MS, University of Cincinnati, 2012, Medicine: Genetic Counseling

Duchenne and Becker muscular dystrophy (DBMD) are progressive and debilitating neuromuscular disorders caused by X-linked recessive mutations in the dystrophin gene. DMD presents in childhood and limits life to the second or third decade. BMD, a relatively milder form, presents in childhood to adulthood. Although improvements in the care of patients with DBMD have enhanced patients' quality and duration of life, there has been no effect on long-term prognosis. Little is known of the psychological morbidity associated with DBMD on families and patients. Birth mothers of children with DBMD, who are themselves carriers of DBMD, may have increased psychological burden. The purpose of this analysis was to describe differences in adaptation and wellbeing between mothers who were carriers and those who were not carriers of DBMD. Data was collected from mothers with biological children with DBMD using a mixed-methods web-based survey. The primary outcome variable, adaptation score, was generated for each participant using the Psychological Adaptation to Illness Scale. Various other scales and investigator-developed questions were also used to measure secondary outcome variables. One hundred twenty-five participants completed the questionnaire and 116 responses were analyzed. Fifty-one (44%) were carriers of a DMD gene mutation, 47 (40%) were not carriers, and 18 (16%) did not know their carrier status. The mean adaptation score was 3.68 (SD=0.9) for carriers and 3.25 (SD=0.9) for non-carriers. Carriers showed better adaptation and higher perceived control than non-carriers (pooled t-test, p=0.02 and p=0.05, respectively). These results were limited by several factors including small sample size, recruitment bias, and increased risk of type 1 error resulting from multiple tests on the data. An open-ended question completed by the carriers revealed various positive and negative effects of being a carrier. In conclusion, carrier status may affect mothers' adaptation to DBMD an (open full item for complete abstract)

Committee: James Collins PhD (Committee Chair); Kathleen Kinnett MSN (Committee Member); Xue Zhang PhD (Committee Member); Martha Walker (Committee Member) Subjects: Genetics

MS, University of Cincinnati, 2013, Medicine: Genetic Counseling

Introduction: Duchenne / Becker muscular dystrophy (DBMD) is an x-linked condition with a wide variation of clinical presentation due to specific gene mutations and the gender of the affected individual. For families of the most severely affected male patients, care needs, natural history, and potential interventions are paramount. In contrast, reproductive risks may be important for less severely affected individuals as in the case of Becker phenotype or DBMD carrier females. The published literature has suggested the caregiver burden and poor prognosis of DBMD has an impact on the biological family as a whole. Additionally, published literature suggests a high disruption rate of adoptions that involve a child with special needs. However, the literature does not currently describe the role of genetic counselors in addressing the needs of families who have adopted a child with DBMD. Purpose: The purpose of this study was to determine the needs of adoptive families with sons diagnosed with DBMD, and how genetic counseling could be tailored to improve this population's experience. Methods: Participants were adoptive parents of males who were under age 18 and had a DBMD Diagnosis. They were recruited through Cincinnati Children's Hospital Medical Center or Duchenne Connect. Semi-structured qualitative interviews were conducted by telephone with use of an interview guide. The interview content was analyzed for recurrent themes using NVivo© software. These themes were organized into categories to summarize the findings. Results: Thirteen adoptive parents were interviewed. Their needs, relative to the diagnosis of DBMD, genetic counseling, and the genetics information, were not specific to adoptive families. In addition to the anticipated themes, 2 adoption specific points were described. Parents of adopted children with DBMD place importance on communicating the diagnostic implication of DBMD to the biological parents. Second, adoptive parents who questioned thei (open full item for complete abstract)

Committee: Robert Hopkin M.D. (Committee Chair); Martha Walker (Committee Member) Subjects: Surgery

