Laminin-111 protein therapy reduces muscle pathology and improves viability of a mouse model of merosin-deficient congenital muscular dystrophy.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, ß1, and γ1) and laminin-221 (ie, α2, ß2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, ß1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A.

Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability in the dy(W) mouse model of merosin-deficient congenital muscular dystrophy type 1A.

Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is a devastating neuromuscular disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. MDC1A is caused by mutations in the LAMA2 gene, which results in the partial or complete loss of laminin-211 and laminin-221, the major laminin isoforms found in the basal lamina of skeletal muscle. MDC1A patients exhibit reduced α7ß1 integrin; however, it is unclear how the secondary loss of α7ß1 integrin contributes to MDC1A disease progression. To investigate whether restoring α7 integrin expression can alleviate the myopathic phenotype observed in MDC1A, we produced transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dy(W⁻/⁻) mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7ß1 integrin to laminin-α2-deficient myofibers, changed the composition of the muscle extracellular matrix, reduced muscle pathology, maintained muscle strength and function and improved the life expectancy of dy(W⁻/⁻) mice. Taken together, these results indicate that enhanced expression of α7 integrin prevents muscle disease progression through augmentation and/or stabilization of the existing extracellular matrix in laminin-α2-deficient mice, and strategies that increase α7 integrin in muscle might provide an innovative approach for the treatment of MDC1A.

Myotendinous junction defects and reduced force transmission in mice that lack alpha7 integrin and utrophin.

The alpha7beta1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of alpha7beta1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and alpha7 integrin (mdx/alpha7(-/-)), or dystrophin and utrophin (mdx/utr(-/-)), exhibit severe muscle pathology and die prematurely. To explore the contribution of the alpha7beta1 integrin and utrophin to muscle integrity and function, we generated mice lacking both alpha7 integrin and utrophin. Surprisingly, mice that lack both alpha7 integrin and utrophin (alpha7/utr(-/-)) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to alpha7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in alpha7/utr(-/-) skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The alpha7/utr(-/-) mouse, therefore, demonstrates the critical roles of alpha7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the alpha7beta1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.

Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The alpha(7)beta(1)-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the alpha(7)-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate alpha(7)-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for alpha(7)-integrin expression. We show that laminin-111 (alpha(1), beta(1), gamma(1)), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased alpha(7)-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of alpha(7)-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.

Laminin-111 restores regenerative capacity in a mouse model for alpha7 integrin congenital myopathy.

Mutations in the alpha7 integrin gene cause congenital myopathy characterized by delayed developmental milestones and impaired mobility. Previous studies in dystrophic mice suggest the alpha7beta1 integrin may be critical for muscle repair. To investigate the role that alpha7beta1 integrin plays in muscle regeneration, cardiotoxin was used to induce damage in the tibialis anterior muscle of alpha7 integrin-null mice. Unlike wild-type muscle, which responded rapidly to repair damaged myofibers, alpha7 integrin-deficient muscle exhibited defective regeneration. Analysis of Pax7 and MyoD expression revealed a profound delay in satellite cell activation after cardiotoxin treatment in alpha7 integrin-null animals when compared with wild type. We have recently demonstrated that the muscle of alpha7 integrin-null mice exhibits reduced laminin-alpha2 expression. To test the hypothesis that loss of laminin contributes to the defective muscle regeneration phenotype observed in alpha7 integrin-null mice, mouse laminin-111 (alpha1, beta1, gamma1) protein was injected into the tibialis anterior muscle 3 days before cardiotoxin-induced injury. The injected laminin-111 protein infiltrated the entire muscle and restored myogenic repair and muscle regeneration in alpha7 integrin-null muscle to wild-type levels. Our data demonstrate a critical role for a laminin-rich microenvironment in muscle repair and suggest laminin- 111 protein may serve as an unexpected and novel therapeutic agent for patients with congenital myopathies.

Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.

The dystrophin glycoprotein complex links laminin in the extracellular matrix to the cell cytoskeleton. Loss of dystrophin causes Duchenne muscular dystrophy, the most common human X-chromosome-linked genetic disease. The alpha7beta1 integrin is a second transmembrane laminin receptor expressed in skeletal muscle. Mutations in the alpha7 integrin gene cause congenital myopathy in humans and mice. The alpha7beta1 integrin is increased in the skeletal muscle of Duchenne muscular dystrophy patients and mdx mice. This observation has led to the suggestion that dystrophin and alpha7beta1 integrin have complementary functional and structural roles. To test this hypothesis, we generated mice lacking both dystrophin and alpha7 integrin (mdx/alpha7(-/-)). The mdx/alpha7(-/-) mice developed early-onset muscular dystrophy and died at 2-4 weeks of age. Muscle fibers from mdx/alpha7(-/-) mice exhibited extensive loss of membrane integrity, increased centrally located nuclei and inflammatory cell infiltrate, greater necrosis and increased muscle degeneration compared to mdx or alpha7-integrin null animals. In addition, loss of dystrophin and/or alpha7 integrin resulted in altered expression of laminin-alpha2 chain. These results point to complementary roles for dystrophin and alpha7beta1 integrin in maintaining the functional integrity of skeletal muscle.