Dystroglycan Matrix Receptor Function in Cardiac Myocytes Is Important for Limiting Activity-Induced Myocardial Damage
Rationale: Genetic mutations in a number of putative glycosyltransferases lead to the loss of glycosylation of dystroglycan and loss of its laminin-binding activity in genetic forms of human muscular dystrophy. Human patients and glycosylation defective myd mice develop cardiomyopathy with loss of dystroglycan matrix receptor function in both striated and smooth muscle.
Objective: To determine the functional role of dystroglycan in cardiac muscle and smooth muscle in the development of cardiomyopathy in muscular dystrophies.
Methods and Results: Using cre/lox–mediated gene targeting, we show here that loss of dystroglycan function in ventricular cardiac myocytes is sufficient to induce a progressive cardiomyopathy in mice characterized by focal cardiac fibrosis, increase in cardiac mass, and dilatation ultimately leading to heart failure. In contrast, disruption of dystroglycan in smooth muscle is not sufficient to induce cardiomyopathy. The specific loss of dystroglycan function in cardiac myocytes causes the accumulation of large, clustered patches of myocytes with membrane damage, which increase in number in response to exercise-induced cardiac stress, whereas exercised mice with normal dystroglycan expression accumulate membrane damage limited to individual myocytes.
Conclusions: Our findings suggest dystroglycan function as an extracellular matrix receptor in cardiac myocytes plays a primary role in limiting myocardial damage from spreading to neighboring cardiac myocytes, and loss of dystroglycan matrix receptor function in cardiac muscle cells is likely important in the development of cardiomyopathy in glycosylation-deficient muscular dystrophies.
Clinically defined cardiovascular disease occurs in more than 70% to 90% of muscular dystrophy patients with defects in the dystrophin–glycoprotein complex (DGC), and heart failure is the second leading cause of death in these patients.1 The DGC contains a key transmembrane glycoprotein, dystroglycan (DG), which is believed to link the intracellular protein dystrophin to the extracellular matrix. DG is composed of α and β subunits. β-DG binds intracellularly to dystrophin, which binds the actin cytoskeleton, and binds extracellularly to α-DG. The heavily glycosylated α-DG binds tightly to extracellular matrix proteins, such as laminin, with high affinity. In muscle cells, the DGC also contains a sarcoglycan–sarcospan complex composed of sarcoglycan proteins (α, β, γ, and δ in striated muscle) and sarcospan. Whereas mutations in dystrophin and each of the four sarcoglycans can cause Duchenne/Becker and limb girdle muscular dystrophies respectively, no mutations in DG have been identified as a cause of human disease.
The distribution of DG and the DGC in both cardiac and vascular smooth muscle have raised questions about whether the cardiomyopathy present in muscular dystrophy is primary to cardiac muscle dysfunction or might be a consequence of changes in vascular function. Sarcoglycan-deficient mice and hamsters show coronary vasospasm,2–4 and coronary dysfunction has been suggested to be the primary cause of cardiomyopathy.2,3 However, it is possible coronary vasospasm or peripheral vasoconstriction could be secondary to the release of factors by diseased cardiac myocytes or skeletal muscle cells.5,6 In support of this hypothesis, transgenic overexpression of δ-sarcoglycan in cardiac myocytes of the δ-sarcoglycan knockout mouse appears to slow or prevent histological evidence of cardiomyopathy.7 The fact that mutations in δ-sarcoglycan can cause dilated cardiomyopathy without skeletal muscle disease,8 and the presence of an alternative sarcoglycan complex in smooth muscle9 suggests sarcoglycans may have a unique function in the cardiovascular system, acting through a mechanism that is distinct from the function of DG and dystrophin glycoprotein complex function.