Bachelor of Science (BS), Ohio University, 2024, Neuroscience

Duchene muscular dystrophy (DMD) is an X-Linked recessive genetic disorder which occurs in approximately 1/5000 XY births and is caused by a mutation in the human dystrophin gene. DMD causes many physiological defects and drastically shortens the lifespan of those afflicted by it. Gene replacement therapies are in clinical trials, but traditional therapies are still needed whilst those are developed. The C. elegans dys-1 gene is highly conserved compared to human dystrophin, and mutations in C. elegans dys-1 produces a clinically relevant phenotype. By use of the C. elegans DMD model, the pathology of DMD can easily be studied in a laboratory setting, allowing various potential traditional treatments to be tested for effectiveness. Zoledronic Acid (ZA) is approved by the U.S. Food and Drug Administration (FDA) to treat osteoporosis in patients with DMD, and preliminary data suggests that ZA could have positive effects for muscular health in DMD patients without osteoporosis. In the present work, it was found that the drug ZA is effective in improving DMD health in the C. elegans DMD model. Additionally, it was found that ZA improves DMD health through lowering the increased Ca2+ levels classically found in dys-1 mutants, which along with further experimentation informs a potential mechanistic pathway through which ZA acts. With this data, it is hoped that a clinical trial for repurposing ZA for use in for DMD patients is conducted in order to lessen the suffering caused by this disorder whilst gene therapeutics are developed.

Committee: Nathaniel Szewczyk (Advisor); Corinne Nielsen (Advisor) Subjects: Neurosciences

Doctor of Philosophy, The Ohio State University, 2024, Molecular Genetics

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutation of the Dystrophin gene resulting in complete absence of the dystrophin protein. Loss of dystrophin destabilizes the muscle membrane and increases muscle's susceptibility to injury. Over time, continuous rounds of muscle insult, muscle degeneration, and regeneration ultimately exhaust muscle's regenerative and reparative capacity and leads to loss of muscle function. There is currently no cure for DMD and there are many significant hurdles in the field for dystrophin-restoration based therapies. Thus, there is a critical need to develop new therapeutics. Genome-wide association studies (GWAS) have uncovered putative genetic modifiers of DMD. One identified class of modifiers are regulators of TGFb signaling. In Chapter 2, we use pharmacological and genetic means to investigate TGFb signaling as a modifier of DMD. Here I show that inhibition of TGFb using TGFb receptor inhibitors LY364947 and SB431542 leads to significant improvement in birefringence intensity, a read out of muscle organization, but causes developmental defects. Modifiers uncovered by GWAS include allelic variants that decrease expression of TGFb regulators latent TGFb binding protein 4 (LTBP4, ltbp4 in zebrafish) and thrombospondin-1 (THBS1, thbs1a and thbs1b in zebrafish) and correlate with decreased TGFb bioavailability and a less severe DMD phenotype. To model the decreased expression alleles of LTBP4 and THBS1 we used zebrafish mutants and CRISPR/Cas-9 F0 'crispants' of ltbp4, thbs1a, and thbs1b and show that mutation of thbs1b or ltbp4 improves survivorship in dmd mutant embryos and combinatorial mutation of thbs1a and ltbp4 leads to improved birefringence intensity, an indiciator of muscle organization. Together, these findings demonstrate that inhibition of TGFb signaling is protective in dmd mutant zebrafish. GWAS for DMD modifiers have also identified SNPS that lie in non-coding putative regulato (open full item for complete abstract)

Committee: Sharon Amacher (Advisor); Martin Haesemeyer (Committee Member); Harold Fisk (Committee Member); Susan Cole (Committee Member) Subjects: Biology; Genetics

MS, University of Cincinnati, 2024, Medicine: Genetic Counseling

Current literature has highlighted a diagnostic delay for Duchenne muscular dystrophy (DMD), clinical and familial needs for an early diagnosis, and increasing support for the inclusion of DMD on newborn screening (NBS) panels. Our aim was to investigate whether early diagnosis is associated with a delay in disease progression outcomes in male siblings with DMD. We conducted a retrospective chart review of 42 siblings sets and compared their disease courses. The primary predictor was age of diagnosis. Primary clinical disease outcomes included ages at loss of ambulation (LoA), first abnormal cardiac finding, first abnormal pulmonary finding, and North Star Ambulatory Assessment (NSAA) scores at age 8 years. The median age at last visit was 14.4 years and 10.5 years for the older and younger sibling cohorts, respectively. Median age of diagnosis in the younger sibling cohort was 2.04 years and was significantly earlier than the median age for the older sibling cohort, 4.96 years (p < 0.001). Corticosteroid treatment was initiated at a median age of 6.40 years for the older siblings and 4.45 years for the younger siblings (p <0.001). Age of diagnosis was not a predictor for the four primary outcomes. Although, advanced diagnostic age may be associated with increased time to perform the Gowers sign at age 8 years (p = 0.059). Though, we were unable to conclude that age of diagnosis is a predictor for disease progression outcomes in this cohort, further studies with longer follow-up times are warranted to provide a more accurate understanding of the impact of early diagnosis on disease course in this population.

