Daniel E. Michele

Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies

Muscle–eye–brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD) are congenital muscular dystrophies with associated, similar brain malformations. The FCMD gene, fukutin, shares some homology with fringe-like glycosyltransferases, and the MEB gene, POMGnT1, seems to be a new glycosyltransferase. Here we show, in both MEB and FCMD patients, that α-dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. We show that this post-translational biochemical and functional disruption of α-dystroglycan is recapitulated in the muscle and central nervous system of mutant myodystrophy (myd) mice. We demonstrate that myd mice have abnormal neuronal migration in cerebral cortex, cerebellum and hippocampus, and show disruption of the basal lamina. In addition, myd mice reveal that dystroglycan targets proteins to functional sites in brain through its interactions with extracellular matrix proteins. These results suggest that at least three distinct mammalian genes function within a convergent post-translational processing pathway during the biosynthesis of dystroglycan, and that abnormal dystroglycan–ligand interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities.

Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD), muscle–eye–brain disease (MEB), and Walker–Warburg syndrome are congenital muscular dystrophies (CMDs) with associated developmental brain defects. Mutations reported in genes of FCMD and MEB patients suggest that the genes may be involved in protein glycosylation. Dystroglycan is a highly glycosylated component of the muscle dystrophin–glycoprotein complex that is also expressed in brain, where its function is unknown. Here we show that brain-selective deletion of dystroglycan in mice is sufficient to cause CMD-like brain malformations, including disarray of cerebral cortical layering, fusion of cerebral hemispheres and cerebellar folia, and aberrant migration of granule cells. Dystroglycan-null brain loses its high-affinity binding to the extracellular matrix protein laminin, and shows discontinuities in the pial surface basal lamina (glia limitans) that probably underlie the neuronal migration errors. Furthermore, mutant mice have severely blunted hippocampal long-term potentiation with electrophysiologic characterization indicating that dystroglycan might have a postsynaptic role in learning and memory. Our data strongly support the hypothesis that defects in dystroglycan are central to the pathogenesis of structural and functional brain abnormalities seen in CMD.

Dystrophic heart failure blocked by membrane sealant poloxamer

Dystrophin deficiency causes Duchenne muscular dystrophy (DMD) in humans, an inherited and progressive disease of striated muscle deterioration that frequently involves pronounced cardiomyopathy. Heart failure is the second leading cause of fatalities in DMD. Progress towards defining the molecular basis of disease in DMD has mostly come from studies on skeletal muscle, with comparatively little attention directed to cardiac muscle. The pathophysiological mechanisms involved in cardiac myocytes may differ significantly from skeletal myofibres; this is underscored by the presence of significant cardiac disease in patients with truncated or reduced levels of dystrophin but without skeletal muscle disease. Here we show that intact, isolated dystrophin-deficient cardiac myocytes have reduced compliance and increased susceptibility to stretch-mediated calcium overload, leading to cell contracture and death, and that application of the membrane sealant poloxamer 188 corrects these defects in vitro. In vivo administration of poloxamer 188 to dystrophic mice instantly improved ventricular geometry and blocked the development of acute cardiac failure during a dobutamine-mediated stress protocol. Once issues relating to optimal dosing and long-term effects of poloxamer 188 in humans have been resolved, chemical-based membrane sealants could represent a new therapeutic approach for preventing or reversing the progression of cardiomyopathy and heart failure in muscular dystrophy.

Disruption of Dag1 in Differentiated Skeletal Muscle Reveals a Role for Dystroglycan in Muscle Regeneration

Striated muscle-specific disruption of the dystroglycan (DAG1) gene results in loss of the dystrophin-glycoprotein complex in differentiated muscle and a remarkably mild muscular dystrophy with hypertrophy and without tissue fibrosis. We find that satellite cells, expressing dystroglycan, support continued efficient regeneration of skeletal muscle along with transient expression of dystroglycan in regenerating muscle fibers. We demonstrate a similar phenomenon of reexpression of functional dystroglycan in regenerating muscle fibers in a mild form of human muscular dystrophy caused by disruption of posttranslational dystroglycan processing. Thus, maintenance of regenerative capacity by satellite cells expressing dystroglycan is likely responsible for mild disease progression in mice and possibly humans. Therefore, inadequate repair of skeletal muscle by satellite cells represents an important mechanism affecting the pathogenesis of muscular dystrophy.

Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats

Non‐technical summary  The force developed by a single fibre in frog muscles is transmitted laterally to the muscle surface with little or no loss. To demonstrate this phenomenon in mammals, a ‘yoke’ apparatus was developed that attached to the surface of whole, parallel‐fibred muscles and permitted measurements of the lateral transmission of forces. We then demonstrated that for wild‐type mice and rats longitudinal and lateral transmission of forces in muscles were not different. In contrast, for skeletal muscles of dystrophic mice and very old rats, in which the dystrophin‐associated glycoprotein complex (DGC) of fibres was disrupted, the forces transmitted laterally were impaired severely. We conclude that during contractions of skeletal muscles, an intact DGC is essential for the lateral transmission of force and disruptions of the DGC lead to sarcomere instability and contraction‐induced injury.

