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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.

Voltage-gated T-type Ca(2+) channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the alpha(1H) voltage-gated T-type Ca(2+) channel (Ca(v)3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload-induced hypertrophy was severely suppressed in mice deficient for Ca(v)3.2 (Ca(v)3.2(-/-)) but not in mice deficient for Ca(v)3.1 (Ca(v)3.1(-/-)). Angiotensin II-induced cardiac hypertrophy was also suppressed in Ca(v)3.2(-/-) mice. Consistent with these findings, cultured neonatal myocytes isolated from Ca(v)3.2(-/-) mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Ca(v)3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Ca(v)3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Ca(v)3.2(-/-), NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Ca(v)3.2(-/-)/NFAT-Luc mice. Our results provide strong genetic evidence that Ca(v)3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy.

The involvement of the sarcoglycan complex in the pathogenesis of muscular dystrophy is becoming increasingly clear. Sarcoglycan gene mutations lead to four forms of autosomal recessive limb-girdle muscular dystrophy. Recent progress has been made with the identification of novel mutations and their correlations with disease. Through this research, a better understanding the molecular pathogenesis of limb-girdle muscular dystrophy has been gained. Finally, animal models are now being used to study viral-mediated gene transfer for the future treatment of this disease.

Primary adhalin (or alpha-sarcoglycan) deficiency due to a defect of the adhalin gene localized on chromosome 17q21 causes an autosomal recessive myopathy. We evaluated 20 patients from 15 families (12 from Europe and three from North Africa) with a primary adhalin deficiency with two objectives: characterization of the clinical phenotype and analysis of the correlation with the level of adhalin expression and the type of gene mutation. Age at onset and severity of the myopathy were heterogeneous: six patients were wheel-chair bound before 15 years of age, whereas five other patients had mild disease with preserved ambulation in adulthood. The clinical pattern was similar in all the patients with symmetric characteristic involvement of trunk and limb muscles, calf hypertrophy, and absence of cardiac dysfunction. Immunofluorescence and immunoblot studies of muscle biopsy specimens showed a large variation in the expression of adhalin. The degree of adhalin deficiency was fairly correlated with the clinical severity. There were 15 different mutations (10 missense, five null). Double null mutations (three patients) were associated with severe myopathy, but in the other cases (null/missense and double missense) there was a large variation in the severity of the disease.

Efforts to understand the function of dystrophin, the protein product for the Duchenne muscular dystrophy gene, resulted in the purification of the dystrophin-glycoprotein complex. Over the past year several novel components of this complex have been identified. Recent studies have extended the number of muscular dystrophies associated with the oligomeric complex to six genetically distinct diseases, including three new forms of limb-girdle muscular dystrophy and one form of congenital muscular dystrophy.

Congenital muscular dystrophies (CMDs) are autosomal recessive, heterogeneous disorders. The most frequent form in the Caucasian population is classic (occidental) CMD, characterized by exclusive muscle involvement, although abnormal brain white matter signals are occasionally observed on MRI. Recently, deficiency of merosin, the laminin isoform in skeletal muscle, has been identified in classic CMD patients. In skeletal muscle, merosin is a native ligand for dystroglycan linking the extracellular matrix and dystrophin. Thus, merosin deficiency could disrupt the attachment of muscle cell to the extracellular matrix and lead to muscle cell necrosis. Since merosin is also expressed in the nervous system and has biologic activities on neurite outgrowth and Schwann cell migration, deficiency of merosin could affect the development of the nervous system. We report here two patients with merosin-negative CMD presenting extensive brain abnormalities characterized by cortical anomaly, polymicrogyria, and abnormal white matter signals.

Recent molecular and biochemical studies have disclosed the detailed molecular organization of the dystrophin-glycoprotein complex, which links the cytoskeleton to the extracellular matrix. Defects in several components of this complex cause different types of muscular dystrophy. This glycoprotein complex is also involved in clustering and anchoring acetylcholine receptors at the postsynaptic membrane.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is a major component of the subsarcolemmal cytoskeleton and exists in a large oligomeric complex tightly associated with several sarcolemmal glycoproteins which provide a linkage to the extracellular matrix protein, laminin. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from 17 DMD patients of various ages. The results revealed a dramatic reduction in all of the dystrophin-associated proteins in the sarcolemma of DMD muscle compared with normal muscle and muscle from a variety of other neuromuscular diseases. This abnormality was common in all 17 DMD patients, irrespective of age. Our results indicate that the absence of dystrophin leads to the loss in all of the dystrophin-associated proteins, which renders DMD muscle fibers susceptible to necrosis. The analysis of dystrophin-associated proteins is important in the assessment of experimental therapies that attempt to replace dystrophin in DMD muscle.

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle.