Committee: Melanie Myers Ph.D. (Committee Chair); Chinmayee Bhimarao Nagaraj M.S. (Committee Member); Cuixia Tian M.D. (Committee Member); Valentina Pilipenko Ph.D. (Committee Member); Niki Armstrong M.S. (Committee Member) Subjects: Medicine

Doctor of Philosophy (PhD), Wright State University, 2023, Biomedical Sciences PhD

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder that affects 1 in 3500 male births worldwide. It is characterized by mutations in the dystrophin gene that results in the loss of functional dystrophin. Dystrophin deficiency leads to instability of the sarcolemma alongside increased inflammation, necrosis and fibrosis resulting in membrane rupture and eventual muscle fiber death. There are currently no effective treatments for DMD. Here we show that Lipin1 expression is significantly downregulated at the mRNA and protein levels in gastrocnemius muscle of mdx mice, the DMD mouse model. Lipin1 has a dual function as a phosphatidic acid phosphatase (PAP) regulating phospholipids and triacylglycerol biosynthesis, but also as a transcriptional cofactor. In this study, we evaluated the role of lipin1 in dystrophic muscle by characterizing two mouse models, Dystrophin/Lipin1-DKO mice and MDX:Lipin1Tg/0 transgenic mice. Further depletion of Lipin1 in our Dystrophin/Lipin1-DKO model showed worsened disease phenotype through increased inflammation, fibrosis, and necroptosis. In contrast, restoration of Lipin1 in our MDX:Lipin1Tg/0 model showed a significant improvement in skeletal muscle health through reduced inflammation, fibrosis, and necroptosis through improved membrane integrity. Altogether, our study showed that Lipin1 could be a potential therapeutic target for DMD.

Committee: Hongmei Ren Ph.D. (Advisor); Weiwen Long Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member); Mark Rich Ph.D. (Committee Member); Michael Leffak Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Duchenne muscular dystrophy (DMD) is a severe and progressive muscular dystrophy that develops in the skeletal muscles because of mutations in the dystrophin gene. Dystrophin stabilizes sarcolemma and assembles neuronal nitric oxide synthase (nNOS) into the dystrophin-associated protein complex on the sarcolemma. The absence of dystrophin triggers the delocalization of nNOS and contributes to the misregulation of muscle development, blood flow, muscle fatigue, and inflammation. Lipin1 was reduced in the skeletal muscles of patients with DMD and the mdx mouse model of DMD. In this study, we explored the role of lipin1 in the regulation of nNOS expression and function. We found that Lipin1 deficiency leads to the downregulation of nNOS protein and gene expression levels, while overexpression of lipin1 elevated nNOS protein and gene expression levels. We also found that Lipin1 upregulated nNOS gene expression by coactivating PPARα and binding to the promoter region of nNOS. Lipin1 deficiency leads to muscle fatigue in lipin1Myf5cKO mice, possibly due to the downregulation of nNOS. Most importantly, overexpressing lipin1 in dystrophic muscle improved muscle fatigue in our mdx:lipin1 transgenic mice which may be through the restoration of nNOS expression.

