京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13354号 / 論医博第2200号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 木村 剛, 教授 羽賀 博典 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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In the heart, sodium (Na+) influx via NaV1.5 channels initiates the action potential, and calcium (Ca2+) influx via CaV1.2 channels has a key role in excitation-contraction coupling and determining the plateau phase of the action potential. Mutations in the genes that encode these ion channels or in proteins that modulate them are linked to arrhythmias and cardiomyopathy, underscoring the need for characterizing mechanisms of regulation. The work presented in this thesis is subdivided into three different chapters, each with a distinct focus on ion channel modulation. The first chapter details our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Physiologic β-adrenergic activation of PKA during the sympathetic “fight or flight” response increases Ca2+ influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. Recent data suggest that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. To test if any non-conserved sites are required for regulation, we generated mice with inducible cardiac-specific expression of α1C with mutations at both conserved and non- conserved predicted PKA phosphorylation sites (35-mutant α1C). Additionally, we createdanother mouse with inducible cardiac-specific expression of β2 with mutations at predicted PKA phosphorylation sites (28-mutant β2B). In each of these mice, β-adrenergic stimulation of Ca²⁺ current was unperturbed. Finally, to test the hypothesis that redundant functional PKA phosphorylation sites exist on the α1C subunit and β2 subunit or that several sites confer incremental regulation, we crossed the 35-mutant α1C mice with the 28-mutant β2B mice to generate offspring expressing both mutant subunits. In these offspring, intact regulation was observed. These results provide the definitive answer that phosphorylation of the α1C subunit or β2 subunit is not required for β-adrenergic regulation of CaV1.2 in the heart. In the second chapter, we study the influence of calmodulin and fibroblast growth homologous factor (FHF) FGF13 on late Na+ current. Studies in heterologous expression systems show that the Ca²⁺-binding protein calmodulin plays a key role in decreasing late Na⁺ current. The effect of loss of calmodulin binding to NaV1.5 on late Na+ current has yet to be resolved in native cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA), disrupting calmodulin binding to the C-terminus. Surprisingly, we found that the IQ/AA mutation did not cause an increase late Na⁺ current in cardiomyocytes. These findings suggest the existence of endogenous protective mechanisms that counteract the increase in late Na+ current that occurs with loss of calmodulin binding. We reasoned that FGF13, a known modulator of late Na+ current that is endogenously expressed in cardiomyocytes but not HEK cells, might play a protective role in limiting late Na+ current. Finally, we coexpressed the IQ/AA mutant NaV1.5 channel in HEK293 cells with FGF13 and found that FGF13 diminished the late Na⁺ currentcompared to cells without FGF13, suggesting that endogenous FHFs may serve to prevent late Na⁺ current in mouse cardiomyocytes. The third chapter of this thesis focuses on the use of proximity labeling and multiplexed quantitative proteomics to define changes in the NaV1.5 macromolecular complex in Duchenne muscular dystrophy (DMD), in which the absence of dystrophin predisposes affected individuals to arrhythmias and cardiac dysfunction.. Standard methods to characterize macromolecular complexes have relied on candidate immunoprecipitation or immunocytochemistry techniques that fall short of providing a comprehensive view of the numbers and types of interactors, as well as the potential dynamic nature of the interactions that may be perturbed by disease states. To provide an inclusive understanding of NaV1.5 macromolecular complexes, we utilize live-cell APEX2 proximity labeling in cardiomyocytes. We identify several proximal changes that align with the electrophysiological NaV1.5 phenotype of young dystrophin-deficient mice, including a decrease in Ptpn3 and Gdp1l and an increase in proteasomal machinery. Whole-cell protein expression fold-change results were used to reveal the altered global expression profile and to place context behind NaV1.5-proximal changes. Finally, we leveraged the neighborhood- specificity of proteins at the lateral membrane, intercalated disc, and transverse tubules of cardiomyocytes to demonstrate that NaV1.5 channels can traffic to all three membrane compartments even in the absence of dystrophin. Thus, the approach of proximity labeling in cardiomyocytes from an animal model of human disease offers new insights into molecular mechanisms of NaV1.5 dysfunction in DMD and provides a template for similar investigations in other cardiac diseases.

