Browsing by Subject "Duchenne muscular dystrophy"
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Item A biochemical and molecular analysis of functional differences between dystrophin and utrophin(2013-11) Belanto, Joseph JohnThe DMD gene encodes the protein dystrophin, a 427kD cytoplasmic protein responsible for linking the actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. Mutations in dystrophin that abolish its expression lead to Duchenne muscular dystrophy (DMD). Patients with DMD become wheelchair bound in their early teens and succumb to fatal cardiac and/or respiratory failure in their mid-twenties to early thirties. There is currently no effective or widely available treatment for DMD beyond ventilatory support and the use of corticosteroids. Many therapies for treating dystrophin deficiency aim at upregulating its autosomal homolog utrophin due to its structural similarity and ability to bind an almost identical repertoire of proteins that dystrophin binds. It was previously shown that utrophin cannot bind neuronal nitric oxide synthase (nNOS) even though dystrophin binds nNOS, establishing for the first time a functional difference between dystrophin and utrophin. Here, we show that transgenic overexpression of utrophin on the mdx mouse background (Fiona-mdx) is not sufficient to rescue the disorganized microtubule network of the mdx mouse. Thus, we have elucidated a second functional difference between dystrophin and utrophin. Additionally, Fiona-mdx mice lack full recovery of cage activity after mild exercise. Our results suggest that any deficiency in nNOS binding or microtubule lattice function caused by loss of dystrophin may not be restored by upregulation of utrophin. Previously, our lab reported that dystrophin directly binds to microtubules and organizes them beneath the sarcolemma. Using in vitro microtubule cosedimentation assays, we show that dystrophin binds to microtubules with strong affinity (KD=0.33µM). Through the use of various recombinant constructs tested via in vitro microtubule cosedimentation we show that spectrin-like repeats 20-22 of the dystrophin central rod are responsible for microtubule binding activity. However, we show that these repeats require flanking regions of dystrophin for proper binding activity, making microtubule binding context-dependent. Additionally, we show that recombinant utrophin does not bind microtubules in vitro, corroborating our in vivo findings of the disorganized subsarcolemmal microtubule lattice of the Fiona-mdx mouse. We also provide evidence showing that dystrophin functions as a molecular guidepost to organize microtubules into a rectilinear lattice.Item Bone's functional and geometric properties in dystrophin-deficient mice and the efficacy of low intensity vibration training to improve musculoskeletal function(2013-03) Novotny, Susan AnneOverall, my dissertation work has shown that bone health is affected in dystrophic mice secondary to the muscle disease (Chapter 3), and both prednisolone and physical inactivity accentuate these declines (Chapter 4). I identified two sets of low intensity, high frequency vibration parameters (45 Hz at 0.6 g and 90 Hz at 0.6 g) that initiated an osteogenic response in mdx mice. Further experiments were performed utilizing the 45 Hz and 0.6 g setting, the results of which indicated that vibration was safe for dystrophic muscle (Chapters 5 and 6). However, long-term training adaptations for musculoskeletal function were not realized (Chapter 6). The lack of adaptations following vibration training in mdx or wildtype mice does not negate the utility of vibration as a potential therapeutic exercise modality for DMD, but further research, utilizing alternative strategies, is needed to determine the full extent of vibration's capacity to improve musculoskeletal health.Item Dystrophin Mutations Associated with Duchenne and Becker Muscular Dystrophy with Thermodynamic Analysis Using Differential Scanning Calorimetry(2015) Botts, Michelle; Hinderliter, AnneItem The Effects Of Beta-1 And -2 Adrenergic Receptor Genotypes On Cardiopulmonary Outcomes In Duchenne Muscular Dystrophy(2019-05) Kelley, EliIntroduction: The main contributor of mortality in Duchenne muscular dystrophy (DMD) patients is cardiorespiratory failure. The beta-1 adrenergic receptor (ADRB1) has been shown to play a functional role in cardiomyocyte function with ADRB1 stimulation increasing cardiac rate, contractility, and work. Multiple polymorphisms of the ADRB1 have