Committee: Hongmei Ren Ph.D. (Advisor); Michael Markey Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biochemistry; Biology

Master of Science (MS), Wright State University, 2022, Biochemistry and Molecular Biology

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that is characterized by severe and progressive muscle wasting (Venugopal & Pavlakis, 2021). This disease is caused by a mutation in the largest known human gene which encodes the protein, dystrophin (Gao & McNally, 2015). Dystrophin connects the inner cytoskeleton to the extracellular matrix and is critical for maintaining the structural stability of muscle cells during contraction (Venugopal & Pavlakis, 2021). Mutations to the dystrophin gene result in myocyte membrane instability, contributing to the structural deterioration of the muscle tissue (Venugopal & Pavlakis, 2021). Progressive muscle degeneration and the replacement of muscle fibers with fibrotic tissue negatively impacts muscle contractility and is particularly detrimental to health when essential muscles such as the diaphragm are affected (Mann et al., 2011). Respiratory failure is a hallmark of DMD and is one of the leading causes of mortality associated with this disease (Venugopal & Pavlakis, 2021). Currently there is no cure for Duchenne Muscular Dystrophy, and gene therapy approaches are limited by the sheer size of the dystrophin gene which spans across 2,200 kb of DNA (Gao & McNally, 2015). Previous data generated from the laboratory has shown that the mdx mouse (used to model DMD) displays reduced expression of lipin1 (Unpublished data). Additionally, other works have shown that skeletal muscle specific lipin1 knockout mice present muscle membrane instability (Sattiraju et al., 2020). Collectively, these findings suggest the potential for lipin1 to serve as an alternative therapeutic target in the dystrophic diaphragm. Within the membrane of the endoplasmic reticulum, lipin1 functions as a phosphatidic acid phosphatase (PAP), which catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DAG), a reaction important for membrane phospholipid and triacylglycerol synthesis (Chen et al., 2014). Current data sugg (open full item for complete abstract)

Committee: Hongmei Ren Ph.D. (Advisor); Weiwen Long Ph.D. (Committee Member); Michael Leffak Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

MS, University of Cincinnati, 2020, Medicine: Clinical and Translational Research

Background: Osteoporosis and vertebral fractures are common in patients with Duchenne Muscular Dystrophy (DMD) treated with glucocorticoids. Bisphosphonates (BP) have been used in pediatric patients for treatment of osteoporosis. However, the long-term effects of oral BP therapy on vertebral morphometry and fractures in patients with DMD and glucocorticoid-induced osteoporosis are not known. Methods: We retrospectively studied patients with DMD who had been treated with oral BP for glucocorticoid-induced osteoporosis at a tertiary-care pediatric center between 2010 and 2017. Demographic data, and glucocorticoid and oral BP treatment histories were obtained from electronic medical records. Treatment outcomes were changes in lumbar (L1-L4) vertebral morphometry and fractures, as assessed by the Genant semi-quantitative method. Patients were included if they had both baseline, within 6 months prior to start of oral BP treatment, and at least one follow-up spine radiograph available for review. The primary outcome was the prevalence of vertebral fractures and the secondary outcome was change in Genant grading during treatment. Results: Fifty-eight patients with DMD (median age 12.2 years, range 5.5-19.6 years) were treated with glucocorticoids for a median duration of 4.7 years (range 1.3-12.6 years) and 33% were non-ambulatory at BP start. Vertebral fractures, defined by Genant grading =1, were present in 6-19% of L1-L4 vertebrae at baseline. Among patients who had radiographs at baseline and 1 year-post BP start, the prevalence of L1-L4 vertebral fractures remained stable post treatment (n=43; 5-18% at baseline vs 8-18% at 1 year, p value 1.00). The prevalence of vertebral fractures at each year up to 5 years was also not statistically different from that of baseline (p-value 0.08-1.00). Among patients who had serial radiographs for comparison, longitudinal examination showed no change in Genant grading in the majority of vertebrae (64-80%) up (open full item for complete abstract)