Although DG mutations have not been linked to human disease, mutation in genes including POMGnT-1, POMT1, POMT1, FKTN, LARGE, and FKRP can cause a broad spectrum of disease severity, ranging from severe congenital muscular dystrophy, such as Walker–Warburg syndrome, to mild LGMD, such as LGMD2I. The proteins encoded by these genes all have homology to glycosyltransferases and mutations in these genes lead to a convergent loss of glycosylation of α-DG and a loss of DG function as a receptor for its known extracellular matrix ligands.10 Myodystrophy (myd) mice, resulting from mutations in LARGE, demonstrate hypoglycosylation of α-DG and abnormal DG ligand binding in skeletal muscle, which is biochemically similar to that of human muscular dystrophy patients with glycosylation-deficient muscular dystrophy.10,11 LARGE is believed to be a “master” regulator of the glycosylation pathway necessary for DG glycosylation and function.12 Studies have shown that myd mice develop histological evidence of cardiomyopathy and a loss of glycosylated DG in the heart, similar to the cardiomyopathy and glycosylation deficiency observed in human patients.13,14 The molecular mechanisms that lead to cardiomyopathy in these disorders are unknown.
In the present study, we show that glycosylation-deficient myd mice have impaired DG glycosylation in smooth muscle, including coronary blood vessels, and a quantitatively comparable loss of high-affinity laminin-binding activity in both cardiac and smooth muscle. To address the significance of loss of DG function with respect to the cardiomyopathy in glycosylation-deficient muscular dystrophies, we used cre/lox technology to disrupt the DG gene in the cardiovascular system in a tissue specific manner. Our results indicate that gene-targeted loss of DG function specifically in cardiac myocytes is sufficient to cause cardiomyopathy in mice. Moreover, in actively contracting myocardium and after increased cardiac stress induced by exercise, DG appears to play an important role in limiting cardiac myocyte membrane damage to individual cells, and loss of its function as an extracellular matrix receptor in mice results in expansion of myocardial damage to nearest neighboring cells.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
B6C3Fe-a/a-LARGEmyd mice were obtained from The Jackson Laboratories. Mice expressing Cre recombinase under control of the myosin light chain 2v regulatory region (MLC2vcre)15 or using the smooth muscle myosin heavy chain (MHC) promoter16 were kind gifts from Dr Kenneth Chien and Dr Gary Owens, respectively. Mice deficient for DAG1 were generated by mating male mice heterozygous for the cre transgene and homozygous for the DAG1 floxed allele, with females that were homozygous for the DAG1 floxed allele.17 The mice from these crosses were born in the predicted Mendelian ratios (see Supplemental Methods). All comparisons were made on age- and sex-matched littermate mice. All procedures were approved by the University Committee for the Use and Care of Animals.
DG Glycosylation, DGC Protein Expression, and Laminin-Binding Activity
DG glycosylation, protein expression, immunolocalization, and laminin-binding activity were analyzed in wheat germ agglutinin–enriched glycoprotein preparations as previously described.10
In Vivo Echocardiography
Mice were analyzed by in vivo echocardiography while conscious as previously described.18
In Vivo Assessment of Myocardial Membrane Damage
Five hours before a 1-hour graded treadmill exercise session, mice (<5 months of age) were given an IP injection of 0.1 mg/g Evan’s blue dye (EBD) in saline. Eighteen hours after injection, with or without exercise, hearts were removed, and 4 cryosections >250 to 300 μm apart were obtained. Immunostaining with anti–laminin 1 antibody (Sigma, 1:1000) was used to show cell boundaries and identify cells with dye uptake.
