DeWayne Townsend

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.

Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma

Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the ~20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild‐type C57BL/10 mice housed under specific pathogen‐free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild‐type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopa‐thology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD.–Chamberlain, J. S., Metzger, J., Reyes, M., Townsend, D., Faulkner, J. A. Dystrophin‐deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyo‐sarcoma. FASEB J. 21, 2195–2204 (2007)

Emergent dilated cardiomyopathy caused by targeted repair of dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) is a fatal disease characterized by deterioration of striated muscle, affecting skeletal and cardiac muscles. Recently, several therapeutic approaches have shown promise for repairing dystrophic skeletal muscles. However, these methods often leave the dystrophic heart untreated. Here we show that, in comparison to fully dystrophin-deficient animals, targeted transgenic repair of skeletal muscle, but not cardiac muscle, in otherwise dystrophin-deficient (mdx) mice paradoxically elicited a fivefold increase in cardiac injury and dilated cardiomyopathy in these animals in vivo. Skeletal muscle repair was shown to increase the voluntary activity of the mdx mice as quantified by voluntary running on the exercise wheel. Because the dystrophin-deficient heart is highly sensitive to increased stress, we hypothesize that increased activity (enabled by the repaired skeletal muscle) provided the stimulus for heightened cardiac injury and heart remodeling. In support of this hypothesis, the primary cellular compliance defect in dystrophin-deficient cardiac myocytes was found to be unchanged by skeletal muscle repair in the mdx mice. These findings provide new information on the evolution of cardiac disease in dystrophin-deficient animals and underscore the importance of implementing global striated muscle therapies for muscular dystrophy.

Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability-dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.

Temperature-sensitive tyrosinase associated with peripheral pigmentation in oculocutaneous albinism.

Several types of autosomal recessive oculocutaneous albinism (OCA) are associated with abnormal tyrosinase function and a generalized reduction in or absence of cutaneous and eye melanin. Each is thought to result from a different mutant allele at the tyrosinase locus, with the mutation producing an enzyme with little or no activity in all involved tissues. In this paper, we report a new type of OCA that results from a tyrosinase allele producing a temperature-sensitive enzyme. The proband had white hair in the warmer areas (scalp and axilla) and progressively darker hair in the cooler areas (extremities) of her body. Melanocyte and melanosome architecture were normal. Quantitative hairbulb tyrosinase (dopa oxidase) assay demonstrated a loss of activity above 35-37 degrees C. Plasma pheomelanin and urine eumelanin intermediates were reduced and correlated with hair melanin content. This is the first temperature-sensitive tyrosinase mutation to be reported in humans and is analogous to the Siamese mutation in the cat and the Himalayan mutation in the mouse.

Designing Heart Performance by Gene Transfer

The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca(2+) handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

Optimized assay for mammalian tyrosinase (polyhydroxyl phenyloxidase)

Tyrosinase is an important enzyme in the formation of pigment throughout the animal kingdom and many assays have been described for measuring its activity. This paper describes an assay which has been optimized to measure the enzymatic activity of mammalian tyrosinase and describes the procedures from which the optimum conditions were determined. The preparation and storage of the stock reagents required to perform the assay are also described in detail as well as some discussion pertaining to the preparation of samples prior to assay.

Elevated urinary dolichol excretion in the hermansky-pudlak syndrome. Indicator of lysosomal dysfunction

The Hermansky-Pudlak syndrome, a triad of albinism, platelets lacking dense bodies, and storage of ceroid-like material in tissues, occurs approximately once in 2,000 northwestern Puerto Ricans. The manifestations of storage disease are variable and include granulomatous colitis, restrictive lung disease, kidney failure, and cardiomyopathy. The autofluorescent material stored in the Hermansky-Pudlak syndrome is histochemically similar to that stored in neuronal ceroid/lipofuscinosis. The material in neuronal ceroid/lipofuscinosis contains dolichols, which are components of lysosomes, and patients show increased urinary excretion of dolichols. This study of 49 patients with the Hermansky-Pudlak syndrome found that urinary dolichol levels are increased in those patients with evidence of ceroid storage in the kidneys but are not elevated when storage occurs in tissues other than the kidneys. The excretion of ceroid was not influenced by the saturation state of dietary fat. A defect in processing of membranes of lysosomes, melanosomes, and dense bodies may be involved in the syndrome.

Hermansky-Pudlak syndrome (HPS): A proposed block in glutathione peroxidase☆

Abstract An unusual form of oculocutaneous albinism associated with a mild hemorrhagic diathesis and the accumulation of a ceroidlike pigment in the reticuloendothelial system was recognized by Hermansky and Pudlak in 1959. Examination of oral mucosal scrapings, urinary sediment, and bone marrow macrophages from patients with Hermansky-Pudlak syndrome (HPS) demonstrates that two forms of lipid accumulate in this disease. Many patients with this disorder have had spontaneous gingival hemorrhage, epistaxis, and prolonged bleeding following tooth extraction, delivery, and surgical procedures. Aspirin administered to such patients may enhance the bleeding diathesis. Analysis of the pigment defect shows that tyrosinase activity is present and pheomelanin pigment accumulates in melanocytes. Platelets lack an adequate storage pool of serotonin and the nonmetabolic nucleotides. A ceriodlike pigment accumulates in reticuloendothelial cells, urinary sediment, and buccal mucosa. The postulated enzymatic block accounting for this traid of signs is a defect in glutathione peroxidase.

Distinct pathophysiological mechanisms of cardiomyopathy in hearts lacking dystrophin or the sarcoglycan complex

Duchenne muscular dystrophy (DMD) and limb girdle muscular dystrophy (LGMD) 2C‐F result from the loss of dystrophin and the sarcoglycans, respectively. Dystrophin, a cytoskeletal protein, is closely associated with the membrane‐bound sarcoglycan complex. Despite this tight biochemical association, the function of dystrophin and the sarcoglycan subunits may differ. The loss of dystrophin in skeletal muscle results in muscle that is highly susceptible to contraction‐induced damage, but the skeletal muscle of mice lacking γ‐ or δ‐sarcoglycan are less susceptible. Using mouse models of DMD, LGMD‐2C, and LGMD‐2F, we demonstrate that isolated cardiac myocytes from mice lacking either γ‐ or δ‐sarcoglycan have normal compliance. In contrast, dystrophin‐deficient myocytes display poor passive compliance and are susceptible to terminal contracture following mild passive extensions. Mice deficient in dystrophin and, less so, δ‐sarcoglycan have reduced survival during in vivo dobutamine stress testing compared to controls. Catheter‐based hemodynamic studies show deficits in both baseline and dobutamine‐stimulated cardiac function in all of the dystrophic mice compared to control mice, with dystrophin‐deficient mice having the poorest function. In contrast, histopathology showed increased fibrosis in the sarcoglycan‐deficient hearts, but not in hearts lacking dystrophin. In summary, this study provides important insights into the unique mechanisms of disease underlying these different models of inherited dystrophic cardiomyopathy and supports a model where dystrophin, but not the sarcoglycans, protects the cardiac myocyte against mechanical damage.—Townsend, D., Yasuda, S., McNally, E., Metzger, J. M. Distinct pathophysiological mechanisms of cardiomyopathy in hearts lacking dystrophin or the sarcoglycan complex. FASEB J. 25, 3106‐3114 (2011). www.fasebj.org