Upregulated miR-224-5p suppresses osteoblast differentiation by increasing the expression of Pai-1 in the lumbar spine of a rat model of congenital kyphoscoliosis.

Congenital scoliosis is defined by the presence of structural anatomical malformations that arise from failures of vertebral formation or segmentation before and after birth. The understanding of genetic background and key genes for congenital scoliosis is still poor. We herein report that the excess expression of plasminogen activator inhibitor-1 (Pai-1) induced by the upregulation of miR-224-5p is involved in the pathogenesis of congenital kyphoscoliosis through impaired osteoblast differentiation. We first investigated the variety and progression of abnormalities of the lumbar spines in Ishibashi (IS) rats, a rat model of congenital kyphoscoliosis. The rats had already shown fusion and division of the primary ossification center at postnatal day 4. Over time, the rats showed various abnormalities of the lumbar spine, including the fusion of the annular epiphyseal nucleus. At postnatal day 42, spinal curvature was clearly observed due to the fusion of the vertebral bodies. Using a microRNA array, we found that the expression of miR-224-5p was increased in the lumbar spine of the rats at postnatal day 4. The expression of Pai-1, which is involved in osteoblast differentiation regulated by miR-224-5p, was also increased, while the levels of type I collagen, a marker of osteoblast differentiation, were decreased in the lumbar spine. These results indicate that the aberrant expression of miRNA-224-5p and its target genes is involved in the impaired osteoblast differentiation and may provide a partial molecular explanation for the pathogenesis of congenital scoliosis.

mRNA expressions of peroxisome proliferator-activated receptor gamma coactivator 1α, tumor necrosis factor-α, and interleukin-6 in paraspinal muscles of patients with lumbar kyphosis: a preliminary study.

BACKGROUND: Kyphosis is a spine deformity that can lead to falls and reduced quality of life. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) regulates mitochondrial biogenesis and is important for proper functioning of skeletal muscle, including the paraspinal muscles, which support and allow movement of the spine. The role of PGC-1α in paraspinal muscles in lumbar kyphosis has not been examined. We also examined the expressions of the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6. METHODS: We obtained paraspinal muscle specimens from 12 patients who underwent posterior lumbar surgery. RNA was isolated from these samples, and quantitative PCR was performed to compare the expression levels of PGC-1α, TNF-α, and IL-6 mRNA between patients with decreased lumbar lordosis (LL) and normal LL patients. RESULTS: TNF-α and IL-6 mRNA expressions in paraspinal muscles were significantly higher in the decreased LL group than in the normal LL group (P=0.048 for both). PGC-1α mRNA expression was slightly increased in the decreased LL group, but the difference was not significant. Age was significantly positively correlated with PGC-1α mRNA expression (P=0.010). PGC-1α mRNA expression was significantly positively correlated with TNF-α mRNA expression (P=0.022). LL was significantly negatively correlated with PGC-1α, TNF-α, and IL-6 mRNA expressions (P=0.015, 0.036, and 0.010, respectively). CONCLUSION: TNF-α and IL-6 mRNA expressions in paraspinal muscles were significantly higher in the decreased LL group than in the normal LL group. LL was significantly negatively correlated with PGC-1α, TNF-α, and IL-6 mRNA expressions. PGC-1α mRNA expression levels in paraspinal muscles may be affected by lumbar kyphosis.

Gene expression of matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor (VEGF) and fibroblast growth factor 23 (FGF23) in femur and vertebra tissues of the hypovitaminosis D kyphotic pig model.

The hypovitaminosis D kyphotic pig provides a reliable model to study the initiation of bone lesions caused by maternal vitamin D (D) deficiencies. Matrix metalloproteinases (MMP; specifically, MMP9 and MMP13) and vascular endothelial growth factor (VEGF) are important in endochondral ossification and are potentially regulated by D. Fibroblast growth factor 23 (FGF23) is interrelated with D homoeostasis and bone mineralisation. Relative mRNA expression of MMP9, MMP13, VEGF and FGF23 was measured in pig femur and vertebra. Sows (n 37) were fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg throughout gestation and lactation. At weaning (3 weeks), pigs were fed diets with 0 (-D) or 7·0 (+D) µg D3/kg, each with 75 and 95 % (LCaP) or 150 and 120 % (HCaP) of the Ca and P requirements. Pigs at birth (n 27), 3 weeks (n 27) and after the nursery period (7 weeks; n 72) were euthanised for analysis. At 3 weeks, femur MMP9 expression of pigs produced by +D or ++D sows was reduced (P<0·05) to 0·5-fold and VEGF expression to 0·4-fold compared with pigs from -D sows. At 7 weeks, MMP9 expression was reduced (P<0·05) to 0·45-fold in femur and 0·58-fold in vertebra from pigs produced by +D or ++D sows compared with pigs from -D sows. Pig femur VEGF expression was reduced to 0·75-fold in pigs produced by ++D sows. MMP9 and VEGF mRNA expression offer potential markers for the initiation of bone lesions in the hypovitaminosis D kyphotic pig model.

