Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

Nutraceutical and functional food bioactive peptides in beef

In recent years, the potential to positively modulate human health through dietary approaches has received considerable attention. Bioactive peptides which are released during the hydrolysis or fermentation of food proteins or following digestion may exert beneficial physiological effects in vivo. The aim of this work was to isolate, characterise and evaluate Angiotensin-І-converting enzyme (ACE-І) inhibitory, antimicrobial and antioxidant peptides from the bovine myofibrillar proteins actin and myosin. In order to generate these peptides, the myofibrillar proteins actin and myosin were hydrolysed with digestive enzymes pepsin, trypsin and α-chymotrypsin, or with the industrial thermolysin-like enzyme “Thermoase”, Amano Inc. It was found that each hydrolysate generated contained peptides which possessed ACE inhibitory, antioxidant and antimicrobial activity. The peptides responsible in part for the observed ACE inhibitory, antioxidant and antimicrobial activity of a number of hydrolysates were isolated using the method of RP-HPLC and the bioactive peptides contained within each active fraction was determined using either MALDI-TOF MS/MS or N-terminal peptide sequencing. During the course of this thesis six ACE inhibitory and five antimicrobial peptides were identified. It was determined that the reported antioxidant activity was a direct result of a number of peptides working in synergy with each other. The IC50 values of the six ACE inhibitory peptides ranged in values of 6.85 to 75.7 µM which compare favourably to values previously reported for other food derived ACE inhibitory peptides, particularly the well known milk peptides IPP and VPP, IC50 values of 5 and 9 µM respectively. All five antimicrobial peptides identified in this thesis displayed activity against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria innocua with MIC values ranging from 0.625 to10 mM. The activity of each antimicrobial peptide was strain specific. Furthermore the role and importance of charged amino acids to the activity of antimicrobial peptides was also determined. Generally the removal of charged amino acids from the sequence of antimicrobial peptides resulted in a loss of antimicrobial activity. In conclusion, this thesis revealed that a range of bioactive peptides exhibiting ACE inhibitory, antioxidant and antimicrobial activities were encrypted in bovine myofibrillar proteins that could be released using digestive and industrial enzymes. Finally enzymatic hydrolysates of muscle proteins could potentially be incorporated into functional foods; however, the potential health benefits would need to be proven in human clinical studies.

Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

Redox remodelling in diaphragm muscle adaptation to chronic sustained hypoxia

Chronic sustained hypoxia (CH) induces functional weakness, atrophy, and mitochondrial remodelling in the diaphragm muscle. Animal models of CH present with changes similar to patients with respiratory-related disease, thus, elucidating the molecular mechanisms driving these adaptations is clinically important. We hypothesize that ROS are pivotal in diaphragm muscle adaptation to CH. C57BL6/J mice were exposed to CH (FiO2=0.1) for one, three, and six weeks. Sternohyoid (upper airway dilator), extensor digitorum longus (EDL), and soleus were studied as reference muscles as well as the diaphragm. The diaphragm was profiled using a redox proteomics approach followed by mass spectrometry. Following this, redox-modified metabolic enzyme activities and atrophy signalling were assessed using spectrophotometric assays and ELISA. Diaphragm isotonic performance was assessed after six weeks of CH ± chronic antioxidant supplementation. Protein carbonyl and free thiol content in the diaphragm were increased and decreased respectively after six weeks of CH – indicative of protein oxidation. These changes were temporally modulated and muscle specific. Extensive remodelling of metabolic proteins occurred and the stress reached the cross-bridge. Metabolic enzyme activities in the diaphragm were, for the most part, decreased by CH and differential muscle responses were observed. Redox sensitive chymotrypsin-like proteasome activity of the diaphragm was increased and atrophy signalling was observed through decreased phospho-FOXO3a and phospho-mTOR. Phospho-p38 MAPK content was increased and this was attenuated by antioxidant treatment. Hypoxia decreased power generating capacity of the diaphragm and this was restored by N-acetyl-cysteine (NAC) but not by tempol. Redox remodelling is pivotal for diaphragm adaptation to chronic sustained hypoxia. Muscle changes are dependent on duration of the hypoxia stimulus, activity profile of the muscle, and molecular composition of the muscle. The working respiratory muscles and slow oxidative fibres are particularly susceptible. NAC (antioxidant) may be useful as an adjunct therapy in respiratory-related diseases characterised by hypoxic stress.

The actinin family of actin crosslinking proteins: natural functions and potential applications in synthetic biology

Actinin and spectrin proteins are members of the Spectrin Family of Actin Crosslinking Proteins. The importance of these proteins in the cytoskeleton is demonstrated by the fact that they are common targets for disease causing mutations. In their most prominent roles, actinin and spectrin are responsible for stabilising and maintaining the muscle architecture during contraction, and providing shape and elasticity to the red blood cell in circulation, respectively. To carry out such roles, actinin and spectrin must possess important mechanical and physical properties. These attributes are desirable when choosing a building block for protein-based nanoconstruction. In this study, I assess the contribution of several disease-associated mutations in the actinin-1 actin binding domain that have recently been linked to a rare platelet disorder, congenital macrothrombocytopenia. I investigate the suitability of both actinin and spectrin proteins as potential building blocks for nanoscale structures, and I evaluate a fusion-based assembly strategy to bring about self-assembly of protein nanostructures. I report that the actinin-1 mutant proteins display increased actin binding compared to WT actinin-1 proteins. I find that both actinin and spectrin proteins exhibit enormous potential as nano-building blocks in terms of their stability and ability to self-assemble, and I successfully design and create homodimeric and heterodimeric bivalent building blocks using the fusion-based assembly strategy. Overall, this study has gathered helpful information that will contribute to furthering the advancement of actinin and spectrin knowledge in terms of their natural functions, and potential unnatural functions in protein nanotechnology.

Diaphragm muscle adaptation to sustained hypoxia: lessons from animal models with relevance to high altitude and chronic respiratory diseases

The diaphragm is the primary inspiratory pump muscle of breathing. Notwithstanding its critical role in pulmonary ventilation, the diaphragm like other striated muscles is malleable in response to physiological and pathophysiological stressors, with potential implications for the maintenance of respiratory homeostasis. This review considers hypoxic adaptation of the diaphragm muscle, with a focus on functional, structural, and metabolic remodeling relevant to conditions such as high altitude and chronic respiratory disease. On the basis of emerging data in animal models, we posit that hypoxia is a significant driver of respiratory muscle plasticity, with evidence suggestive of both compensatory and deleterious adaptations in conditions of sustained exposure to low oxygen. Cellular strategies driving diaphragm remodeling during exposure to sustained hypoxia appear to confer hypoxic tolerance at the expense of peak force-generating capacity, a key functional parameter that correlates with patient morbidity and mortality. Changes include, but are not limited to: redox-dependent activation of hypoxia-inducible factor (HIF) and MAP kinases; time-dependent carbonylation of key metabolic and functional proteins; decreased mitochondrial respiration; activation of atrophic signaling and increased proteolysis; and altered functional performance. Diaphragm muscle weakness may be a signature effect of sustained hypoxic exposure. We discuss the putative role of reactive oxygen species as mediators of both advantageous and disadvantageous adaptations of diaphragm muscle to sustained hypoxia, and the role of antioxidants in mitigating adverse effects of chronic hypoxic stress on respiratory muscle function.

Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

The identification and characterisation of Rab4 interacting proteins

Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11.

Fractionation and compositional studies of rabbit skeletal muscle membranes

The observations of Hooke (1665), Schleiden & Schwann (1839) and Virchow (1855) led to the identification of the cell as the basic structural unit of living material. In the intervening years, it has been firmly established that the chemical processes which underlie the proper functioning, development and reproduction of the organism are cellular activities. The development of the electron microscope has enabled cell structure to be studied in detail. A picture of the cell as an entity with a complex and highly organised internal structure has emerged from the work of Palade, Porter, Fernandez-Moran and many others. Although cells from different tissues and organisms differ in aspects of their structure and consequently in function, they have several features in common. A retentive membrane encloses a number of cell constituents, which include membrane-enclosed subcellular structures known as organelles. The cells of most tissues also contain a reticulum or system of branching tubules. The interplay of the biochemical activities of these structures enables the cell to function. Almost thirty years ago, Claude, Palade, Schneider, Hogeboom, de Duve and others set out to analytically fractionate the subcellular components obtained after the fragmentation of liver cells. This approach has become known as subcellular fractionation, and signalled a major conceptual breakthrough in biochemistry (reviewed by de Duve, 1964, 1967, 1971). The significance of this breakthrough has been underlined by the award of the 1974 Nobel Prize in Medicine to de Duve, Palade and Claude. This thesis is concerned with the application of subcellular fractionation techniques to the separation and characterisation of the membrane systems of the rabbit skeletal muscle cell.

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Isolation, characterization, and functional assessment of novel smooth muscle like stem progenitors

Vascular smooth muscle cells (VSMC) are one of the key players in the pathogenesis of cardiovascular diseases. The origin of neointimal VSMC has thus become a prime focus of research. VSMC originate from multiple progenitors cell types. In embryo the well-defined sources of VSMC include; neural crest cells, proepicardial cells and EPC. In adults, though progenitor cells from bone marrow (BM), circulation and tissues giving rise to SMC have been identified, no progress has been made in terms of isolating highly proliferative clonal population of adult stem cells with potential to differentiate into SMC. Smooth muscle like stem progenitor cells (SMSPC) were isolated from cardiopulmonary bypass filters of adult patients undergoing CABG. Rat SMSPC have previously been isolated by our group from the bone marrow of Fischer rats and also from the peripheral blood of monocrotaline induced pulmonary hypertension (MCT-PHTN) animal model. Characterization of novel SMSPC exhibited stem cell characteristics and machinery for differentiation into SMC. The expression of Isl-1 on SMSPC provided unique molecular identity to these circulating stem progenitor cells. The functional potential of SMSPC was determined by monitoring adoptive transfer of GFP+ SMSPC in rodent models of vascular injury; carotid injury and MCT-PHTN. The participation of SMSPC in vascular pathology was confirmed by quantifying the peripheral blood, and engrafted levels of SMSPC using RT-PCR. In terms of translating into clinical practice, SMSPC could be a good tool for detecting the atherosclerotic plaque burden. The current study demonstrates the existence of novel adult stem progenitor cells in circulation, with the potential role in vascular pathology.

Characterization of the degradation of wild-type and mutant HFE proteins during stress signalling in the endoplasmic reticulum

HFE is a transmembrane protein that becomes N-glycosylated during transport to the cell membrane. It acts to regulate cellular iron uptake by interacting with the Type 1 transferrin receptor and interfering with its ability to bind iron-loaded transferrin. There is also evidence that HFE regulates systemic iron levels by binding to the Type II transferrin receptor although the mechanism by which this occurs is still not well understood. Mutations to HFE that disrupt this function, or physiological conditions that decrease HFE protein levels, are associated with increased iron uptake, and its accumulation in tissues and organs. This is exemplified by the point mutation that results in conversion of cysteine residue 282 to tyrosine (C282Y), and gives rise to the majority of HFE-related hemochromatoses. The C282Y mutation prevents the formation of a disulfide bridge and disrupts the interaction with its co-chaperone β2-microglobulin. The resulting misfolded protein is retained within the endoplasmic reticulum (ER) where it activates the Unfolded Protein Response (UPR) and is subjected to proteasomal degradation. The absence of functional HFE at the cell surface leads to unregulated iron uptake and iron loading. While the E3 ubiquitin ligase involved in the degradation of HFE-C282Y has been identified, the mechanism by which it is targeted for degradation remains relatively obscure. The primary objective of this project was to further our understanding of how the iron regulatory HFE protein is targeted for degradation. Our studies suggest that the glycosylation status, and the active process of deglycosylation, are central to this process. We identified a number of additional factors that can contribute towards degradation and explored their regulation during ER stress conditions.