Glycosylation-Deficient Muscular Dystrophy Mice Show Loss of DG Glycosylation in Both Cardiac and Smooth Muscle
Hearts from myd mice at 10 months of age displayed evidence of significant but focal interstitial myocardial collagen deposition, consistent with cardiac remodeling that occurs following myocardial damage (Figure 1A). To address how the loss of LARGE activity affects DG glycosylation in myd mice across tissues, the apparent molecular weight shifts of α-DG protein from tissues of wild-type (WT) and myd mice were compared using an antibody against the core α-DG protein. In addition to skeletal muscle and brain as previously reported,10 both cardiac muscle and smooth muscle α-DG from myd mice (using bladder as a source of tissue composed primarily of smooth muscle), were also markedly reduced in molecular weight compared to their littermate WT controls (Figure 1B and 1D). In contrast, other tissues such as kidney, lung, testes, spleen, and liver did not show as large a shift in molecular weight of α-DG, although more subtle shifts were still observed in the absence of LARGE activity (Online Figure I). By comparing the amounts of IIH6-reactive α-DG to the amount of α-DG recognized by the core α-DG antibody, levels of the highly glycosylated form of α-DG (recognized by IIH6) were high in WT skeletal muscle, brain, heart, smooth muscle, and kidney but barely detectable in lung, testes, spleen and liver (Figure 1B and 1C; Online Figure I). The reactivity of IIH6 antibody was tightly correlated with laminin-binding activity of α-DG detected by laminin overlay assay (Figure 1C and Online Figure I). In each adult tissue, where the IIH6-reactive, laminin-binding form of α-DG was highly expressed in WT mice, the IIH6 reactive α-DG and laminin-binding activity by overlay assay was undetectable in samples from myd mice. Loss of α-DG glycosylation did not appear to markedly impact the expression levels of the DG peptide in these tissues. Staining of tissues from myd mice, including the myocardium, demonstrate that the glycosylated form of α-DG is lost from all tested vascular smooth muscle including coronary blood vessels (Figure 1E). This loss of DG glycosylation results in a significant loss of total high-affinity laminin-binding activity in skeletal, cardiac and smooth muscle, and brain (Figure 1F). Together, these results demonstrate that LARGE activity is necessary for the type of DG glycosylation that is required for its function as an extracellular matrix receptor in: striated muscle (including the heart); smooth muscle; the nervous system; and, to a lesser extent, the kidney.
Genetic Disruption of the DG Gene in the Cardiac Myocytes or Smooth Muscle Leads to a Tissue-Specific Loss of DG Expression
To determine the significance that the loss of DG function in cardiac or vascular smooth muscle has for the development of cardiomyopathy, mice bearing an allele of the DG gene (DAG1) flanked by loxP sites (L/L) were crossed with mice that express cre-recombinase in either cardiac ventricular muscle (MLC2vcre-L/L) or in all smooth muscle (SMMHCcre-L/L). In MLC2vcre-L/L mice, there was loss of DG expression in nearly all ventricular cardiac myocytes, whereas coronary vascular smooth muscle expression of DG was maintained (Figure 2A). A small percentage of cardiac myocytes (21 of 2350 myocytes in random fields from 3 animals) showed residual DG expression (Figure 2A). Conversely, in the SMMHCcre-L/L mice showed complete loss of DG staining in vascular smooth muscle in coronary vessels, the aorta (although largely collagen-based tunica externa remained positive), and bladder smooth muscle (Figure 2A; Online Figure II). Whereas nearly all ventricular myocytes in the SMMHCcre-L/L mice exhibited normal DG expression, a small subset of ventricular myocytes in this mouse was targeted by this promoter (Figure 2A). These targeted myocytes were observed in sample sections of 2 of 4 SMMHCcre-L/L hearts examined, but only 18 and 27 myocytes were disrupted in an entire cross section of the heart in those samples. This is likely attributable to the short-lived, developmental expression of the smooth muscle MHC gene in a subset of myocytes that has been characterized previously.16
To determine how loss of DG protein expression impacted the expression of the DGC in cardiac muscle, DGC protein expression in ventricles of MLC2vcre-L/L mice was examined (Figure 2B). DG expression was reduced 93.8±1.5% (means±SEM, n=4, P<0.05). Somewhat surprisingly, the loss of DG protein expression in cardiac myocytes by gene targeting failed to cause a corresponding marked reduction in dystrophin expression, and the expressed dystrophin was retained at the sarcolemma (Figure 2C), even though DG is a direct binding partner of dystrophin in the DGC. In contrast, sarcoglycan expression levels were reduced to a much greater extent than the dystrophin expression levels, although not lost completely in MLC2vcre-L/L mice. These results suggest that dystrophin may interact with other proteins in cardiac muscle cells to confer dystrophin protein stability. There was also a slight increase in dysferlin expression (37% increase, P<0.05), but no change in α7 or β1 integrin expression (P>0.05), as measured by densitometry, in MLC2vcre-L/L animals. In SMMHCcre-L/L mice, immunofluorescence staining also revealed a reduction of β-sarcoglycan immunofluorescence staining in vascular smooth muscle and bladder smooth muscle accompanying loss of DG expression in SMMHCcre-L/L mice (Online Figure II).
