Nitric oxide donors improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm

In Duchenne muscular dystrophy (DMD), palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide synthase activity in dystrophic mice promotes regeneration, the outcome of two nitric oxide (NO) donor drugs, MyoNovin (M) and isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug prednisone (P) in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as controls or with an NO donor ± P. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. P decreased body weight gain, M increased quadriceps mass, and I increased heart mass. P increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO donors + P (M+P, I+P) reduced %EBD+ fibers and calcification vs. P alone. %EBD+ fibers in M+P diaphragm did not differ from control. NO donor treatment reduced proliferation and the population of c-met+ cells and accelerated fiber regeneration. Concurrent with P, NO donor treatment suppressed two important detrimental effects of P in mice, possibly by accelerating regeneration, rebalancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest that NO donors could improve current therapy for DMD.     

Severe muscle dysfunction precedes collagen tissue proliferation in mdx mouse diaphragm.

After extensive necrosis, progressive diaphragm muscle weakness in the mdx mouse is thought to reflect progressive replacement of contractile tissue by fibrosis. However, little has been documented on diaphragm muscle performance at the stage at which necrosis and fibrosis are limited. Diaphragm morphometric characteristics, muscle performance, and cross-bridge (CB) properties were investigated in 6-wk-old control (C) and mdx mice. Compared with C, maximum tetanic tension and shortening velocity were 37 and 32% lower, respectively, in mdx mice (each P < 0.05). The total number of active CB per millimeter squared (13.0 +/- 1.2 vs. 18.4 +/- 1.7 x 10(9)/mm(2), P < 0.05) and the CB elementary force (8.0 +/- 0.2 vs. 9.0 +/- 0.1 pN, P < 0.01) were lower in mdx than in C. The time cycle duration was lower in mdx than in C (127 +/- 18 vs. 267 +/- 61 ms, P < 0.05). Percentages of fiber necrosis represented 2.8 +/- 0.6% of the total muscle fibers, and collagen surface area occupied 3.6 +/- 0.7% in mdx diaphragm. Our results pointed to severe muscular dysfunction in mdx mouse diaphragm, despite limited necrotic and fibrotic lesions.     

Genetic modifiers of muscular dystrophy: implications for therapy

The genetic understanding of the muscular dystrophies has advanced considerably in the last two decades. Over 25 different individual genes are now known to produce muscular dystrophy, and many different "private" mutations have been described for each individual muscular dystrophy gene. For the more common forms of muscular dystrophy, phenotypic variability can be explained by precise mutations. However, for many genetic mutations, the presence of the identical mutation is associated with marked phenotypic range that affects muscle function as well as cardiac function. The explanation for phenotype variability in the muscular dystrophies is only now being explored. The availability of genetically engineered animal models has allowed the generation of single mutations on the background of highly inbred strain. Phenotypic variation that is altered by genetic background argues for the presence of genetic modifier loci that can ameliorate or enhance aspects of the dystrophic phenotype. A number of individual genes have been implicated as modifiers of muscular dystrophy by studies in genetically engineered mouse models of muscular dystrophy. The value of these genes and products is that the pathways identified through these experiments may be exploited for therapy.     

In situ study of the sarcoplasmic reticulum function in control and mdx mouse diaphragm muscle.

Sarcoplasmic reticulum (SR) calcium handling in diaphragm was compared between mdx mice (7-8 weeks old) and age-matched controls. The total SR Ca2+ load was released from the SR by rapidly cooling muscle bundles from 22 to -1 degree C. The plateau amplitude of the rapid cooling contracture (RCC) was considered as an index of the SR Ca2+ content. The steady-state RCC amplitude was significantly lower by 50% in mdx bundles mainly because of a decreased capacity of the dystrophic diaphragm to generate maximal tension. There was no significant difference between either RCC time to peak or the time to half-relaxation of the transient, spike-like, contractile response induced by muscle rewarming. The recovery process of RCC was studied by using a paired RCC protocol. In both groups, at the shortest interval (10 s) between two RCCs, the amplitude of the second RCC was decreased by 25% compared with the first RCC. Increasing the time interval led to progressive monoexponential recovery of the second RCC with similar time constants in control and mdx diaphragm. These results indicate that the dystrophic process does not significantly alter SR Ca2+ uptake nor Ca2+ redistribution within the muscular cell.     

