R. Mark Grady

Skeletal and Cardiac Myopathies in Mice Lacking Utrophin and Dystrophin: A Model for Duchenne Muscular Dystrophy

Dystrophin is a cytoskeletal protein of muscle fibers; its loss in humans leads to Duchenne muscular dystrophy, an inevitably fatal wasting of skeletal and cardiac muscle. mdx mice also lack dystrophin, but are only mildly dystrophic. Utrophin, a homolog of dystrophin, is confined to the postsynaptic membrane at skeletal neuromuscular junctions and has been implicated in synaptic development. However, mice lacking utrophin show only subtle neuromuscular defects. Here, we asked whether the mild phenotypes of the two single mutants reflect compensation between the two proteins. Synaptic development was qualitatively normal in double mutants, but dystrophy was severe and closely resembled that seen in Duchenne. Thus, utrophin attenuates the effects of dystrophin deficiency, and the double mutant may provide a useful model for studies of pathogenesis and therapy.

Role for |[alpha]|-dystrobrevin in the pathogenesis of dystrophin-dependentmuscular dystrophies

A dystrophin-containing glycoprotein complex (DGC) links the basal lamina surrounding each muscle fibre to the fibre’s cytoskeleton, providing both structural support and a scaffold for signalling molecules. Mutations in genes encoding several DGC components disrupt the complex and lead to muscular dystrophy. Here we show that mice deficient in α-dystrobrevin, a cytoplasmic protein of the DGC, exhibit skeletal and cardiac myopathies. Analysis of double and triple mutants indicates that α-dystrobrevin acts largely through the DGC. Structural components of the DGC are retained in the absence of α-dystrobrevin, but a DGC-associated signalling protein, nitric oxide synthase, is displaced from the membrane and nitric-oxide-mediated signalling is impaired. These results indicate that both signalling and structural functions of the DGC are required for muscle stability, and implicate α-dystrobrevin in the former.

Maturation and maintenance of the neuromuscular synapse: genetic evidence for roles of the dystrophin--glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) links the cytoskeleton of muscle fibers to their extracellular matrix. Using knockout mice, we show that a cytoplasmic DGC component, alpha-dystrobrevin (alpha-DB), is dispensable for formation of the neuromuscular junction (NMJ) but required for maturation of its postsynaptic apparatus. We also analyzed double and triple mutants lacking other cytoskeletal DGC components (utrophin and dystrophin) and myotubes lacking a alpha-DB or a transmembrane DGC component (dystroglycan). Our results suggest that alpha-DB acts via its linkage to the DGC to enhance the stability of postsynaptic specializations following their DGC-independent formation; dystroglycan may play additional roles in assembling synaptic basal lamina. Together, these results demonstrate involvement of distinct protein complexes in the formation and maintenance of the synapse and implicate the DGC in the latter process.

Syne-1, A Dystrophin- and Klarsicht-related Protein Associated with Synaptic Nuclei at the Neuromuscular Junction

We describe a novel protein, Syne-1, that is associated with nuclear envelopes in skeletal, cardiac, and smooth muscle cells. Syne-1 contains multiple spectrin repeats similar to those found in dystrophin and utrophin, as well as a domain homologous to the carboxyl-terminal of Klarsicht, a protein associated with nuclei and required for a subset of nuclear migrations inDrosophila. In adult skeletal muscle fibers, levels of Syne-1 are highest in the nuclei that lie beneath the postsynaptic membrane at the neuromuscular junction. These nuclei are transcriptionally specialized, expressing genes for synaptic components at higher levels than extrasynaptic nuclei in the same cytoplasm. Syne-1 is the first protein found to be selectively associated with synaptic nuclei. Syne-1 becomes concentrated in synaptic nuclei postnatally. It remains synaptically enriched following denervation or degeneration/regeneration, and is also present at high levels in the central nuclei of dystrophic myotubes. The location and structure of Syne-1 suggest that it may participate in the migration of myonuclei in myotubes and/or their anchoring at the postsynaptic apparatus. Finally, we identify a homologous gene, syne-2, that is expressed in an overlapping but distinct pattern.

