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.

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.

Effects of gestational and postnatal exposure to chronic intermittent hypoxia on diaphragm muscle contractile function in the rat

Alterations to the supply of oxygen during early life presents a profound stressor to physiological systems with aberrant remodeling that is often long-lasting. Chronic intermittent hypoxia (CIH) is a feature of apnea of prematurity, chronic lung disease, and sleep apnea. CIH affects respiratory control but there is a dearth of information concerning the effects of CIH on respiratory muscles, including the diaphragm—the major pump muscle of breathing. We investigated the effects of exposure to gestational CIH (gCIH) and postnatal CIH (pCIH) on diaphragm muscle function in male and female rats. CIH consisted of exposure in environmental chambers to 90 s of hypoxia reaching 5% O2 at nadir, once every 5 min, 8 h a day. Exposure to gCIH started within 24 h of identification of a copulation plug and continued until day 20 of gestation; animals were studied on postnatal day 22 or 42. For pCIH, pups were born in normoxia and within 24 h of delivery were exposed with dams to CIH for 3 weeks; animals were studied on postnatal day 22 or 42. Sham groups were exposed to normoxia in parallel. Following gas exposures, diaphragm muscle contractile, and endurance properties were examined ex vivo. Neither gCIH nor pCIH exposure had effects on diaphragm muscle force-generating capacity or endurance in either sex. Similarly, early life exposure to CIH did not affect muscle tolerance of severe hypoxic stress determined ex vivo. The findings contrast with our recent observation of upper airway dilator muscle weakness following exposure to pCIH. Thus, the present study suggests a relative resilience to hypoxic stress in diaphragm muscle. Co-ordinated activity of thoracic pump and upper airway dilator muscles is required for optimal control of upper airway caliber. A mismatch in the force-generating capacity of the complementary muscle groups could have adverse consequences for the control of airway patency and respiratory homeostasis.

Early life exposure to chronic intermittent hypoxia primes increased susceptibility to hypoxia-induced weakness in rat sternohyoid muscle during adulthood

Intermittent hypoxia is a feature of apnea of prematurity (AOP), chronic lung disease, and sleep apnea. Despite the clinical relevance, the long-term effects of hypoxic exposure in early life on respiratory control are not well defined. We recently reported that exposure to chronic intermittent hypoxia (CIH) during postnatal development (pCIH) causes upper airway muscle weakness in both sexes, which persists for several weeks. We sought to examine if there are persistent sex-dependent effects of pCIH on respiratory muscle function into adulthood and/or increased susceptibility to re-exposure to CIH in adulthood in animals previously exposed to CIH during postnatal development. We hypothesized that pCIH would cause long-lasting muscle impairment and increased susceptibility to subsequent hypoxia. Within 24 h of delivery, pups and their respective dams were exposed to CIH: 90 s of hypoxia reaching 5% O2 at nadir; once every 5 min, 8 h per day for 3 weeks. Sham groups were exposed to normoxia in parallel. Three groups were studied: sham; pCIH; and pCIH combined with adult CIH (p+aCIH), where a subset of the pCIH-exposed pups were re-exposed to the same CIH paradigm beginning at 13 weeks. Following gas exposures, sternohyoid and diaphragm muscle isometric contractile and endurance properties were examined ex vivo. There was no apparent lasting effect of pCIH on respiratory muscle function in adults. However, in both males and females, re-exposure to CIH in adulthood in pCIH-exposed animals caused sternohyoid (but not diaphragm) weakness. Exposure to this paradigm of CIH in adulthood alone had no effect on muscle function. Persistent susceptibility in pCIH-exposed airway dilator muscle to subsequent hypoxic insult may have implications for the control of airway patency in adult humans exposed to intermittent hypoxic stress during early life.

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.

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.

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.

Immune and stress factors in the pathophysiology of the mdx mouse model of Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal multi-system neuromuscular disease caused by loss of dystrophin. The loss of dystrophin from membranes of contractile muscle cells and the dysregulation of the DAPC, induces chronic inflammation due to tissue necrosis and eventual replacement with collagen which weakens muscular force and strength. Dystrophin deficiency may cause under-diagnosed features of DMD include mood disorders such as depression and anxiety and dysfunction of the gastrointestinal tract. The first study in the thesis examined mood in the dystrophin-deficient mdx mouse model of DMD and examined the effects of the tri-cyclic antidepressant, amitriptyline on behaviours. Amitriptyline had anti-depressant and anxiolytic effects in the mdx mice possibly through effects on stress factors such as corticotrophin-releasing factor (CRF). This antidepressant also reduced skeletal muscle inflammation and caused a reduction in circulating interleukin (IL)-6 levels. In the second and third studies, we specifically blocked IL-6 signalling and used Urocortin 2, CRFR2 agonist to investigate their potential as therapeutic targets in mdx mice pathophysiology. Isometric and isotonic contractile properties of the diaphragm, were compared in mdx mice treated with anti IL-6 receptor antibodies (anti IL-6R) and/or Urocortin 2. Deficits in force production, work and power detected in mdx mice were improved with treatment. In study three I investigated contractile properties in gastrointestinal smooth muscle. As compared to wild type mice, mdx mice had slower faecal transit times, shorter colons with thickened muscle layers and increased contractile activity in response to recombinant IL-6. Blocking IL-6 signalling resulted in an increase in colon length, normalised faecal output times and a reduction in IL-6-evoked contractile activity. The findings from these studies indicate that for both diaphragm and gastrointestinal function in a dystrophin-deficient model, targeting of IL-6 and CRFR2 signalling has beneficial therapeutic effects.