Brain tumour stem cells: implications for cancer therapy and regenerative medicine

The cancer relapse and mortality rate suggest that current therapies do not eradicate all malignant cells. Currently, it is accepted that tumorigenesis and organogenesis are similar in many respects, as for example, homeostasis is governed by a distinct sub-population of stem cells in both situations. There is increasing evidence that many types of cancer contain their own stem cells: cancer stem cells (CSC), which are characterized by their self-renewing capacity and differentiation ability. The investigation of solid tumour stem cells has gained momentum particularly in the area of brain tumours. Gliomas are the most common type of primary brain tumours. Nearly two-thirds of gliomas are highly malignant lesions with fast progression and unfortunate prognosis. Despite recent advances, two-year survival for glioblastoma (GBM) with optimal therapy is less than 30%. Even among patients with low-grade gliomas that confer a relatively good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells endowed with features of primitive neural progenitor cells and a tumour-initiating function. In general, this fraction is characterized for forming neurospheres, being endowed with drug resistance properties and often, we can isolate some of them using sorting methods with specific antibodies. The molecular characterization of these stem populations will be critical to developing an effective therapy for these tumours with very dismal prognosis. To achieve this aim, the development of a mouse model which recapitulates the nature of these tumours is essential. This review will focus on glioma stem cell knowledge and discuss future implications in brain cancer therapy and regenerative medicine.     

Myotonic dystrophy mouse models: towards rational therapy development

DNA repeat expansions can result in the production of toxic RNA. RNA toxicity has been best characterised in the context of myotonic dystrophy. Nearly 20 mouse models have contributed significant and complementary insights into specific aspects of this novel disease mechanism. These models provide a unique resource to test pharmacological, anti-sense, and gene-therapy therapeutic strategies that target specific events of the pathobiological cascade. Further proof-of-principle concept studies and preclinical experiments require critical and thorough analysis of the multiple myotonic dystrophy transgenic lines available. This review provides in-depth assessment of the molecular and phenotypic features of these models and their contribution towards the dissection of disease mechanisms, and compares them with the human condition. More importantly, it provides critical assessment of their suitability and limitations for preclinical testing of emerging therapeutic strategies.     

Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy

Attempts to develop gene therapy for Duchenne muscular dystrophy (DMD) have been complicated by the enormous size of the dystrophin gene. We have performed a detailed functional analysis of dystrophin structural domains and show that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, reveal that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins are fully protected against damage caused by muscle activity and are not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice results in a striking reversal of histopathological features of this disease. These results demonstrate that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.     

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy

Duchenne muscular dystrophy (DMD)--which is caused by mutations in the dystrophin gene-is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA--miR-31--that represses dystrophin expression by targeting its 3' untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.     

In vivo MRI characterization of progressive cardiac dysfunction in the mdx mouse model of muscular dystrophy

Aims

The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice.

Methods and Results

Serial measurements of cardiac morphology and function were made in the same group of mdx mice and controls (housed in a non-SPF facility) using MRI at 1, 3, 6, 9 and 12 months after birth. Left ventricular (LV) and right ventricular (RV) systolic and diastolic function, response to dobutamine stress and myocardial fibrosis were assessed. RV dysfunction preceded LV dysfunction, with RV end systolic volumes increased and RV ejection fractions reduced at 3 months of age. LV ejection fractions were reduced at 12 months, compared with controls. An abnormal response to dobutamine stress was identified in the RV of mdx mice as early as 1 month. Late-gadolinium-enhanced MRI identified increased levels of myocardial fibrosis in 6, 9 and 12-month-old mdx mice, the extent of fibrosis correlating with the degree of cardiac remodeling and hypertrophy.

Conclusions

MRI could identify cardiac abnormalities in the RV of mdx mice as young as 1 month, and detected myocardial fibrosis at 6 months. We believe these to be the earliest MRI measurements of cardiac function reported for any mice, and the first use of late-gadolinium-enhancement in a mouse model of congenital cardiomyopathy. These techniques offer a sensitive and clinically relevant in vivo method for assessment of cardiomyopathy caused by muscular dystrophy and other diseases.     

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research.     

Multiplicity of infection does not accelerate infectivity evolution of viral parasites in laboratory microcosms

Coinfection with multiple parasite genotypes [multiplicity of infection (MOI)] creates within-host competition and opportunities for parasite recombination and is therefore predicted to be important for both parasite and host evolution. We tested for a difference in the infectivity of viral parasites (lytic phage Φ2) and resistance of their bacterial hosts (Pseudomonas fluorescens SBW25) under both high and low MOI during coevolution in laboratory microcosms. Results show that MOI has no effect on infectivity and resistance evolution during coevolution over ～80 generations of host growth, and this is true when the experiment is initiated with wild-type viruses and hosts, or with viruses and hosts that have already been coevolving for ～330 generations. This suggests that MOI does not have a net effect of accelerating parasite adaptation to hosts through recombination, or slowing adaptation to hosts through between-parasite conflict in this system.     

Dietary iron loading does not influence biliary iron excretion in rats

The effect of dietary iron loading on biliary iron excretion was investigated with male Wistar rats aged 6 wk. The rats were fed purified diets with either 174 or 1740 mg FeSO₄. 7H₂O/kg diet and demineralized water for 6 wk. Blood haemoglobin, hematocrit, and iron concentrations in kidney and heart were not affected and iron concentrations in liver, spleen, and tibia were significantly raised after feeding the high-iron diet. The high-iron diet did not raise biliary iron excretion, suggesting that biliary iron excretion does not play an important role in regulating iron metabolism in rat after dietary iron loading.     

Light fractionation does not enhance the efficacy of methyl 5-aminolevulinate mediated photodynamic therapy in normal mouse skin.

Previous work demonstrated that fractionated illumination using two fractions separated by a dark interval of 2 h, significantly enhanced the clinical efficacy of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA). Considering the increasing clinical use of methyl 5-aminolevulinate (MAL) and the expected gain in efficacy by light fractionation we have investigated the response to MAL-PDT using a single and a two-fold illumination scheme and compared that with ALA-PDT. Our results show that fractionated illumination does not enhance the efficacy of PDT using MAL as it does using ALA despite the comparable fluorescence intensities at the end of the first light fraction and at the start of the second light fraction. Only the initial rate of photobleaching was slightly greater during ALA-PDT although the difference was small. Previously we hypothesized that cells surviving the first fraction are more susceptible to the second fraction. Since this is not true for MAL-PDT our data suggest that the distribution of MAL and ALA in tissues, and therefore the site of PDT induced damage, is an important parameter in the mechanism underlying the 2-fold illumination scheme.     

Respiratory loading intensity and diaphragm oxidative stress: N-acetyl-cysteine effects.

We hypothesized that resistive breathing of moderate to high intensity might increase diaphragm oxidative stress, which could be partially attenuated by antioxidants. Our objective was to assess the levels of oxidative stress in the dog diaphragm after respiratory muscle training of a wide range of intensities and whether N-acetyl-cysteine (NAC) might act as an antioxidant. Twelve Beagle dogs were anesthetized with 1% propophol, tracheostomized, and subjected to continuous inspiratory resistive breathing (IRB) (2 h/day for 2 wk). They were further divided into two groups (n = 6): NAC group (oral NAC administration/24 h for 14 days) and control group (placebo). Diaphragm biopsies were obtained before (baseline biopsy) and after (contralateral hemidiaphragm) IRB and NAC vs. placebo treatment. Oxidative stress was evaluated in all diaphragm biopsies through determination of 3-nitrotyrosine immunoreactivity, protein carbonylation, hydroxynoneal protein adducts, Mn-SOD, and catalase, using immunoblotting and immunohistochemistry. Both protein tyrosine nitration and protein carbonylation were directly related to the amount of the respiratory loads, and NAC treatment abrogated this proportional rise in these two indexes of oxidative stress in response to increasing inspiratory loads. A post hoc analysis revealed that only the diaphragms of dogs subjected to high-intensity loads showed a significant increase in both protein tyrosine nitration and carbonylation, which were also significantly reduced by NAC treatment. These results suggest that high-intensity respiratory loading-induced oxidative stress may be neutralized by NAC treatment during IRB in the canine diaphragm.     

Gibberellin does not accelerate rates of cell division in the dwarf pea shoot apical meristem

Although GA₃ doubled the numbers of cells in dwarf pea internodes,it caused no significant acceleration of cell division ratesin the apical meristem, estimated using cell doubling times,mitotic indices, or percentage labelled mitoses data. Increasedcell numbers in GA₃-treated pea stems must be generated withinthe extending internodes. Key words: Cell division cycle, gibberellin, pea, Pisum, shoot apical meristem     

Polyglutamine expansion in ataxin-3 does not affect protein stability: implications for misfolding and disease

Polyglutamine proteins that cause neurodegenerative disease are known to form proteinaceous aggregates, such as nuclear inclusions, in the neurons of affected patients. Although polyglutamine proteins have been shown to form fibrillar aggregates in a variety of contexts, the mechanisms underlying the aberrant conformational changes and aggregation are still not well understood. In this study, we have investigated the hypothesis that polyglutamine expansion in the protein ataxin-3 destabilizes the native protein, leading to the accumulation of a partially unfolded, aggregation-prone intermediate. To examine the relationship between polyglutamine length and native state stability, we produced and analyzed three ataxin-3 variants containing 15, 28, and 50 residues in their respective glutamine tracts. At pH 7.4 and 37 degrees C, Atax3(Q50), which lies within the pathological range, formed fibrils significantly faster than the other proteins. Somewhat surprisingly, we observed no difference in the acid-induced equilibrium and kinetic un/folding transitions of all three proteins, which indicates that the stability of the native conformation was not affected by polyglutamine tract extension. This has led us to reconsider the mechanisms and factors involved in ataxin-3 misfolding, and we have developed a new model for the aggregation process in which the pathways of un/folding and misfolding are distinct and separate. Furthermore, given that native state stability is unaffected by polyglutamine length, we consider the possible role and influence of other factors in the fibrillization of ataxin-3.     

An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy

The Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) is a severe blistering disease inherited in an autosomal-dominant fashion. Here we report the generation of a mouse model that allows focal activation of a mutant keratin 14 allele in epidermal stem cells upon topical administration of an inducer, resulting in EBS phenotypes in treated areas. Using laser capture microdissection, we show that induced blisters healed by migration of surrounding nonphenotypic stem cells into the wound bed. This observation provides an explanation for the lack of mosaic forms of EBS-DM. In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin. Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.