Calsequestrin is a calcium-binding protein known to sequester calcium accumulated in the sarcoplasmic reticulum (SR) of muscle cells during relaxation. In the present study, we used affinity-purified antibodies to chicken cardiac calsequestrin to identify a 60,000-Da calsequestrin in frog myocardium. Like previously identified cardiac calsequestrins, it is enriched in cardiac microsomes, it is enriched by biochemical procedures previously used to purify cardiac and skeletal calsequestrins, and it exhibits a pH-dependent shift in its apparent Mr on a two-dimensional gel system. Finally, the NH2-terminal amino acid sequence of this 60,000-Da immunoreactive protein purified by fast protein liquid chromatography was identical to that of rabbit skeletal and canine cardiac calsequestrin. Thus, we conclude that this protein corresponds to the calsequestrin isoform in frog ventricular muscle. Frog calsequestrin was localized in discrete foci present at the periphery but absent from the central regions of frog ventricular myocytes as determined by immunofluorescence labeling. Immunoelectron microscopic labeling demonstrated that calsequestrin was confined to the lumen of two structurally distinct regions of the SR, where it was localized in the subsarcolemmal region of the myofibers. One of these appeared to correspond to the terminal SR previously reported to be closely apposed to the sarcolemma of frog myofibers. The other region, although close to the sarcolemma, was not physically joined to it and appeared to correspond to corbular SR. It generally is believed that frog cardiac SR does not provide activator Ca2+ required for excitation-contraction coupling. However, the identification of a calsequestrin isoform very similar to mammalian cardiac calsequestrin that is confined to specialized regions of frog cardiac SR lends support to the idea that frog cardiac SR has the ability to store Ca2+ and thus function in some capacity in frog cardiac muscle contraction.

The nitrendipine receptor of the voltage-dependent Ca2+ channel purified from rabbit skeletal muscle has been shown to contain four polypeptide components of 175,000, 170,000, 52,000, and 32,000 daltons. Despite the existence of a substantial amount of data on the composition of the nitrendipine receptor, little is known about the relationship between the 175,000 and 170,000 dalton subunits of the receptor and the lower molecular weight components of the receptor. A monoclonal antibody specific to the 52,000 dalton component of the receptor has now been produced. The monoclonal antibody is capable of specifically immunoprecipitating the [3H]dihydropyridine-labeled nitrendipine receptor from detergent-solubilized membranes. Immunoprecipitation experiments with 32P-labeled nitrendipine receptor have demonstrated a tight association between the 170,000 dalton nitrendipine binding subunit and the 52,000 dalton polypeptide of the receptor. Immunoblotting experiments have shown that the 52,000 dalton polypeptide copurifies with the 175,000 and 170,000 dalton subunits of the nitrendipine receptor at all stages of the purification. In addition, the higher molecular weight subunits of the receptor were not labeled by the antibody. Densitometric scanning of Coomassie blue stained SDS-polyacrylamide gels of the purified nitrendipine receptor has shown that the 175,000, 170,000, 52,000, and 32,000 dalton subunits of the nitrendipine receptor exists in a 1:1:1:1 stoichiometric ratio. In conclusion, we have demonstrated that the 52,000 dalton polypeptide is an integral and distinct subunit of the purified nitrendipine receptor of the voltage-dependent Ca2+ channel.

High-affinity antibodies specific for the 1,4-dihydropyridine Ca2+ channel blockers have been produced in sheep and affinity purified using a dihydropyridine-Sepharose affinity column. Dihydropyridine-Sepharose affinity matrix was synthesized by reaction of aminohexyl-Sepharose with an affinity analogue of nifedipine, dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridine-dicarbo xylate. Residual amine groups were then blocked by carbodiimide-catalyzed acetylation. [3H]Nitrendipine-binding activity in serum was specifically absorbed by the dihydropyridine-Sepharose affinity column. The bound antibody was eluted with diethylamine (pH 11.5) in 10% dioxane or with a low-affinity dihydropyridine ligand (diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate), pH 7.4. Thirty-six milligrams of highly pure IgG antibody, as demonstrated by sodium dodecyl sulfate-gel electrophoresis, was isolated from 50 ml hyperimmune sheep serum. The affinity-purified anti-dihydropyridine antibodies have been shown to have high affinity (Kd approximately 0.1 nM) and specificity for the 1,4-dihydropyridine Ca2+ channel blockers and, therefore, exhibit dihydropyridine-binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+ channel blockers. Immunoblot staining of an azidopine-bovine serum albumin conjugate with affinity-purified antidihydropyridine antibodies demonstrated that the anti-dihydropyridine antibodies recognize the 1,4-dihydropyridine Ca2+ channel blockers when covalently coupled to protein and, therefore, should be useful in the identification and purification of receptors covalently labelled with 1,4-dihydropyridine Ca2+ channel blockers.

Localization of calsequestrin in sheep Purkinje fibers was determined by indirect immunofluorescence labeling of cryostat sections of sheep myocardium from the intraventricular wall. The results presented show that calsequestrin is present in discrete foci at the peripheral, as well as the interior regions of the cytoplasm. Since Purkinje fibers lack transverse tubules, the presence of calsequestrin at specific foci in the interior regions of the cytoplasm in these cells suggests that calsequestrin is localized in the lumen of peripheral junctional sarcoplasmic reticulum, as well as in the lumen of corbular sarcoplasmic reticulum present in the I band region of the myofibrils. Assuming that the function of calsequestrin is to sequester calcium into the lumen of the sarcoplasmic reticulum, these results imply that two structurally different regions of the sarcoplasmic reticulum function as calcium storage sites in mammalian Purkinje fibers and raises the possibility that calcium storage and/or release from these two sites might be regulated differently.