Figure 2. Increased viral titers and localization of virus and Evans blue dye (EBD).
a, Viral titers in the hearts of CVB3 infected mice 6 d after infection. The viral titers in the hearts of C57mdx/C3H were significantly higher than in C57wt/C3H mice (*, P < 0.05, n = 3). b, Localization of EBD staining and viral capsid proteins from CVB3 infected C57wt/C3H mice (top row) and C57mdx/C3H (bottom row) 6 d after infection (scale bar, 100 m). Fluorescent images of the EBD stain alone (red, left), CVB3 immunostain alone (CVB; green, center) and superimposed (right). Bright staining for CVB3 colocalizes with the EBD-positive myocytes in the C57wt/C3H mice (top, arrow). In the section from C57mdx/C3H mice, however, most EBD-positive cells have only remnants of viral proteins (bottom, arrows). Most CVB3-positive cells are adjacent to EBD-positive cells, but most CVB3-infected cells are EBD negative or span an EBD-positive and an EBD-negative cell (bottom right, arrowhead). c, EBD staining and immunostaining for viral protein from the heart of uninfected C57mdx/C3H mouse. EBD and viral protein staining are essentially negative in uninfected C57mdx/C3H mice. Scale bar, 100 M.
To determine the mechanism by which dystrophin deficiency causes increased viral replication, we assessed whether dystrophin deficiency enhanced virus exit from infected myocytes and subsequent infection of adjacent cells. We compared the location of myocytes staining positive with Evans blue dye with the location of cells staining positive for coxsackievirus. In the hearts of C57wt/C3H mice infected with coxsackievirus, we saw bright staining for coxsackievirus in the rare Evans blue dye−positive cells (Fig. 2b, top). In the hearts of C57mdx/C3H mice infected with coxsackievirus, however, the majority of Evans blue dye−positive cells contained only remnants of viral proteins. Immediately adjacent to the Evans blue dye−positive cells were myocytes that also stained brightly for coxsackievirus (Fig. 2b). We also saw occasional foci of bright viral staining that overlapped an Evans blue dye−positive cell and an Evans blue dye-negative cell, consistent with release of the virus from the dye−positive cell and infection of the adjacent dye-negative cell (Fig. 2b). We did not detect viral protein or disruption of the sarcolemmal membrane in uninfected C57wt/C3H mice (data not shown). In the hearts of uninfected C57mdx/C3H mice, we saw rare Evans blue dye−positive cells but did not detect viral protein (Fig. 2c).
These observations are consistent with the notion that the membranes of infected myocardial cells are more easily disrupted in the absence of dystrophin, resulting in more efficient release of the virus from infected cells and subsequent infection of adjacent myocytes.
Dystrophin inhibits cytopathic effects and release of virus To further understand the results from infected dystrophin-deficient mice, we sought to determine the effect of both wild-type and cleavage-resistant dystrophin expression on virally mediated cytopathic effects and release of virus in CVB3-infected cultured cells. We generated a cleavage-resistant dystrophin expression vector by mutating the P4 and P3 amino-acid residues (2424 and 2425, respectively) of the protease 2A cleavage site in the hinge 3 region of mouse dystrophin from leucine and serine to proline and aspartate, respectively (L2424P, S2425D) (Fig. 3a). These amino acids were chosen because they are the same as those present in the naturally occurring rabbit dystrophin, which is resistant to cleavage by protease 2A (data not shown). To demonstrate that the mutant dystrophin was resistant to cleavage, we transiently transfected expression vectors containing wild-type or mutant full-length mouse dystrophin cDNA into HeLa cells and infected the cells 36 hours later with CVB3. We harvested the cells eight hours after infection and prepared protein extracts. Immunoblotting of the extracts with Dy4/6D3, an antibody against dystrophin, indicated that the L2424P and S2425D mutations in the hinge 3 region prevented cleavage of dystrophin in CVB3-infected cells (Fig. 3b).
Figure 3. Mutation of dystrophin in hinge 3 (H3) region prevents cleavage of dystrophin by enteroviral protease 2A.
a, The leucine and serine at amino-acid residues 2424 and 2425 were changed to proline and aspartate, respectively. b, HeLa cells were transfected with either a wild-type dystrophin expression vector (wt) or the mutant dystrophin expression vector (mut). After infection with CVB3 (+), cell extracts were immunoblotted with an anti-dystrophin antibody, Dy4/6D3 and compared with extracts from untransfected cells (-). The 280-kD cleavage fragment was present only in CVB3-infected cells that expressed wild-type dystrophin.