Sammanfattning  Bakgrund: Duchennes muskeldystrofi (DMD) är en ovanlig genetisk muskelsjukdom som beräknas drabba cirka tio pojkar i Sverige per år. Sjukdomen påverkas av en brist på proteinet dystrofin, vilket leder till en progressiv nedbrytning av skelett- och respiratoriska muskler samt myocardium. Detta leder till en successiv försämring av muskelstyrka samt ledrörlighet. Fysioterapeutiska åtgärder vid arbete med patientgruppen är framförallt inriktade mot att fördröja försämringen av sjukdomsförloppet. Syfte: Syftet med denna studie var att ge ökad förståelse för hur den fysioterapeutiska behandlingen ser ut för patienter med DMD, samt hur fysioterapeuter upplever arbetet med patientgruppen.  Metod: Fyra semistrukturerade intervjuer genomfördes med fysioterapeuter som hade erfarenhet av arbete med patientgruppen. Materialet från intervjuerna analyserades därefter med kvalitativ innehållsanalys enligt Graneheim och Lundman. Resultat: Analysen resulterade i tre huvudkategorier: “Mångfacetterad fysioterapeutisk behandling”, “Samarbete för att bidra till ett normalt liv” och “Komplex diagnos påverkar fysioterapeutens upplevelser”. Slutsats: Fysioterapeuterna uttryckte att huvudmålet för den fysioterapeutiska behandlingen var att ligga steget före med insatser samt att fördröja försämring. Att stötta patienten till ett delaktigt och självständigt liv ansågs som en nyckelkomponent i behandlingen. Arbetet med patientgruppen kunde stundom upplevas som svårt och sorgsamt, men också fridsamt och positivt. Fysioterapeuters upplevelser kan ge en inblick i vårdprocessen och därav bidra till utveckling av en bättre vård.

Dysferlin deficient muscular dystrophy is a devastating disease that leads to loss of mobility and quality of life in patients. Dysferlin is a 230 kD protein primarily expressed in skeletal muscle that functions in membrane resealing. Dysferlin loss of function leads to a decrease in the membrane resealing response after injury in skeletal muscle, which is thought to cause degeneration of the musculature over time. Dysferlin cDNA is 7.4 kb and exceeds AAV packaging capacity of ~ 5kb. This thesis focuses on the generation of mini dysferlin mutants that can be packaged in AAV for downstream testing of therapeutic efficacy. In addition, this thesis creates the groundwork for preclinical studies in mice that can potentially be translated to human patients. A mouse model for dysferlin deficiency was characterized and key disease phenotypes were identified. In addition, cell lines carrying a genetically encoded calcium indicator protein, gCaMP, were established to measure mini dysferlin resealing capacity and for downstream testing in vivo.

The extracellular matrix (ECM) of the skeletal muscle provides the framework for the muscle structure and plays a key role in the repair and maintenance of myofibers through the resident fibroblasts and muscle satellite cells. However, excessive production of ECM components, notably collagen, leads to fibrosis which impedes muscle function, impairs the natural repair process, and leads to muscle weakness. Fibrosis is a hallmark of muscular dystrophies, including Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy is a terminal, x-linked disorder characterized by progressive muscle wasting as muscle fibers are replaced by fibrosis and fat. There are approximately 300,000 DMD patients worldwide, and the few disease modifying treatments are genotype specific, only helping a small percentage of the patient population. Myostatin is a member of the transforming growth factor beta (TGF-β) family of ligands, is a negative regulator of muscle mass, and may also contribute to the fibrotic environment in dystrophic muscle through myofibroblast proliferation and survival. Therefore, myostatin blockade could potentially ameliorate muscle weakness in DMD patients by increasing skeletal mass and function while also reducing the accumulation of fibrosis. A murine anti-myostatin antibody, mRK35, and its humanized analogue, domagrozumab, are specific and potent inhibitors of myostatin. mRK35 was tested in multiple mouse models, from healthy C57Bl/6 and C57Bl/10, mildly dystrophic C57Bl/10.mdx, and severely dystrophic D2.mdx mice, for changes in muscle mass, muscle function, and fibrotic content. Additionally, inflammatory, fibrotic, and myogenic gene expression changes were analyzed in the severely dystrophic animals treated with mRK35. Domagrozumab was tested in non-human primates (NHPs) for changes in skeletal muscle mass. Myostatin blockade with mRK35 resulted in muscle anabolic and functional improvements in healthy murine models and NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume. However, as mice age or as the dystrophic severity of the model increases, the anabolic effect of myostatin inhibition is diminished. The extensor digitorum longus (EDL) muscle escapes this trend and is the most responsive to myostatin inhibition across all mouse strains and disease severities. However, analysis of the fibrotic content in the triceps and diaphragms of D2.mdx mice treated with mRK35 for 8 weeks does not reveal any change in fibrotic content. Gene expression changes in the muscles within these mice appear to be tightly tied to their healthy or dystrophic state and myostatin inhibition has minimal effect. In sum, while specific myostatin inhibition with mRK35 increases muscle weight and function in mice, there is no conclusive evidence of reduced muscle fibrosis. / 2023-06-22T00:00:00Z