Committee: Patrick Ryan Ph.D. (Committee Chair); Jane Khoury Ph.D. (Committee Member); Meilan Rutter (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Duchenne muscular dystrophy is a severe childhood-onset striated muscle disease that has no cure. The current treatment for skeletal muscle weakness has substantial side-effects. We have previously shown that treatment with drugs that inactivate the mineralocorticoid receptor (MR) improves skeletal muscle function in muscular dystrophy mice. To determine if these MR antagonists work through direct mechanisms in the skeletal muscle, we conditionally knocked out the myofiber MR in muscular dystrophy mice. Genetic ablation of MR improved skeletal muscle force and reduced fibrosis in muscular dystrophy mice similar to that observed with MR antagonist drugs. Additionally, MR antagonists stabilize fragile dystrophic skeletal muscle membranes in a MR independent manner, suggesting that these drugs have many benefits for skeletal muscle in muscular dystrophy. We then evaluated previously identified candidate MR responsive genes for their role in muscular dystrophy. None of the evaluated genes appeared to be direct myofiber specific MR targets. To investigate the role of the myofiber MR in normal muscle biology, we acutely injured the skeletal muscle of myofiber MR conditional knockout mice on a wild-type background. We found for the first time that acutely injured skeletal muscle has MR hormonal regulation and genetic ablation of the MR temporarily stabilized damaged myofibers at four days after acute muscle injury. Pharmacological inhibition of the MR with MR antagonist treatment delayed normal muscle repair in acute injury, suggesting additional roles for MR in other cell types in skeletal muscle contribute to regeneration after acute injury. These results have implications for MR modulation after acute and chronic skeletal muscle injuries.

Committee: Jill Rafael-Fortney (Advisor); Lawrence Kirschner (Committee Member); Sharon Amacher (Committee Member); Paul Martin (Committee Member) Subjects: Cellular Biology; Molecular Biology; Physiology

Doctor of Philosophy, The Ohio State University, 2016, Molecular, Cellular and Developmental Biology

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein as a result of truncating mutations in the DMD gene. Initial exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) because when skipped, an ORF can be restored and produce an internally deleted, yet partially functional protein. In contrast, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild type protein. To test this approach, we have developed a mouse model which contains a duplication of exon 2 (Dup2), the most common single exon duplication in patients. We are developing exon-skipping therapies for duplication mutations, which account for around 6%-11% of all duplication mutations by testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA) and antisense oligomer-induced skipping in the Dup2 mouse. Dose escalation studies of both intramuscular and systemic delivery of scAAV9.U7.ACCA show efficient skipping of exon 2, along with increased expression of properly localized dystrophin that restores muscle function. Furthermore, intramuscular injections of antisense oligomers results in widespread skipping and properly localized dystrophin protein. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Complete skipping of exon 2 results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin allowing for cap-independent translation from an alternative initiation site within exon 6. Each of the approximately 5% of patients harboring mutations 5' of the IRES in exon 5 have the potential to benefit from this highly functional dystrophin isoform.

Committee: Kevin Flanigan (Advisor); Scott Harper (Committee Member); Louise Rodino-Klapac (Committee Member); Denis Guttridge (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2014, Medicine: Molecular and Developmental Biology

Duchenne Muscular Dystrophy (DMD) and many of the limb girdle muscular dystrophies form a family of diseases called sarcoglycanopathies. In these diseases, mutation of any of a host of membrane and membrane associated proteins leads to increased stretch induced damage, aberrant signaling, and increased activity of non-specific cation channels, inducing muscle necrosis. Due to ongoing necrosis, DMD follows a progressive clinical course that leads to death in the mid-twenties. This course is slowed only modestly by high dose corticosteroids, which cause a plethora of harsh side effects. Targeted therapies are needed to ameliorate this disease until a more permanent therapy such as replacement of the mutated gene can be routinely performed. Here, we identified sodium calcium exchanger 1 (NCX1) as a potential therapeutic target. We started from the observation that sodium calcium exchanger 1 (NCX1) was upregulated during the necrotic phase of the disease in Sgcd-/- mice, which have similar pathology and mechanism of disease to boys with DMD. To test the causal effect of NCX1 overexpression on disease, we generated mice that overexpress NCX1 specifically in skeletal muscle. By Western blotting and immunofluorescence, we showed that NCX1 transgenic mice express more NCX1 protein in a similar localization pattern as endogenous NCX1. Sodium calcium exchange activity was also shown to be increased in NCX1 TG mice using an in situ sodium calcium exchange assay. When we examined the histology of NCX1 TG mice, we found that overexpression of NCX1 caused pathologic changes in skeletal muscle of the hindlimb. We then crossed the NCX1 trangene into Sgcd-/-, mdx, and Dysf-/- dystrophic backgrounds and found that the disease of these models was increased by the presence of the NCX1 transgene. Interestingly, NCX1 mediated opposing effects in the diaphragm, where NCX1 transgenic overexpression was protective in dystrophic backgrounds. To determine the effect of endogeno (open full item for complete abstract)