Polymethylmethacrylate distribution is associated with recompression after vertebroplasty or kyphoplasty for osteoporotic vertebral compression fractures: A retrospective study.

BACKGROUND: Osteoporotic vertebral compression fracture, always accompanied with pain and height loss of vertebral body, has a significant negative impact on life quality of patients. Vertebroplasty or kyphoplasty is minimal invasive techniques to reconstruct the vertebral height and prevent further collapse of the fractured vertebrae by injecting polymethylmethacrylate into vertebral body. However, recompression of polymethylmethacrylate augmented vertebrae with significant vertebral height loss and aggressive local kyphotic was observed frequently after VP or KP. The purpose of this study was to investigate the effect of polymethylmethacrylate distribution on recompression of the vertebral body after vertebroplasty or kyphoplasty surgery for osteoporotic vertebral compression fracture. METHODS: A total of 281 patients who were diagnosed with vertebral compression fracture (T5-L5) from June 2014 to June 2016 and underwent vertebroplasty or kyphoplasty by polymethylmethacrylate were retrospectively analyzed. The X-ray films at 1 day and 12 months after surgery were compared to evaluate the recompression of operated vertebral body. Patients were divided into those without recompression (non-recompression group) and those with recompression (recompression group). Polymethylmethacrylate distribution pattern, including location and relationship to endplates, was compared between the two groups by lateral X-ray film. Multivariate logistic regression analysis was performed to assess the potential risk factors associated with polymethylmethacrylate distribution for recompression. RESULTS: One hundred and six (37.7%) patients experienced recompression after surgery during the follow-up period. The polymethylmethacrylate distributed in the middle of vertebral body showed significant differences between two groups. In non-recompression group, the polymethylmethacrylate in the middle portion of vertebral body were closer to endplates than that in the recompression group (upper: t = 31.41, p<0.001; lower: t = 12.19, p<0.001). The higher percentage of the height of polymethylmethacrylate in the middle portion of vertebral body indicates the lower risk of recompression (odds ratio [OR]<0.01, p<0.001). The recompression group and non-recompression group showed significant difference in "contacted" polymethylmethacrylate distribution pattern (polymethylmethacrylate contacted to the both upper/lower endplates) (χ2 = 66.23, p<0.001). The vertebra with a "contacted" polymethylmethacrylate distribution pattern has lower risk of recompression (OR = 0.09, p<0.001). CONCLUSIONS: Either more polymethylmethacrylate in the middle portion of vertebral body or "contacted" polymethylmethacrylate distribution pattern had a significantly less incidence of recompression. The findings indicated that the control of polymethylmethacrylate distribution during surgery may reduce the risks of recompression after vertebroplasty or kyphoplasty.

Surgical Outcomes of Single-Level Transforaminal Lumbar Interbody Fusion for Degenerative Spondylolisthesis With and Without Kyphotic Alignment.

OBJECTIVE: We sought to investigate the impact of single-level transforaminal lumbar interbody fusion (TLIF) on lumbar sagittal profile in degenerative spondylosis (DS) patients with or without kyphotic alignment, as well as compare radiologic and clinical outcomes based on preoperative sagittal alignment. BACKGROUND: DS with a kyphotic alignment at an involved segment constitutes a distinct subgroup. However, previous studies concerning surgical outcomes often lump all patients together without focusing on this distinct subgroup. METHODS: This study reviewed a consecutive series of patients who received single-level TLIF for DS between 2009 and 2016. They were assigned to Groups K and L. All patients were followed up for >2 years. Then demographics and radiographic and clinical outcomes were compared between the groups. RESULTS: There were 19 and 115 patients in Group K and Group L, respectively. Compared with Group L, Group K was characterized by loss of lumbar lordosis and anterior shifting of L1 axis S1 distance. After surgery, lumbar lordosis and L1 axis S1 distance was significantly improved in Group K, while no significant change occurred in group L. The mean reduction rate was significantly higher in Group K, which had less slippage after surgery, but the differences in slip angle, anterior disk height, and posterior disk height were not significant. The preoperative Oswestry Disability Index and visual analog scale for back pain scores were significantly higher in Group K, while no differences were found in postoperative evaluation. CONCLUSIONS: Despite effective changes radiographic parameters and clinical outcomes, our findings suggest that the 2 groups behave differently in response to single-level TLIF procedure. Reconstruction of lumbar sagittal profile can be achieved in DS with a kyphotic alignment after surgery.

GdX/UBL4A null mice exhibit mild kyphosis and scoliosis accompanied by dysregulation of osteoblastogenesis and chondrogenesis.

GdX, also named ubiquitin-like protein 4A, is a ubiquitin-domain protein characterized by a ubiquitin-like domain that regulates the movement of misfolded proteins from the endoplasmic reticulum membrane to proteasome. However, its function in skeletal biology remains unclear. Here, we report that GdX plays a crucial role in skeletal development as mice lacking GdX exhibit skeletal dysplasias, mild kyphosis, and scoliosis. During embryonic stage, GdX knockout mice display decreased bone mineral density and trabecular bone accompanied by delayed osteogenic formation. GdX knockout mice also have blended spine and small body size. At the molecular level, GdX knockout mice showed perturbed expression of osteogenesis-related genes and cartilage developmental genes, indicative of altered differentiation of mesenchymal cell lineage. Collectively, our results uncovered GdX as a novel regulator in bone development and a potential candidate gene for skeletal dysplasias.

Serum and tissue 25-OH vitamin D3 concentrations do not predict bone abnormalities and molecular markers of vitamin D metabolism in the hypovitaminosis D kyphotic pig model.

The hypovitaminosis D kyphotic pig provides a model to study maternal vitamin D (D) carryover on gross and molecular characteristics of bone abnormalities in offspring. Excess maternal D is proposed to protect offspring under nutritional challenges from developing bone abnormalities. Relationships between D sufficiency parameters and bone abnormalities were characterised. Sows (n 37) were fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg throughout gestation and lactation. At weaning (3 weeks) pigs were fed diets with 0 (-D) or 7·0 (+D) µg D3/kg, each with 75 and 95 % (LCaP) or 150 and 120 % (HCaP) of the Ca and P requirements. Pigs were euthanised before colostrum consumption at birth (n 27), 3 weeks (n 27) or after the nursery period (7 weeks, n 71) for tissue analysis. At 7 weeks, differences due to maternal D were detected (P≤0·05) in pig growth, serum parameters and mRNA expression regardless of nursery diet. Prevalence of kyphosis in pigs at 13 weeks was affected by maternal D, but not prevented by only HCaP or +D nursery diets. Increased (P≤0·05) serum 25-OH-D3 concentrations in sows fed +D or ++D diets were not reflected by similar magnitudes of 25-OH-D3 in colostrum, 18-d milk, or serum and tissue concentrations in pigs. The mode of action by which maternal dietary D influences development of skeletal abnormalities warrants further investigation.

Volumetric and Fatty Infiltration Imbalance of Deep Paravertebral Muscles in Adolescent Idiopathic Scoliosis.

BACKGROUND Several studies have described the differences in electromyographic activity and histological changes of paravertebral muscles in patients with adolescent idiopathic scoliosis (AIS). However, there is little knowledge about the muscle volumetric and fatty infiltration imbalance of patients with AIS. MATERIAL AND METHODS Thirty-four patients with AIS were evaluated with standardized anteroposterior (AP) and lateral standing films for the location and direction of the apex of scoliosis, coronal Cobb angle, apex vertebra translation, and thoracic kyphosis; and with magnetic resonance imaging (MRI) scan of the spine at the level of T4-L1. The muscle volume and fatty infiltration rate of bilateral deep paravertebral muscles at the level of upper end, apex, and lower end vertebra were measured. RESULTS All patients had major thoracic curve with apex of curves on the right side. The muscle volume on the convex side was larger relative to the concave side at the three levels, while the fatty infiltration rate was significantly higher on the concave side. The difference index of the muscle volume was significantly larger at the apex vertebra level than at the upper end vertebra level (p=0.002) or lower end vertebra level (p<0.001). The difference index of muscle volume correlated with apex vertebra translation (r=-0.749, p=0.032), and the difference index of fatty involution correlated with apex vertebra translation (r=0.727, p=0.041) and Cobb angle (r=0.866, p=0.005). CONCLUSIONS Our findings demonstrated significant imbalance of muscle volume and fatty infiltration in deep paravertebral muscles of AIS patients. Moreover, these changes affected different vertebra levels, with the most imbalance of muscle volume at the apex vertebra. We interpreted this as morphological changes corresponding with known altered muscle function of AIS.

Mice with an N-Ethyl-N-Nitrosourea (ENU) Induced Tyr209Asn Mutation in Natriuretic Peptide Receptor 3 (NPR3) Provide a Model for Kyphosis Associated with Activation of the MAPK Signaling Pathway.

Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.

The Trk family of neurotrophin receptors is downregulated in the lumbar spines of rats with congenital kyphoscoliosis.