Rare conditions can be catastrophic for families and the implications for public health can be substantial. Our study compared basic surveillance through active medical record review with a linked administrative data file to assess the number of cases of two rare conditions, fragile X syndrome (FXS) and muscular dystrophy (MD) in a population. Methods: Two methods of data collection were used to collect information from five counties comprising two standard metropolitan statistical areas of South Carolina. The passive system relied mostly on health claims data using ICD-9 CM diagnostic codes. The active system relied on a nurse abstracting records from a list of all licensed physicians with specialties in neurology, orthopedics, and genetics. Results: There were 141 FXS cases and 348 MD cases that met the case definitions using active surveillance. Additional cases were found for both conditions but they were determined to not be true cases. After linking the actively collected MD and FXS cases to passive datasets, we found that the estimated total numbers of cases were similar to using capture-recapture analysis; the positive predictive values for cases identified in the passive system were 56.6% for MD and 75.7% for FXS. Conclusions: Applying capture-recapture methods to passively collected surveillance data for rare health conditions produced an estimate of the number of true cases that was similar to that obtained through active data collection.

In this retrospective cohort study, we characterize the health profile of preterm males with Duchenne muscular dystrophy. Major clinical milestones (ambulation cessation, assisted ventilation use, and onset of left ventricular dysfunction) and corticosteroids use in males with Duchenne muscular dystrophy identified through a population-based surveillance system were analyzed using Kaplan-Meier survival curves and Cox proportional hazards modeling. The adjusted risk of receiving any respiratory intervention among preterm males with Duchenne muscular dystrophy was 87% higher than among the corresponding full-term males with Duchenne muscular dystrophy. The adjusted risks for ambulation cessation and left ventricular dysfunction were modestly elevated among preterm compared to full-term males, but the 95% confidence intervals contained the null. No difference in the start of corticosteroid use between preterm and full-term Duchenne muscular dystrophy males was observed. Overall, the disease course seems to be similar between preterm and full-term males with Duchenne muscular dystrophy; however, pulmonary function seems to be affected earlier among preterm males with Duchenne muscular dystrophy.

The objective of this study was to investigate whether males who were born preterm took longer to receive a Duchenne muscular dystrophy diagnosis than term males. Data for males with Duchenne muscular dystrophy identified through a population-based surveillance system were analyzed using a Kaplan-Meier estimator. The first signs and symptoms were noted at a median age of 2 years in both groups. Median age when first signs and symptoms prompted medical evaluation was 2.59 years among preterm and 4.01 years among term males. Median age at definitive diagnosis was 4.25 years and 4.92 years for preterm and term males, respectively. Neither difference was statistically significant. Preterm males tended to be seen for their initial medical evaluation earlier than term males, though they were not diagnosed significantly earlier. It may take clinicians longer after the initial evaluation of preterm males to arrive at a Duchenne muscular dystrophy diagnosis.

No description available.