Committee: Jeff Molkentin Ph.D. (Committee Chair); James Lessard Ph.D. (Committee Member); Jerry Lingrel Ph.D. (Committee Member); Joshua Waxman Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Molecular Biology

MS, University of Cincinnati, 2014, Medicine: Clinical and Translational Research

Background: Duchenne and Becker muscular dystrophy (DBMD) are caused by mutations in dystrophin. Cardiac manifestations vary broadly across the population, making prognosis difficult. Current dystrophin genotype-cardiac phenotype correlations are limited. For skeletal muscle, the reading-frame rule suggests that in-frame mutations tend to yield milder phenotypes. Methods: A translational model was applied to patient-specific deletion, indel, and nonsense mutations to predict exons and protein domains present within the truncated dystrophin protein. Patients were dichotomized into predicted present and predicted absent groups for exons and protein domains of interest. Development of myocardial fibrosis (represented by late gadolinium enhancement (LGE)) and depressed left ventricular ejection fraction (LVEF) on CMR was compared. Findings: Patients (total n=274) with predicted present cysteine-rich domains (CRD) (n=34), C-terminal domains (CTD) (n=30), and both actin-binding domain 1 and cysteine-rich domain (ABD1+CRD) (n=21) had a decreased risk of LGE and trended toward greater freedom from LGE. Patients with in-frame mutations exactly overlapped with those with CTD predicted present. Patients with predicted present C-terminal domain/in-frame mutations and N-terminal actin-binding+cysteine-rich domains trended towards decreased risk of and greater freedom from depressed LVEF. Interpretation: Genotypes previously implicated in altering DBMD cardiac phenotype were not significantly related to LGE and depressed LVEF. Patients with predicted present cysteine-rich domain, C-terminal domain/in-frame mutations, and N-terminal actin-binding+cysteine-rich domains trended toward greater freedom from LGE and depressed LVEF, suggesting that the DBMD reading-frame rule may be applicable to the cardiac phenotype. Genotype-phenotype correlations may help predict the cardiac phenotype for DBMD patients and guide future therapies.

Committee: Erin Nicole Haynes Dr. P.H. (Committee Chair); Zhiqian Gao Ph.D. MSPH (Committee Member); Michael Taylor M.D. Ph.D. (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2011, Molecular, Cellular and Developmental Biology

Duchenne muscular dystrophy (DMD) is the most common form of the inherited muscular dystrophies. Cardiomyopathy is present in about 90% of patients and heart failure accounts for at least 20% of DMD-associated deaths. Dystrophin-deficient cardiomyopathy recapitulates many of the contractile phenotypes found in the majority of patients with end-stage dilated heart failure stemming from a variety of etiologies. Therefore, treatment targeting dystrophin-deficient cardiomyopathy may also be useful beyond the field of muscular dystrophy. New types of therapy can not be developed without understanding the molecular and cellular processes that contribute to heart failure. We set out to assess how contractility is dynamically regulated in healthy myocardium. We also used animal models of DMD to explore novel therapeutic treatments of cardiomyopathy. We investigated the beat-to-beat regulation of cardiac contractility in healthy myocardium under near physiological conditions. We developed a random cycle length (CL) approach, during which the twitch contractions of 5 different CLs were randomized around a physiological stimulation baseline. It was shown that the history of at least 3 CLs prior to a contraction influences myocardial contractility. The pattern of CL contribution to a given twitch is different between rat and dog, which have substantial differences in calcium handling. This suggests that investigation of calcium handling on a beat-to-beat basis will provide us with more insights into dynamic regulation of cardiac contractility. Calcium indicator bis-fura-2 was used to acquire calcium transients. Our data indicates that the changes in calcium transients are minor compared to the dramatic changes in contractile force in the cycle lengths protocol. The absolute systolic Ca2+ ion concentration is not the sole determinant in the calcium-force relationship in the isometric twitch of an intact muscle. We also explored novel therapeutic treatments of cardiomyopathy in (open full item for complete abstract)