Tissue-Specific Loss of DG Expression in Cardiac Myocytes Is Sufficient to Cause a Cardiomyopathic Phenotype in Mice
Ten-month-old MLC2vcre L/L mice showed marked cardiac pathology, including myocardial cell degeneration marked by eosinophilic myocytes, infiltration of mononuclear cells in a subset of cases with severe pathology, and significant focal collagen deposition (Figure 3A). All hearts from MLC2vcre-L/L mice examined appeared enlarged in cross section and a subset showed focal thinning of the left ventricular wall. Consistent with these histological observations, MLC2vcre-L/L mice showed a 65% increase in heart weight (HW)/body weight (BW) ratios compared to L/L mice (Figure 3B) and a significant increase in cardiac fibrosis, with nearly 9% of the myocardium being replaced with focal fibrotic tissue in 10-month-old mice (Figure 3C). Despite the focal nature of the fibrosis, microfil perfusion experiments did not reveal evidence of coronary vasospasms (Online Figure III). SMMHCcre-L/L mice at 10 months of age failed to show alterations in cardiac mass or any pathology by histological staining (Figure 3).
Consistent with the morphological findings, murine echocardiography showed the cardiac mass and end diastolic volume were significantly increased in MLC2vcre-L/L mice (Figure 4). The ratio of the end diastolic volume to mass was also increased, confirming these mice experienced pathological chamber dilation as opposed to a mere increase in overall heart size. MLC2vcre-L/L mice also showed a significant impairment of systolic function, as evidenced by a reduction in the left ventricular ejection fraction. In contrast, the loss of DG function in smooth muscle had no measureable impact on cardiac function as measured by echocardiography. Together, these echocardiography findings support the conclusion that the loss of DG function in cardiac myocytes is necessary and sufficient to cause a functional dilated cardiomyopathy.
Examining mice at several ages indicated that the first detectable signs of cardiomyopathy by histology and pathological markers in MLC2vcre-L/L mice occurs around seven moths of age. MLC2vcre-L/L mice at 3 months of age appear histologically and morphologically normal (Figure 5). MLC2vcre-L/L mice at 7 months begin to show small focal areas of fibrosis and a small increase in HW/BW ratio. Furthermore, MLC2vcre-L/L mice at 7 months of age show significant activation of β-MHC gene expression, which as important molecular marker of cardiomyopathic remodeling associated with both cardiac hypertrophy and fibrosis,19 but mice at 3 months of age showed no activation of β-MHC or ANF (Figure 5). These data suggest that cardiomyopathy develops progressively in adult animals because of cumulative effects of lifetime loss of DG in cardiac myocytes, rather than a congenital defect in young animals.
DG Expression in Cardiac Myocytes Is Necessary for Limiting Stress-Induced Cardiac Myocyte Damage From Spreading to Nearest Neighbor Cells
Because loss of DG expression in cardiac myocytes and loss of DG laminin-binding activity in myd mice is sufficient to cause a progressive cardiomyopathy characterized by focal fibrosis, we hypothesized that the function of DG as an extracellular matrix receptor in cardiac myocytes might play a primary role in preventing myocardial damage in young animals that accumulates with age, leading to cardiomyopathic remodeling. To test this hypothesis, young WT and MLC2vcre-L/L mice (<5 months old, before detectable fibrosis or cardiac histopathology) were examined for sarcolemma damage in cardiac myocytes, by EBD uptake in sedentary and acutely exercised animals. In WT, unexercised animals, very few EBD-positive cells were observed, with most sections devoid of any dye uptake. Interestingly, in WT animals, EBD-positive myocytes were consistently observed after exercise, but the sarcolemma damage was almost always limited to single isolated myocytes. In contrast, MLC2vcre-L/L animals had a markedly different pattern of EBD uptake both in exercised and unexercised mice, where EBD uptake seemed to occur in large groups of neighboring cells, or cell “patches”(Figure 6). We also noted the patches of membrane damage in MLC2vcre-L/L mice appeared to be limited to cardiac myocytes that were organized in similar orientations of their longitudinal, or mechanical axis (Figure 6). To examine this curious pattern of dye uptake in more detail, the total number of patches of damaged cells as small as one cell, and the size of those patches were quantified (Figure 7). These results showed that acute exercise