To assess the effect of wild-type and cleavage-resistant dystrophin on virally mediated cytopathic effects and release of the virus, we used Chinese hamster ovary (CHO) cells, which do not express dystrophin and are normally resistant to infection by coxsackievirus. Transfection of CHO cells with mouse coxsackie−adenovirus receptor (CAR) allows infection of only the successfully transfected cells with coxsackievirus19. Cotransfection with a green fluorescent protein (GFP) expression vector identifies cells that have been successfully transfected. Accordingly, we cotransfected CHO cells with either wild-type mouse dystrophin (Dys(wt))20 or cleavage-resistant mutant dystrophin (Dys(mut)), mouse CAR and GFP expression vectors. The control cells were transfected with empty expression vector, mouse CAR and GFP. Western-blot analysis of dystrophin-transfected CHO cells showed that dystrophin was localized to the membrane fraction, as previously seen in cardiac myocytes13 and dystrophin-transfected COS cells21 (data not shown). To ensure that expression of dystrophin did not prevent CAR expression in cotransfected cells, we stained cotransfected CHO cells for the expression of CAR. There was no significant difference between the groups in the percentage of GFP-positive cells that expressed CAR (94%, 92% and 93% for control, Dys(wt) and Dys(mut), respectively) (Fig. 4a).
Figure 4. Dystrophin decreases the enterovirus-induced cytopathic effect and viral release in cultured cells.
a, Colocalization of GFP (top row) and CAR (bottom row) expression. CHO cells were cotransfected with GFP and CAR expression vectors and with wild-type or mutant dystrophin (Dys(wt) and Dys(mut)). Almost all of the GFP-positive cells expressed CAR in control and Dys(wt) or Dys(mut) transfected cells. b, Effects of wild-type and cleavage-resistant dystrophin on cytopathic effect in CVB3-infected CHO cells that expressed CAR. The virus-induced cytopathic effect was determined over time by quantifying the decrease in GFP-positive cells, reported as a percentage of cells at time zero (n = 5 random fields in each of 3 separate samples). Expression of Dys(wt) () resulted in a significantly smaller viral-mediated cytopathic effect at 6 h, 8 h and 10 h after infection as compared with control (). This effect was greater in cells that expressed Dys(mut) (). *, P < 0.05 as compared to control cells; †, P < 0.05 Dys(wt) as compared to Dys(mut). c, Photomicrographs of GFP-positive cells described in (b) were obtained at 0 h (left) and 10 h (right) after infection. The cytopathic effect, as assessed on the basis of the number of GFP-positive cells and the overall shape of the cells, was markedly less in cells that expressed either mutant or wild-type dystrophin than in cells transfected with empty vector (control). d, The ratio of viral titers in the culture medium to titers in the cell was markedly higher in cells that did not express dystrophin (control). As compared to Dys(wt), expression of Dys(mut) further prevented the release of virus (n = 3; *, P < 0.05 compared with control cells; †, P < 0.05 Dys(wt) as compared to Dys(mut)). Scale bars, 100 M
We infected the CHO cells 48 hours after transfection and determined the cytopathic effect of coxsackievirus infection over time by quantifying the percentage of GFP-positive cells remaining after infection. This was higher in cells that expressed wild-type dystrophin than in those transfected with the empty expression vector. The protective effect was even greater with expression of cleavage-resistant dystrophin (Fig. 4b). In addition, most of the remaining dystrophin-transfected cells were larger and had a more normal shape than the smaller, rounded appearance of the remaining control-transfected cells.
To determine whether the decrease in cytopathic effect was associated with a change in viral replication, we measured viral titers in the cell culture medium and the cell pellet at ten hours after infection. The sums of the viral titers were not significantly different for dystrophin-transfected and control cells (data not shown). Notably, however, we found that the ratio of viral titers in the culture medium, as compared with the ratio of titers in the cell pellet, was significantly lower after transfection of wild-type dystrophin and even lower in cells that expressed the cleavage-resistant dystrophin (Fig. 4d).
Thus, the total viral titers in cultures of dystrophin-expressing and control cells in vitro were the same, whereas in vivo viral titers were higher in dystrophin-deficient mice. This is probably a result of differences in the model systems. In the intact mouse heart, only a subset of cells are initially infected, and the extent of viral propagation is dependent on several rounds of viral replication, so that release of the virus is required for the infection of adjacent myocytes. In cultured cells, essentially all the transfected cells are simultaneously infected; therefore, virus propagation is not dependent on secondary infection of adjacent cells. The data from cell culture demonstrate that expression of dystrophin decreases the cytopathic effect and inhibits release of virus from infected cells. This protective effect was independent of a cellular immune response and is consistent with our data from intact hearts indicating that dystrophin protects the infected cells from virus−induced disruption. It is also consistent with previously published data showing that dystrophin stabilizes the cell membrane22. Furthermore, it indicates that the cleavage of dystrophin by the viral protease is important for the release of the virus from the cell.
Discussion Our results demonstrate that the hearts of dystrophin-deficient mice are more susceptible to enterovirus-induced cardiomyopathy, as indicated by the higher proportion of Evans blue dye−positive myocytes and higher viral titers after infection. The pattern of coxsackieviral staining in vivo and the effect of wild-type and cleavage-resistant dystrophin on virus-infected cultured cells provide compelling evidence that dystrophin stabilizes the cell membrane in infected myocytes. This is consistent with findings that dystrophin-deficient myocytes are more susceptible to mechanical injury22 and that the absence of dystrophin increases the release of intracellular proteins from the cytoplasm23. The dystrophin-mediated stabilizing effect on cell-membrane permeability decreases the extent of viral propagation and viral-mediated injury.
Our findings also are suggestive of mechanisms by which dystrophin deficiency may increase the severity of enteroviral cardiomyopathy. Dystrophin is a subsarcolemmal rod-shaped protein that is believed to stabilize the sarcolemma by attaching the actin cytoskeleton to the extracellular matrix through the dystrophin-associated glycoprotein complex6,
24,
25. This connection protects muscle cells from contraction-induced damage22. Enteroviruses are typically released from the cell by disruption of the cell membrane or cell lysis26. Because dystrophin provides mechanical support to the sarcolemma, and the absence of dystrophin results in increased susceptibility to sarcomere rupture, the virus exits from the infected cell more efficiently in the absence of dystrophin. This allows more rapid propagation of the virus to adjacent myocytes, resulting in higher viral titers and greater cardiomyopathy. In the presence of dystrophin, the rate and extent of viral release may be slowed by the requirement that protease 2A cleave dystrophin. This would result in lower viral titers than are seen in the absence of dystrophin.
Our cell-culture experiments demonstrated that when protease 2A−mediated cleavage of dystrophin is prevented, the cytopathic effect is further decreased and the release of virus from the cell is inhibited. This is consistent with the fact that proteases from other viruses can also cleave cytoskeletal proteins27,
28,
29,
30,
31. One would therefore expect that if a cleavage-resistant dystrophin were present in the heart, propagation of virus within the heart would be very inefficient. In addition, the markedly increased susceptibility of dystrophin-deficient mice to viral infection of the heart indicates that one of the mechanisms whereby the hereditary dystrophin deficiency of Duchenne muscular dystrophy leads to cardiomyopathy may be through increased susceptibility to virally mediated cardiomyopathy. It is less clear whether viral infection is involved in the dystrophin remodeling that occurs in end-stage cardiomyopathies32.
We conclude that dystrophin deficiency increases host susceptibility to coxsackieviral infection. This probably results from a more efficient release of the virus from the infected myocyte, and is associated with an increase in virus-mediated cytopathic effects. Although there are undoubtedly a number of mechanisms by which dystrophin deficiency causes cardiomyopathy in individuals with Duchenne muscular dystrophy or X-linked dilated cardiomyopathy, our data support the notion that the absence of dystrophin increases the susceptibility of such individuals to virally mediated cardiomyopathy.
Methods Virus. CVB3 was derived from the infectious cDNA copy of the cardiotropic H3 of CVB3 (ref. 16). The titer of the virus was determined by a plaque-forming assay in HeLa cells16.
Cells. HeLa cells were a kind gift from S.A. Huber, University of Vermont, and were cultured in DMEM medium with 10% FBS. CHO-K1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland) and grown in F-12K medium containing 2 mM l-glutamine and 10% FBS.
Antibodies. Immunostaining and immunoblotting for dystrophin was done with a monoclonal antibody recognizing the mid-rod domain of dystrophin (Dy4/6D3, NovoCastra, Newcastle, UK). The polyclonal rabbit antibody against CVB3 was a generous gift from A. Henke33. CAR expression was determined using an antibody against Flag-M2 (Stratagene, La Jolla, California) directed against a carboxy-terminal Flag tag on transfected CAR.
CVB3 infection in vivo. F1 male C57mdx/C3H mice and F1 male C57wt/C3H mice were inoculated by intraperitoneal injection with 5 104 plaque-forming units of CVB3 at 4 weeks of age. They were subsequently injected with Evans blue dye 12 h before they were killed. Mouse hearts were analyzed at days 2, 4 and 6 after infection. All animal protocols were approved by the UCSD Animal Subjects Committee.
Analysis of disruption of sarcolemmal membrane integrity and localization of viral protein. Heart tissue was embedded in OCT Tissue Tek (Sakura Finetechnical, Torrance, California) and snap-frozen in isopentane chilled in liquid nitrogen, and 6-m sections were cut by cryosection. Fluorescent staining of Evans blue dye was used to assess the impairment of sarcolemmal membrane integrity. Under fluorescent microscope, 5 areas of each cryosection were blindly and randomly selected and imaged. The images were imported into an image-analysis system (ImageJ http://rsb.info.nih.gov/ij). The area of Evans blue dye−positive cells was quantified and the extent of sarcolemmal disruption was expressed as the percentage of the area of dyed cells compared with the total area of the heart in the microscope field. Viral proteins in the hearts of CVB3-infected mice were detected by immunofluorescence using a rabbit polyclonal antibody against CVB313,
33.
Viral titers. To measure viral titers in mouse hearts, the heart tissues were weighed, homogenized in DMEM medium (Gibco-BRL, Grand Island, New York) and then subjected to 3 cycles of freezing and thawing to release the virus from the homogenates. Cellular debris was removed by centrifugation and the titer of virus in the supernatant was determined by standard plaque-formation assays on HeLa monolayers16. To measure viral titers in infected, cotransfected CHO cells, the culture medium was collected and the cells were first washed 3 times with PBS to remove viruses on the cell surface and then harvested. Viral titers in the medium and in cell cultures were also measured by plaque-formation assays.
Determination of cardiac troponin I. Serum concentrations of cardiac troponin I (cTnI) were measured by an enzyme sandwich immunoassay using two cTnI-specific monoclonal antibodies with independent epitopes for cTnI. The normal range of serum troponin concentration is <0.2 ng/ml.
Statistical analysis. Data are expressed as mean s.e.m. A Student's t-test was used for comparisons of two groups and an analysis of variance (ANOVA) for comparison of three groups.
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Acknowledgments We thank R. Myers and D. Rappaport for providing equipment and advice for cryosections, immunostaining and imaging; J. Chen and S. Evans for helpful discussions; A. Henke for the anti-CVB3 antibody; and P.R. Clemens and C.C. Lee for providing the mouse dystrophin expression vector. This work was supported in part by a US National Institutes of Health grant (5 R01 HL57365-03) and an American Heart Association Established Investigator Award (to K.U.K.); by Our Lady of Mercy Hospital, Catholic University of Korea (to G.H.L.); and by a training grant from the Deutsche Forschungsgemeinschaft (Ba 1668/1-1) (to C.B.).
Competing interests statement:
The authors declare that they have no competing financial interests.
m). c, Quantification of sarcolemmal disruption. Disruption of cardiomyocyte sarcolemmal membrane integrity was significantly greater in C57mdx/C3H mice (
) than in C57wt/C3H (l) at days 2 (n = 3), 4 (n = 3) and 6 (n = 6) after infection (*, P < 0.05). d, Serum concentration of cardiac troponin I (cTnI) after infection with CVB3. Concentrations of cTnI were significantly higher in C57mdx/C3H than C57wt/C3H mice (*, P < 0.05, n = 4). e, H&E staining of the heart from CVB3 infected C57wt/C3H mice (top) and C57mdx/C3H mice (bottom) (scale bar, 100 
) resulted in a significantly smaller viral-mediated cytopathic effect at 6 h, 8 h and 10 h after infection as compared with control (
). This effect was greater in cells that expressed Dys(mut) (
). *, P < 0.05 as compared to control cells; †, P < 0.05 Dys(wt) as compared to Dys(mut). c, Photomicrographs of GFP-positive cells described in (b) were obtained at 0 h (left) and 10 h (right) after infection. The cytopathic effect, as assessed on the basis of the number of GFP-positive cells and the overall shape of the cells, was markedly less in cells that expressed either mutant or wild-type dystrophin than in cells transfected with empty vector (control). d, The ratio of viral titers in the culture medium to titers in the cell was markedly higher in cells that did not express dystrophin (control). As compared to Dys(wt), expression of Dys(mut) further prevented the release of virus (n = 3; *, P < 0.05 compared with control cells; †, P < 0.05 Dys(wt) as compared to Dys(mut)). Scale bars, 100 
104 plaque-forming units of CVB3 at 4 weeks of age. They were subsequently injected with Evans blue dye 12 h before they were killed. Mouse hearts were analyzed at days 2, 4 and 6 after infection. All animal protocols were approved by the UCSD Animal Subjects Committee.
s.e.m. A Student's t-test was used for comparisons of two groups and an analysis of variance (ANOVA) for comparison of three groups.