Committee: Paul Janssen (Advisor); Jonathan Davis (Committee Member); Jill Rafael-Fortney (Committee Member); Mark Ziolo (Committee Member) Subjects: Biomedical Research; Medicine; Physiology

Doctor of Philosophy, The Ohio State University, 2006, Chemistry

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of functional dystrophin. Despite research focused on the replacement of the defective gene and the structure and function of dystrophin, there is no effective treatment of cure for this inevitably fatal disease. Rescue of dystrophin deficient, mdx, and utrophin/dystrophin deficient, dko, mouse models by the reintroduction of dystrophin has validated gene therapy as a therapeutic approach for DMD. Unfortunately the gene exceeds the capacity of adeno-associated viral (AAV) vectors. We investigated the expression of required dystrophin domains from two molecules as a means of expanding AAV capacity. Although the dystrophin proteins co-localize at the membrane, no improvement of dystrophic pathology is observed. Therefore, trans introduction of overlapping, truncated dystrophin proteins cannot be used to overcome cloning capacity limitations. The signaling mechanisms that lead from dystrophin deficiency to clinical DMD are largely unknown. We also conducted a proteomic analysis of phosphorylation differences between mdx and dko skeletal muscle and an immunoblot based examination of known signaling pathways. A serine phosphoprotein was observed in dko mitochondrial and microsome fractions but absent from corresponding mdx preparations. Unfortunately protein identification was inhibited by the abundance of actin in muscle. The nuclear factor-κB inhibitor, IκBβ, was observed to exhibit an increase presence in dko muscle hinged upon the additional presence of both a nuclear and cytosolic form of the protein. Finally, we examined the neuromuscular junction (NMJ) protein, Calmodulin-associated serine/threonine kinase (CASK) in the cellular components of the NMJ. CASK exhibits a developmentally regulated localization in the C2C12 myogenic cell line. It is observed in the nuclei of proliferating myoblasts, but is excluded to the cytosol in differentiating myotubes. CASK also exhibits a nucl (open full item for complete abstract)

Committee: Jill Rafael-Fortney (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, The Ohio State University, 2004, Educational Studies: Hums, Science, Tech and Voc

Duchenne Muscular Dystrophy (DMD) is a genetic disease that results from absence of the dystrophin protein, affects 1 in 3,500 male births, and leads to death by late teens to early twenties. Absence of the dystrophin protein leads to muscle wasting and cardiomyopathy due to compromised plasma membrane structure and impaired force transduction. Endurance exercise training involving cardiovascular overload may aid in maintenance and function of muscle. Hypotheses were tested to determine if endurance exercise training altered: a.) skeletal muscle fiber type composition; b.) regeneration and collagen accumulation in skeletal muscle; and c.) cardiomyopathic features in the mdx and delta-cys/mdx animals. Methods: Six groups of nine animals were studied: C57/BL10 (C57) sedentary, C57 swim, mdx-sedentary, mdx –swim, delta-cys/mdx-sedentary, and delta-cys/mdx-swim. Mice were progressively swim-trained five days/week for 10 weeks, while forelimb grip strength measurements were taken weekly and swim time to exhaustion was recorded at each session. Following training, soleus, extensor digitorum longus, quadriceps, diaphragm, and cardiac muscles were excised. Myosin heavy chain isoform composition, regeneration of myofibers and collagen content were examined. Cardiac tissue cross sections were examined for evidence of cardiomyopathy. Results: Exercise training led to an increase in forelimb grip strength in mdx, delta-cys/mdx, and C57 mice compared with non-swim trained mice. Swimming resulted in increases of type IIa and decreases of type IIx myosin heavy chain proportions in diaphragm muscles of mdx mice. Swim training did not cause increased damage in any skeletal muscle examined from mdx, delta-cys/mdx, or C57 animals. Furthermore, swimming did not cause significant increases in parameters of cardiomyopathy in dystrophic animals. Conclusion: The swim training protocol used in the present investigation resulted in training adaptations in mdx and delta-cys/mdx mice, and did (open full item for complete abstract)

Committee: Steven Devor (Advisor) Subjects: