Dean J. Burkin

Severe muscular dystrophy in mice that lack dystrophin and α7 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 α7β1 integrin is a second transmembrane laminin receptor expressed in skeletal muscle. Mutations in the α7 integrin gene cause congenital myopathy in humans and mice. The α7β1 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 α7β1 integrin have complementary functional and structural roles. To test this hypothesis, we generated mice lacking both dystrophin and α7 integrin (mdx/α7-/-). The mdx/α7-/- mice developed early-onset muscular dystrophy and died at 2-4 weeks of age. Muscle fibers from mdx/α7-/- 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 α7-integrin null animals. In addition, loss of dystrophin and/or α7 integrin resulted in altered expression of laminin-α2 chain. These results point to complementary roles for dystrophin and α7β1 integrin in maintaining the 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 α7β1-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 α7-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate α7-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for α7-integrin expression. We show that laminin-111 (α1, β1, γ1), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased α7-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of α7-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.

Role for the α7β1 integrin in vascular development and integrity

The α7β1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the α7β1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the α7 integrin in these tissues during embryonic development, α7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the α7 promoter is reported by β‐galactosidase. From embryonic day (ED) 11.5 to ED14.5, β‐galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the α7 integrin gene resulted in partial embryonic lethality. Several α7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The α7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of α5 and α6B integrin chains was detected in the cerebral arteries of α7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the α7β1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity. Developmental Dynamics 234:11–21, 2005.

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, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, 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.

Laminin-111 Restores Regenerative Capacity in a Mouse Model for α7 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.

Valproic Acid Activates the PI3K/Akt/mTOR Pathway in Muscle and Ameliorates Pathology in a Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a lethal neuromuscular disease that currently has no effective therapy. Transgenic overexpression of the alpha7 integrin in mdx/utrn(-/-) mice, a model of Duchenne muscular dystrophy ameliorates the disease. We have isolated and used alpha7(+/-) muscle cells expressing beta-galactosidase, driven by the endogenous alpha7 promoter, to identify compounds that increase alpha7 integrin levels. Valproic acid (VPA) was found to enhance alpha7 integrin levels, induce muscle hypertrophy, and inhibit apoptosis in myotubes by activating the Akt/mTOR/p70S6K pathway. This activation of the Akt pathway occurs within 1 hour of treatment and is mediated by phosphatidylinositol 3-OH kinase. To evaluate the potential use of VPA to treat muscular dystrophy, mdx/utrn(-/-) mice were injected with the drug. Treatment with VPA lowered collagen content and fibrosis, and decreased hind limb contractures. VPA-treated mice also had increased sarcolemmal integrity and decreased damage, decreased CD8-positive inflammatory cells, and higher levels of activated Akt in their muscles. Thus, VPA has important biological effects that may be applicable for the treatment of muscular dystrophy.

Exercise promotes α7 integrin gene transcription and protection of skeletal muscle

The alpha7beta1 integrin is increased in skeletal muscle in response to injury-producing exercise, and transgenic overexpression of this integrin in mice protects against exercise-induced muscle damage. The present study investigates whether the increase in the alpha7beta1 integrin observed in wild-type mice in response to exercise is due to transcriptional regulation and examines whether mobilization of the integrin at the myotendinous junction (MTJ) is a key determinant in its protection against damage. A single bout of downhill running exercise selectively increased transcription of the alpha7 integrin gene in 5-wk-old wild-type mice 3 h postexercise, and an increased alpha7 chain was detected in muscle sarcolemma adjacent to tendinous tissue immediately following exercise. The alpha7B, but not alpha7A isoform, was found concentrated and colocalized with tenascin-C in muscle fibers lining the MTJ. To further validate the importance of the integrin in the protection against muscle damage following exercise, muscle injury was quantified in alpha7(-/-) mice. Muscle damage was extensive in alpha7(-/-) mice in response to both a single and repeated bouts of exercise and was largely restricted to areas of high MTJ concentration and high mechanical force near the Achilles tendon. These results suggest that exercise-induced muscle injury selectively increases transcription of the alpha7 integrin gene and promotes a rapid change in the alpha7beta integrin at the MTJ. These combined molecular and cellular alterations are likely responsible for integrin-mediated attenuation of exercise-induced muscle damage.

Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice

Native volume-sensitive outwardly rectifying anion channels (VSOACs) play a significant role in cell volume homeostasis in mammalian cells. However, the molecular correlate of VSOACs has been elusive to identify. The short isoform of ClC-3 (sClC-3) is a member of the mammalian ClC gene family and has been proposed to be a molecular candidate for VSOACs in cardiac myocytes and vascular smooth muscle cells. To directly test this hypothesis, and assess the physiological role of ClC-3 in cardiac function, we generated a novel line of cardiac-specific inducible ClC-3 knock-out mice. These transgenic mice were maintained on a doxycycline diet to preserve ClC-3 expression; removal of doxycycline activates Cre recombinase to inactivate the Clcn3 gene. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the knock-out mice at both 1.5 and 3 weeks off doxycycline. In mice off doxycycline, time-dependent inactivation of ClC-3 gene expression was confirmed in atrial and ventricular cells by qRT-PCR and Western blot analysis. Electrophysiological examination of native VSOACs in isolated atrial and ventricular myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, VSOAC current densities were significantly reduced, compared to age-matched control mice maintained on doxycycline. These results indicate that ClC-3 is a key component of native VSOACs in mammalian heart and plays a significant cardioprotective role against cardiac hypertrophy and failure.

Loss of the α7 Integrin Promotes Extracellular Signal-Regulated Kinase Activation and Altered Vascular Remodeling

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the &agr;7&bgr;1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the &agr;7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the &agr;7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in &agr;7 integrin–deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to &agr;7 integrin–null cells in a manner dependent on Ras signaling. &agr;7 Integrin–null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the &agr;7&bgr;1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.

Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability in the dyW 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 α7β1 integrin; however, it is unclear how the secondary loss of α7β1 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 dyW−/− mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7β1 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 dyW−/− 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.