was able to produce a significant number of EBD-positive cells in WT animals, but these patches were typically observed as one isolated cardiac myocyte (Figure 7). In contrast, loss of DG function resulted in slightly greater numbers of patches both in sedentary and exercised animals but, more significantly, a dramatic increase in the size of those patches up to patch sizes that exceeded 25 contacting cells in cross sections of myocardium from MLC2vcre-L/L mice (Figure 7). Note that the measurements of patch size were sampled in cross-sections, suggesting that these patches likely contain a significantly larger number of cells in 3 dimensions. Also, the greater number of patches observed in MLC2vcre-L/L animals could, in part, be attributable to the increased size of the patches increasing likelihood of observing a patch in a random section. The mean patch size in each genotype was not statistically different in control and exercised animals, indicating the size of the patch was not influenced by exercise stress. Taken together, these results suggest that cardiac membrane damage and uptake of EBD can occur even in WT mice in response to increased activity, but the primary role of DG in cardiac myocytes is to protect against the expansion of that initial myocyte damage to neighboring cardiac myocytes. Myocytes that take up EBD also take up large proteins from the extracellular space including immunoglobulin (Figure 8). By marking membrane damage with immunoglobulin, Figure 8 shows that sedentary myd mice also have large patches of neighboring myocytes with membrane damage similar to that observed in sedentary MLC2vcre-L/L animals. Therefore, loss of the function of DG as an extracellular matrix receptor in cardiac myocytes appears critical to preventing expansion of initial myocyte membrane damage to neighboring cardiac myocytes.
The mechanistic role of DG function in muscular dystrophy and cardiomyopathy, and the exact function of DG in striated muscle and smooth muscle, is still debated. Although cytoskeletal to matrix linkages through DG and other receptors have been proposed to be critical for muscle cell survival,20 the association of the dystrophin glycoprotein complex with signaling molecules such as kinase cascades and nitric oxide synthase suggest several possible significant roles in striated and vascular smooth muscle.21,22 Our results indicate that the loss of DG function in cardiac myocytes results in increased myocyte membrane damage in response to increased activity and is sufficient to cause a progressive dilated cardiomyopathy and that loss of DG function in smooth muscle does not cause a detectable cardiac phenotype.
Despite the uniform genetic deletion of DG function in myocytes (Figures 2 and 7⇑), the membrane damage observed in MLC2vcre-L/L mice is not uniform and occurs in distinct focal patches of adjacent myocytes, rather than just randomly membrane-damaged myocytes throughout the myocardium. Patchy areas of cell membrane damage were also observed in myd mice in the presence of uniform loss of DG laminin-binding function (Figures 1 and 8⇑). Previous studies in sarcoglycan deficient animals suggested the cardiomyopathy was correlated with coronary vasospasm and loss of sarcoglycan expression in smooth muscle.2,3 However, we did not observe evidence of coronary vasospasm by microfil perfusion in MLC2vcre-L/L mice, and DG and DGC expression in smooth muscle was normal in MLC2vcre-L/L mice. Furthermore, no patches of myocardial damage or fibrosis were observed in SMMHCcre-L/L mice. We also demonstrate that exercise can induce surprisingly significant numbers of EBD-positive cells in WT mice compared to unexercised WT mice, but the damage is always limited to 1 or 2 cells. Taken together, these data suggest that the interaction of DG with the extracellular matrix is required for stabilizing neighboring cells against sarcolemma injury and preventing expansion of membrane damage to neighboring cells in response to an individual cell becoming damaged. Because significant focal patches of membrane damage are observed in younger ML2vcre-L/L mice, even in the absence of exercise, the accumulation of focal myocardial damage even under sedentary conditions is likely playing a primary role in the development of DG-deficient cardiomyopathy and the development of focal myocardial fibrosis. The relatively slow progression of the cardiac disease in MLC2vcre-L/L mice may be attributable to many cells in the patches with membrane damage undergoing successful plasma membrane repair resulting in fewer cells with permanent damage.
