Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

It is a well established phenomenon that the epithelial lining of the urinary bladder (urothelium) when implanted into skeletal muscle induces ectopic ossification. However, despite numerous observations, this reaction is poorly understood. This research further studied this reaction by - (a) demonstrating the reaction in a suitable small animal model; (b) attempting to induce the reaction by implanting urothelial cells purified by cell culture techniques; and (c) comparing the bone forming reaction induced by implanted urothelium to the reaction induced by implanting Bone Marrow Stem Cells (BMSC's) and Osteophyte Stem Cells (OSC's). By demonstrating newly formed bone after the implantation of guinea pig urothelium into the skeletal muscle of a Severe Combined Immuno-Deficient Mouse (SCID-Mouse) this research demonstrated that a suitable small animal model had been established. This is despite inherent difficulties (particularly bacterial contamination) associated with establishing a primary cell culture of guinea pig urothelial cells. Additionally, the intramuscular ectopic osteoinductive potential of human BMSC's (hBMSC's) in the SCID-mouse has also been demonstrated. Confirming that the injection of cultured cells in suspension is an adequate intramuscular delivery technique, this research demonstrates that hBMSC's induce ectopic ossification by non-immunological means. This research has demonstrated a number of differences between urothelium induced ectopic ossification and ectopic ossification induced by BMSC's, suggesting they are two separate processes. This is important because the chemotaxis and subsequent osteogenic differentiation of BMSC's has previously been one of the more popular postulated mechanisms of urothelium induced ectopic ossification. Finally, this research has demonstrated the ectopic osteoinductive potential of stem cells isolated from the marrow of human osteophytes (human Osteophyte Stem Cells, hOSC's). This observation has not been previously reported, and will hopefully provide a valuable contribution to a body of knowledge that has important ramifications in both the treatment of osteoarthritis, and the use of BMSC's in tissue engineering.

[Truncated abstract] Mutations in the skeletal muscle -actin gene (ACTA1) have been shown to be one cause of a broad group of muscle disorders all termed the congenital myopathies. Over 170 different mutations have now been identified across all 6 coding exons of ACTA1 in patients presenting with muscle weakness and any one or more of the following histopathological features: nemaline rods, intranuclear rods, fibre-type disproportion, excess of thin filaments and central cores. While the identification of the causative gene has been of great comfort for affected patients and their families, with pre-natal genetic testing becoming available, the ultimate aim is to develop a therapy for these disorders. Of the therapies currently being explored for the muscular dystrophies, up-regulation of an alternative gene seemed to be one of the most promising avenues for treatment of the ACTA1 diseases. Up-regulation of utrophin, the foetal homologue of dystrophin, has been shown to be a promising therapy for the treatment of Duchenne muscular dystrophy. The main aim of my research was to determine whether up-regulation of cardiac -actin, the predominant -actin expressed in foetal skeletal muscle and in the adult heart, could be used as a therapy for the ACTA1 diseases. A proof-of-concept experiment was performed whereby skeletal muscle -actin knock-out (KO) mice (all of which die by postnatal day 9) were crossed with transgenic mice over-expressing cardiac -actin (known as Coco mice) in postnatal skeletal muscle. ... While patients that are ACTA1 nulls have been identified in a number of mainly consanguineous populations, the majority of ACTA1 mutations result in dominant disease in which the mutant protein interferes with the function of the wild-type skeletal muscle -actin. Research described in this thesis also focuses on characterizing two transgenic mouse models of dominant ACTA1 disease at the ultra-structural, cellular and functional level; this is the first step towards a proof-of-concept experiment to determine whether cardiac -actin up-regulation can dilute out the pathogenesis of dominant ACTA1 disease. It has long been noted that patients with ACTA1 disease do not have ophthalmoplegia, even in the most-severely affected individuals. Protein analysis performed on extraocular muscle (EOM) biopsies obtained from humans, sheep and pigs showed that the EOMs co-express cardiac and skeletal muscle -actin, with cardiac -actin comprising 70 % of the striated -actin pool. Thus we propose that sparing of the EOMs in ACTA1 disease is at least in part due to cardiac -actin diluting out the pathogenesis associated with expression of the mutant skeletal muscle -actin. This finding provides further support for the hypothesis that dilution of mutant skeletal muscle -actin in dominant ACTA1 disease by up-regulation of cardiac -actin may be a viable therapy for this group of devastating muscle diseases. The research contained herein has advanced the understanding of the pathobiology of skeletal muscle -actin diseases and provides strong evidence in support of cardiac -actin up-regulation as a promising therapy for these diseases.

The dystrophinopathies are a group of disorders characterised by cellular absence of the membrane stabilising protein, dystrophin. Duchenne muscular dystrophy is the most severe disorder clinically. The deficiency of dystrophin, in the muscular dystrophy X-linked (mdx) mouse causes an elevation in intracellular calcium in cardiac myocytes. Potential mechanisms contributing to increased calcium include enhanced influx, sarcoplasmic reticular calcium release and\or reduced sequestration or sarcolemmal efflux. This dissertation examined the potential mechanisms that may contribute to an intracellular calcium overload in a murine model of muscular dystrophy. The general cardiomyopathy of the mdx myocardium was evident, with the left atria from mdx consistently producing less force than control atria. This was associated with delayed relaxation. The role of the L-type calcium channels mediating influx was initially investigated. Dihydropyridines had a lower potency in contracting left atria corresponding to a redued dihydropyridine receptor affinity in radioligand binding studies of mdx ventricular homogenates (P<0.05). This was associated with increased ventricular dihydropyridine receptor protein and mRNA levels (P<0.05). The function of the sarcoplasmic reticulum in terms of release and also sequestration of calcium via the sarco-endoplasmic reticulum ATPase were investigated. A lower force of contraction was evident in mdx left atria in response to a range of stimulation frequencies (P<0.05) and concentrations of extracellular calcium (P<0.05). However, in the presence of 1 nM Ryanodine to block sarcoplasmic reticular calcium release, increased stimulation frequency caused similar forces to those obtained in control mice suggesting enhanced calcium influx via L-type calcium channels in mdx. Rapid cooling contractures showed a reduced contracture in mdx compared to control in response to cooling. This suggests some dysfunction in SR storage, which may be associated with the delayed relaxation time. Concentration-response curves to inhibitors of the sarco-endoplasmic reticulum showed no difference in function of the enzyme responsible for calcium uptake into the sarcoplasmic reticulum. Although sarco-endoplasmic reticulum ATPase mRNA was upregulated, no functional benefit was evident. This study indicates that a deficiency of dystrophin leads to upregulation of L-type calcium channels that contribute to increased calcium influx, with no functional change in sarcoplasmic reticular sequestration. Upregulation of the influx pathway is a potential mechanism for the calcium overload observed in mdx cardiac muscle.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.