Browsing by Subject "skeletal muscle"
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(2023)The group has identified two rare, previously uncharacterized missense variants in the YBX3 gene in a Finnish patient presenting with an unusual form of nemaline myopathy. The patient also inherited two biallelic TPM3 variants, one RYR1 variant from the father and one SRPK3 variant from the mother. TPM3 and RYR1 are known nemaline myopathy causing genes and the other variants identified in the patients, including the YBX3 variants, are thought to have a modifying effect on the phenotype. YBX3 encodes Y-box binding protein 3 (YB-3) and, YB-3 is a member of the Y-box binding (YB) protein family, that in addition to YB-3 consists of YB-1 and YB-2. The YB-proteins have mainly been studied in the context of cancer, with most studies focusing on YB-1. Studies indicate the ability of YB-proteins to compensate for the loss of one homolog suggesting functional redundancy between YB-3 and YB-1, and YB-3 and YB-2. Compared to its homologs, YB-3 is highly expressed in skeletal muscle. The aim of this thesis was to try out a new cell culturing method when investigating the role of YB-3 in the differentiation of myoblasts into myotubes. MSY-3 is the murine orthologue of YB-3. MSY3-knockdown mouse C2C12 myoblast lines were established using GIPZ lentiviral short hairpin constructs and by selection with puromycin. The success of transfection was determined using qPCR. The myoblasts were differentiated for 20 days on a gelatin hydrogel surface to support long-term culture and to provide phenotypes of higher physiological relevance with improved contractile maturity. Myoblasts cultured on coverslips were immunofluorescently stained for MSY-3. HeLa cells were transfected with a construct encoding N-terminally FLAG-tagged human YB-3 in a pcDNA-vector. YB-3-FLAG was purified using anti-FLAG magnetic beads. The eluated immunoprecipitation sample was sent to N-terminal sequencing to obtain information on post-translational modifications, to support further experiments regarding the post-translational cleavage of YB-3. N-terminal sequencing revealed an enrichment of YB-3 and YB-1 in the immunoprecipitation sample but not of YB-2, and previously undescribed post-translational modifications were identified. The MSY3-knockdown myotubes exhibited no spontaneous twitching on the hydrogel, while the control C2C12 myotubes twitched frequently. Misalignment of the MSY3-knockdown myotubes and changes in morphology was also observed in one of the MSY3-knockdown cell lines. This suggests that differentiating myoblasts on gelatin hydrogel is a potential strategy for studying the functions of YB-3 in myoblast differentiation and to elucidate its role in skeletal muscle.
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(2021)Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.
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