been identified such as the Gly49 polymorphism that includes at least one glycine (Gly) for serine (Ser) substitution at amino acid 49 resulting in either homozygous for Gly (Gly49Gly) or heterozygous (Gly49Ser) polymorphisms and a Gly for Arg substitution at amino acid 389 resulting in either homozygous for Gly (Gly389) or heterozygous (Arg389). Heart failure patients with these polymorphisms (Gly49 and Arg389) have been shown to have improved cardiac function and decreased mortality risk. Furthermore, the beta-2 adrenergic receptor (ADRB2) has been shown to influence respiratory muscle strength and function. Multiple polymorphisms of the ADRB2 have been identified as including a glycine (Gly) for arginine (Arg) substitution at amino acid 16. The Gly16 polymorphism has been shown to have higher receptor density on lymphocytes, be more resistant to receptor downregulation, and functionally demonstrate improved respiratory function when compared with the Arg16 genotype in humans. Purpose: The purpose of this dissertation was to assess the functional consequences of ADRB1 genotypes on cardiac function and ADRB2 genotypes on pulmonary function in patients with DMD. We hypothesize DMD patients with the Gly389 polymorphism would have a lower incidence of cardiac events compared with those expressing Arg389 polymorphism and that DMD patients with the Gly16 polymorphism would have a reduced risk of nocturnal ventilation (NV) use at any given age compared with those patients expressing the Arg16 polymorphism. Methods: For study 1, we performed genotyping of the ADRB1 (amino acid 49) and high-intensity, steady-state exercise on 71 healthy subjects (Ser49Ser = 52, Gly49Ser = 19). For study 2, we performed genotyping of the ADRB2 (amino acid 16) and high-intensity, steady-state exercise on 77 healthy subjects (AA = 18, AG = 25, GG = 34). Data from CINRG-DNHS including 175 DMD patients (ages 3-25 yrs) with up to 9.7 years follow-up were analyzed focusing on ADRB1 and ADRB2 functional variants for studies 3 and 4. We performed Cox proportional hazard and Kaplan-Meier time to event analyses for the age of NV use and the age of cardiac outcomes and interventions. Results: There were no differences between ADRB1 genotype groups in age, height, weight, BMI, or watts achieved in the healthy patients. Additionally, there were no differences between genotype groups for cardiac output (CO), systolic blood pressure (BPsys), or diastolic blood pressure (BPdias) at rest, maximal exercise, or in change from rest to maximal exercise. There were, however, differences between genotype groups for resting CI and SVR and for HR at peak exercise (HRmax) with the Gly49Ser genotype presenting improved CI and a lower SVR at rest, and a higher HR at peak exercise. There was a trend towards significance (p = 0.058) for the change in stroke volume from rest to peak exercise (∆SV) with the Ser49Ser genotype demonstrating a larger change in SV. There were no differences between ADRB2 genotype groups in age, height, weight, or BMI in the healthy patients. The genotype groups differed significantly in watts, and watts/VO2 with heavy exercise with the Gly16 genotype achieving higher workloads. There was a trend towards significance (p=0.058) for watts/kg. There were no differences between ADRB1 genotype groups in age, height, weight, number of ambulatory patients, or age of loss of ambulation in our DMD cohort. The Arg389 polymorphism demonstrated a higher mean corticosteroid use compared with the Gly389 polymorphism. The genotype groups differed significantly (P<0.05) in the risk of diuretics use with the Gly389 polymorphism demonstrating a 5.01-fold increased risk of diuretics use at any age compared with the Arg389 polymorphism. There were no differences between ADRB2 genotype groups in age, height, weight, corticosteroid use, number of ambulatory patients, or age of loss of ambulation in our DMD cohort. The Gly16 polymorphism demonstrated a higher probability (P<0.05) for the use of NV assistance at any given age compared with the Arg16 polymorphism. The genotype groups differed significantly in the risk of NV use with the Gly16 polymorphism demonstrating a 2.77-fold increased risk of using NV at any given age compared with the Arg16 polymorphism. Conclusion: These data suggest genetic variation in the ADRB1 gene may influence the age of diuretics use in DMD patients with DMD patients expressing the Gly389 polymorphism being more likely to use diuretics compared with patients expressing the Arg389 polymorphism. Additionally, these data suggest genetic variation in the ADRB2 gene may also influence the age of NV use in DMD patients. Specifically, DMD patients expressing the Gly16 polymorphism were more likely to use NV at any given age compared with patients expressing the Arg16 polymorphism.Item Modeling and rescue of Duchenne muscular dystrophy cardiomyopathy using human induced pluripotent-derived cardiomyocytes(2021-01) Kamdar, ForumDuchenne muscular dystrophy (DMD) is the most common muscular dystrophy and affects 1:5000 boys born in the United States. DMD is a result of mutations in the dystrophin (DMD) gene that leads to the absence of the full length cytoskeletal protein dystrophin, which is expressed in skeletal muscle, brain, and heart. The absence of dystrophin leads to weakness of not only the skeletal muscle but also the heart. With advances in treatment for DMD, patients are living longer but a cardiomyopathic phenotype has been uncovered. DMD associated cardiomyopathy is nearly ubiquitous and is the leading cause of death with adults with DMD. There have been limited studies and therapies for dystrophic heart failure thus far, and there is a critical need to identify the pathophysiology and develop effective therapeutic regimens. In this thesis, I hypothesized that DMD cardiomyopathy could be modeled using DMD patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs) to identify physiological changes and future drug therapies. To explore and define therapies for DMD cardiomyopathy, we used DMD patient-specific and dystrophin null isogenic hiPSC-derived CMs to examine the physiological response to adrenergic agonists and -blocker treatment. We further examined these agents in vivo using wildtype and the mdx mouse model. At baseline and following adrenergic stimulation, DMD hiPSC-derived CMs had a significant increase in arrhythmic calcium traces compared to isogenic controls. Further, these arrhythmias were significantly decreased with propranolol treatment. Using telemetric monitoring, we observed that mdx mice, which lack dystrophin, and were stimulated with isoproterenol had an arrhythmic death and the lethal arrhythmias were rescued, in part, by propranolol pretreatment. Using single cell and bulk RNA-seq, we compared DMD and control hiPSC-derived CMs, mdx mice and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized following propranolol treatment in the mdx model. We also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived CMs have similar dysregulated pathways as the human DMD heart. We further determined that relatively few DMD patients see a cardiovascular specialist or receive β-blocker therapy. The results of these experiments highlight important mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. Importantly, these results may serve as a platform to elucidate further mechanisms of DMD cardiomyopathy and serve as a platform for testing novel therapies. Our results also provide a rationale for an adequately powered clinical study that examines the impact of β-blocker therapy in patients with dystrophinopathies.Item The sparing of the extraocular muscles in Duchenne muscular dystrophy: intrinsic differences in myogenic precursor cells(2013-10) McDonald, Abby AnnThe extraocular muscles (EOM) are both morphologically and functionally spared in the absence of dystrophin, which results in the fatal disease Duchenne muscular dystrophy (DMD). It is currently thought that intrinsic differences between the EOM and other non-cranial skeletal muscles account for this sparing. The work of this thesis examines the sparing of the EOM in different mouse models of DMD and differences between progenitor cells of the EOM and those of non-cranial skeletal muscles. Results of functional and morphological studies indicate that the mdx:utrophin+/- mouse model may serve as a more useful model of disease than the mdx mouse model, and that EOM sparing is not due to autosomal homolog utrophin up-regulation, as the EOM are spared in mouse models that lack both dystrophin and utrophin. Finally, gamma irradiation studies suggest that there is a population of progenitor cells in the EOM that is either better able to survive in the diseased muscle or is more highly proliferative than the progenitor population found in limb. Greater understanding of this population of progenitors in the EOM may provide insight into EOM sparing in DMD, and possibly even therapeutic advancements for treatment of this fatal disease.