Effects of Myosin Heavy Chain Isoform Switching on Ca2+-Activated Tension Development in Single Adult Cardiac Myocytes

Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca2+ sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the alpha-MHC isoform to exclusively the beta-MHC isoform. We found a significant desensitization in the Ca2+ sensitivity of tension development in beta-MHC-expressing ventricular myocytes (pCa50=5. 51+/-0.03, where pCa is -log[Ca2+], and pCa50 is pCa at which tension is one-half maximal) compared with that in predominantly alpha-MHC-expressing myocytes (pCa50=5.68+/-0.05). No differences between the 2 groups were observed in the steepness of the tension-pCa relationship or in the maximum isometric force generated. Instantaneous stiffness measurements were made that provide a relative measure of changes in the numbers of myosin crossbridges attached to actin during Ca2+ activation. Results showed that the relative stiffness-pCa relationship was shifted to the right in beta-MHC-expressing myocytes compared with the alpha-MHC-expressing cardiac myocytes (pCa50=5.47+/-0.05 versus 5.76+/-0.05, respectively). We conclude that MHC isoform switching in adult cardiac myocytes alters the Ca2+ sensitivity of stiffness and tension development. These results suggest that the activation properties of the thin filament are in part MHC isoform dependent in cardiac muscle, indicating an additional role for MHC isoforms in defining cardiac contractile function.

Direct, convergent hypersensitivity of calcium-activated force generation produced by hypertrophic cardiomyopathy mutant |[alpha]|-tropomyosins in adult cardiac myocytes

Familial hypertrophic cardiomyopathy is a clinically and genetically diverse autosomal dominant disorder characterized by ventricular hypertrophy and myocyte disarray in the absence of known hypertrophic stimuli. It has been linked to many cardiac contractile proteins, including four point mutations in α-tropomyosin (Tm). Here we use adenoviral-mediated gene transfer into adult cardiac myocytes in vitro to show that all four hypertrophic cardiomyopathy α-Tm proteins can be expressed and incorporated into normal sarcomeric structures in cardiac myocytes at similar levels as normal α-Tm proteins after 5–6 days in culture. Isometric force recordings of single cardiac myocytes demonstrated inappropriate increased force output at submaximal calcium concentration with a specific mutant allele hierarchy. These data indicate that the severity of direct calcium-sensitizing effect of mutations in α-Tm may predict the clinical severity of mutant α-Tm-associated hypertrophic cardiomyopathy.

In vivo acceleration of heart relaxation performance by parvalbumin gene delivery

Defective cardiac muscle relaxation plays a causal role in heart failure. Shown here is the new in vivo application of parvalbumin, a calcium-binding protein that facilitates ultrafast relaxation of specialized skeletal muscles. Parvalbumin is not naturally expressed in the heart. We show that parvalbumin gene transfer to the heart in vivo produces levels of parvalbumin characteristic of fast skeletal muscles, causes a physiologically relevant acceleration of heart relaxation performance in normal hearts, and enhances relaxation performance in an animal model of slowed cardiac muscle relaxation. Parvalbumin may offer the unique potential to correct defective relaxation in energetically compromised failing hearts because the relaxation-enhancement effect of parvalbumin arises from an ATP-independent mechanism. Additionally, parvalbumin gene transfer may provide a new therapeutic approach to correct cellular disturbances in calcium signaling pathways that cause abnormal growth or damage in the heart or other organs.

COL4A1 Mutations Cause Ocular Dysgenesis, Neuronal Localization Defects, and Myopathy in Mice and Walker-Warburg Syndrome in Humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.

Cardiac Dysfunction in Hypertrophic Cardiomyopathy Mutant Tropomyosin Mice Is Transgene-Dependent, Hypertrophy-Independent, and Improved by β-Blockade

Abstract— Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in proteins of the cardiac contractile apparatus, including &agr;-tropomyosin (Tm). Mice expressing &agr;Tm in the heart were developed to determine the effects of FHC mutant Tm on cardiac structure and function from single cardiac myocytes to whole organ function in vivo. Expression of E180G mutant Tm did not produce cardiac hypertrophy or detectable changes in cardiac muscle morphology. However, E180G mutant Tm expression increased the Ca2+ sensitivity of force production in single cardiac myocytes in a transgene expression–dependent manner. Contractile dysfunction in single myocytes manifested organ level dysfunction, as conductance-micromanometry showed E180G Tm mice had significantly slowed relaxation (diastolic dysfunction) under physiological conditions. The diastolic dysfunction in E180G Tm mice was no longer evident during &bgr;-blockade because propranolol eliminated the effect of E180G Tm to slow myocardial relaxation. Cellular and organ level dysfunction were evident in E180G Tm mice in the absence of significant cardiac structural abnormalities normally associated with FHC. These findings therefore suggest that diastolic dysfunction in FHC may be a direct consequence of FHC mutant protein expression. In addition, because diastolic dysfunction in E180G Tm mice is dependent on inotropic status, cardiovascular stress may play an important role in FHC pathogenesis.