Neurotransmitter Receptor Dynamics Studied In Vivo by Reversible Photo-Unbinding of Fluorescent Ligands

We show that fluorescently tagged ligands with high affinity for their targets can be reversibly unbound by focused laser excitation. By sequential unbinding and relabeling with different colors of alpha-bungarotoxin, we selectively labeled adjacent pools of acetylcholine receptors (AChRs) at neuromuscular junctions of adult mice. Timelapse imaging in vivo revealed that synaptic AChRs completely intermingle over approximately 4 days and many extrasynaptic AChRs are incorporated into the synapse each day. In mice that lacked alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex, rates of AChR turnover, and intermingling were increased approximately 4- to 5-fold. These results demonstrate remarkable molecular dynamism underlying macroscopic stability of the postsynaptic membrane, and establish alpha-dystrobrevin as a key control point for regulation of mobility and turnover.

Tyrosine-phosphorylated and nonphosphorylated isoforms of α-dystrobrevin: Roles in skeletal muscle and its neuromuscular and myotendinous junctions

α-Dystrobrevin (DB), a cytoplasmic component of the dystrophin–glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking αDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main αDB isoforms, αDB1 and αDB2, with common NH2-terminal but distinct COOH-terminal domains. αDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. αDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in αDB−/− mice prevented muscle fiber degeneration; however, only αDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of αDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, αDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, αDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Cerebellar Synaptic Defects and Abnormal Motor Behavior in Mice Lacking α- and β-Dystrobrevin

The dystrobrevins (αDB and βDB) bind directly to dystrophin and are components of a transmembrane dystrophin–glycoprotein complex (DGC) that links the cytoskeleton to extracellular proteins in many tissues. We show here that αDB, βDB, and dystrophin are all concentrated at a discrete subset of inhibitory synapses on the somata and dendrites of cerebellar Purkinje cells. Dystrophin is depleted from these synapses in mice lacking both αDB and βDB, and DBs are depleted from these synapses in mice lacking dystrophin. In dystrophin mutants and αDB,βDB double mutants, the size and number of GABA receptor clusters are decreased at cerebellar inhibitory synapses, and sensorimotor behaviors that reflect cerebellar function are perturbed. Synaptic and behavioral abnormalities are minimal in mice lacking either αDB or βDB. Together, our results show that the DGC is required for proper maturation and function of a subset of inhibitory synapses, that DB is a key component of this DGC, and that interference with this DGC leads to behavioral abnormalities. We suggest that motor deficits in muscular dystrophy patients, which are their cardinal symptoms, may reflect not only peripheral derangements but also CNS defects.

Rational approach to use of heparin during cardiac catheterization in children

OBJECTIVES We sought to determine an anticoagulation protocol for use during cardiac catheterization in children. BACKGROUND There are few data to indicate which dose of heparin represents adequate anticoagulation or how best to monitor its efficacy. In this study, adequate anticoagulation was defined as the amount of heparin needed to prevent a significant increase in serum fibrinopeptide A, a sensitive marker for thrombin activity. The degree of heparinization was estimated by the activated clotting time. METHODS Thirty-six children (1 month to 19.5 years old) with congenital heart disease underwent diagnostic cardiac catheterization; 13 of these 36 patients had an additional interventional procedure. Sheaths and catheters were flushed with heparinized saline (1 IU/ml); during the procedure, 33 of the 36 patients received either a 50- or a 100-IU/kg heparin bolus. Paired fibrinopeptide A and activated clotting time samples were obtained throughout each procedure. RESULTS Increasing the activated clotting time with heparin resulted in a dose-related decrease in fibrinopeptide A levels. A single heparin bolus of either 50 or 100 IU/kg elevated the activated clotting time above baseline level (209 +/- 52 s after 50 IU/kg, 270 +/- 57 s after 100 IU/kg vs. 133 +/- 20 s at baseline [p < 0.0001]) and reduced fibrinopeptide A levels below baseline (7.9 +/- 14 ng/ml after 50 IU/kg, 4.8 +/- 3.7 ng/ml after 100 IU/kg vs. 38 +/- 59 ng/ml at baseline [p < 0.0001]). Heparin flush alone did not increase the activated clotting time above baseline and failed to suppress an increase in fibrinopeptide A levels. There were no differences in activated clotting time and fibrinopeptide A values between patients undergoing diagnostic or interventional procedures. CONCLUSIONS Administration of a heparin bolus to maintain an activated clotting time > 200 s prevented a significant increase in thrombin activity. Heparin flush alone did not provide adequate anticoagulation. Patients undergoing an interventional procedure did not require more heparin than that needed for a diagnostic procedure.