In the working myocardium, cardiac myocytes are attached to neighboring cells by interacting laterally with matrix that separates adjacent cells and longitudinally through intercalated disks that mechanically and electrically connect neighboring cells. If isolated membrane damage in a single cell results in transient or permanent contractile failure, the surrounding myocytes may experience increased mechanical load. In the absence of stabilizing lateral interactions of those myocytes with extracellular matrix, this may result in inappropriate stretch or strain on neighboring myocytes that leads to damage of the sarcolemma. The localization of DG to costameres in striated muscle places DG at an important location for stabilizing myocytes, the sarcolemma, and their sarcomeres against mechanical activity or stretch. Dystrophin-deficient skeletal muscle has been shown to be particularly sensitive to the damaging effects of lengthening contractions.23 Other matrix receptors in muscle, such as integrins, may also provide a parallel pathway toward in stabilizing cardiac myocytes. The combined loss of dystrophin and integrin in myocytes results in a more severe histological phenotype than the loss of either gene alone, especially under conditions of stress such as isoproterenol infusion.24 It is possible that other mechanisms may be involved, including increased sensitivity of DG-deficient myocytes to circulating factors released by myocytes with membrane damage. We noted the patches of membrane damage also appeared to be limited to myocytes that were organized in similar orientations of their longitudinal axis (Figure 6), suggesting that the damage was related to orientation of the mechanical strain or stretch and not simply diffusion of molecules to neighboring cells.
A qualitative comparison of the histopathologic cardiac phenotypes of MLC2vcre-L/L mice (Figure 3), the phenotypes of myd mice (Figure 1 and Online Figure IV), and previous studies in dystrophin-deficient mdx mice suggests that MLC2vcre-L/L mice may have a more severe cardiac phenotype than both myd and mdx mice. This could be attributable to the fact that myd mice retain DG protein expression and preserve some of the intracellular functions of the DGC21,22 Alternatively, the lack of skeletal muscle disease in MLC2vcre mice may result in increased locomotor activity and added stress on the heart as compared to other dystrophic mouse models. Dystrophic mdx mice fatigue very quickly,22 and myd mice have observable reduced mobility and hindlimb paralysis caused by severe dystrophy and peripheral nerve dysfunction. Rescue of skeletal muscle disease in mdx mice by a skeletal muscle–specific minidystrophin transgene has been shown to lead to increased voluntary activity and a worse cardiac phenotype, possibly caused by increased stress placed on these hearts.25 Therefore, there may be a paradoxical inverse relationship between cardiac disease severity in dystrophic models and the good health of skeletal muscle. This would fit well with our observation that acute exercise appears to increase the number of patches of cell membrane damage in MLC2vcre mice. Although myd mice are a genetic model of the relatively rare congenital muscular dystrophy MDC1D, the loss of DG glycosylation and the function of DG as an extracellular matrix receptor is so far biochemically identical to many forms of glycosylation-deficient muscular dystrophy including the relatively common LGMD2I.10 Our results suggest that if a specific genetic disruption of this glycosylation pathway affected DG matrix receptor function in cardiac myocytes, this would likely lead to increased susceptibility to activity-induced myocardial damage and cardiovascular disease in human patients.
We thank Sally Prouty for technical assistance and the University of Iowa Hybridoma Facility for preparing the antibody IIH6. We thank the Nathan Shock Center In Vivo Functionality Core and Center for Integrative Genomics at the University of Michigan for support. We thank members of the Michele laboratory and Campbell laboratory for critical comments on the manuscript.
Sources of Funding
This work is funded by a Grant-in-Aid from the American Heart Association and NIH grant R01-HL080388 (to D.E.M.); an American Heart Association Postdoctoral Fellowship (to Z.K.); and NIH grant RR017369 (to R.M.W.). This work was supported in part by the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center grant 1 U54 NS053672. K.P.C. is an investigator of the Howard Hughes Medical Institute.
Original received April 20, 2009; revision received September 8, 2009; accepted September 10, 2009.
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