Lung transplantation in children with idiopathic pulmonary arterial hypertension: An 18-year experience

BACKGROUND The natural history of idiopathic pulmonary arterial hypertension (IPAH) in patients of all ages is one of relentless progression. For those who fail medical therapy, lung transplantation remains the ultimate palliation. In the USA, IPAH is the second leading indication for lung transplantation in children and first for children 1 to 5 years of age. In this study, we report our 18-year experience with lung transplantation in children with IPAH. METHODS We performed a retrospective chart review of children with IPAH listed for lung transplant at our center between 1991 and 2009. Our data reflect a total of 26 children ranging in age from 1.6 to 18.9 years. Nineteen were transplanted and 7 died while waiting (27%). The impact of a number of pre-transplant variables on survival was evaluated. RESULTS Median survival for those transplanted was 5.8 years, with 1- and 5-year survival rates of 95% and 61%, respectively. Survival was independent of pre-transplant considerations such as age, weight, need for intravenous (IV) inotropes, use of IV pulmonary vasodilators, year of transplant and severity of right-sided cardiac pressures. There was 1 hospital death. Compared with the transplanted group, children who died waiting had a significantly higher incidence of supra-systemic right heart pressures (p = 0.02) and hemoptysis (p = 0.01). CONCLUSIONS Our study is the largest to date to look at outcomes for lung transplantation in children with IPAH. Their median survival compares favorably with that of all pediatric lung transplant recipients, 5.8 years vs 4.5 years, respectively. We did not identify any pre-transplant variables that presaged a poorer outcome. Thus, survival seemed more related to factors that influence long-term outcomes in all transplant recipients such as rejection and infection. Lung transplantation remains a viable option for children with IPAH, especially for those with supra-systemic right heart pressures despite maximal medical therapy.

First experience with a paracorporeal artificial lung in a small child with pulmonary hypertension

Stabilizing children with decompensating refractory pulmonary hypertension may require extracorporeal circulatory support. Currently, options in this regard are limited to use of venoarterial extracorporeal membrane oxygenation (ECMO), but this approach is frequently fraught with complications. Alternatively, the interventional pumpless lung-assist membrane ventilator (Novalung GmbH, Heilbronn, Germany) can be used through a femoral artery to femoral vein shunt in patients with end-stage lung disease and hypercapnia or hypoxemia refractory to mechanical ventilation and through a pulmonary artery to left atrium shunt in patients with refractory pulmonary hypertension. The latter configuration has several advantages for children with pulmonary hypertension: (1) It provides a parallel low resistance circuit that drastically decreases right ventricular (RV) afterload and native pulmonary pressure, allowing RV recovery and pulmonary vascular remodeling. (2) It may serve as a long-term pulmonary assist device or as a bridge to lung transplant or a bridge to recovery. (3) It allows physical rehabilitation. Here we report the first successful use of the Novalung surgical lung-assist (sLA) device in a small child with pulmonary hypertension.