Congenital scoliosis is a condition characterized by spinal curvature beyond the physiological norm. The molecular mechanisms underlying the pathogenesis of congenital scoliosis are beginning to be clarified; however, the genes related to congenital scoliosis are still unknown. We herein report the results of a comprehensive analysis of gene expression in the spines from a rat model of congenital kyphoscoliosis obtained using DNA microarrays. The rats (Ishibashi rats, IS) showed decreased expression levels of genes associated with bone formation, such as those associated with retinol metabolism and type I collagen. Interestingly, the flexion sites of the IS rats showed low expression levels of tropomyosin receptor kinases (Trks: TrkA, TrkB, and TrkC), which belong to the neurotrophic receptor tyrosine kinase family. Moreover, this phenomenon was observed only in the flexion sites of the spine, and the expression levels of Trks in other parts of the spine in these rats were normal. The decreased expression levels of Trks were observed at both the mRNA and protein levels. We also observed that the number of Trk-immunopositive cells in the lumbar spine in the IS rats was lower than that in wild-type rats. These findings indicate that the Trks have an important function in regulating normal bone formation, and provide a molecular explanation for the pathogenesis of congenital kyphoscoliosis.

Loss of Nfkb1 leads to early onset aging.

NF-κB is a major regulator of age-dependent gene expression and the p50/NF-κB1 subunit is an integral modulator of NF-κB signaling. Here, we examined Nfkb1-/- mice to investigate the relationship between this subunit and aging. Although Nfkb1-/- mice appear similar to littermates at six months of age, by 12 months they have a higher incidence of several observable age-related phenotypes. In addition, aged Nfkb1-/- animals have increased kyphosis, decreased cortical bone, increased brain GFAP staining and a decrease in overall lifespan compared to Nfkb1+/+. In vitro, serially passaged primary Nfkb1-/- MEFs have more senescent cells than comparable Nfkb1+/+ MEFs. Also, Nfkb1-/- MEFs have greater amounts of phospho-H2AX foci and lower levels of spontaneous apoptosis than Nfkb1+/+, findings that are mirrored in the brains of Nfkb1-/- animals compared to Nfkb1+/+. Finally, in wildtype animals a substantial decrease in p50 DNA binding is seen in aged tissue compared to young. Together, these data show that loss of Nfkb1 leads to early animal aging that is associated with reduced apoptosis and increased cellular senescence. Moreover, loss of p50 DNA binding is a prominent feature of aged mice relative to young. These findings support the strong link between the NF-κB pathway and mammalian aging.

Constitutive expression of Yes-associated protein (Yap) in adult skeletal muscle fibres induces muscle atrophy and myopathy.

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5-7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20-25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34-40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.

Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0)) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal.

Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders.

Osteocrin is a specific ligand of the natriuretic Peptide clearance receptor that modulates bone growth.

Osteocrin (Ostn) is a recently discovered secreted protein produced by cells of the osteoblast lineage that shows a well conserved homology with members of the natriuretic peptide (NP) family. We hypothesized that Ostn could interact with the NP receptors, thereby modulating NP actions on the skeleton. Ostn binds specifically and saturably to the NP peptide receptor-C (NPR-C) receptor with a Kd of approximately 5 nM with no binding to the GC-A or GC-B receptors. Deletion of several of the residues deemed important for NP binding to NPR-C led to abolition of Ostn binding, confirming the presence of a "natriuretic motif." Functionally, Ostn was able to augment C-type natriuretic peptide-stimulated cGMP production in both pre-chondrocytic (ATDC5) and osteoblastic (UMR106) cells, suggesting increased NP levels due to attenuation of NPR-C associated NP clearance. Ostn-transgenic mice displayed elongated bones and a marked kyphosis associated with elevated bone cGMP levels, suggesting that elevated natriuretic peptide activity contributed to the increased bone length possibly through an increase in growth plate chondrocyte proliferation. Thus, we have demonstrated that Ostn is a naturally occurring ligand of the NPR-C clearance receptor and may act to locally modulate the actions of the natriuretic system in bone by blocking the clearance action of NPR-C, thus locally elevating levels of C-type natriuretic peptide.

Correlation between cytogenetic abnormalities in cells and metabolic shifts in children with spinal deformities.

We studied the relationship between cytogenetic abnormalities in buccal epithelial cells and metabolic shifts in children with scoliosis and kyphosis. The incidence of nucleus abnormalities and the corresponding metabolic shifts were found to depend on the presence of spinal deformities and ecological factors. The problem of formation of risk groups for spinal deformities in ecologically unfavorable regions is discussed.

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS.

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease.

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.

Esophageal transit of the weekly film-coated risedronate (Actonel) placebo tablet in subjects with Kyphosis.

Risedronate sodium is a pyridinyl bisphosphonate of proven effectiveness for the treatment and prevention of osteoporosis and Paget's disease of the bone. The aim of this study was to compare the esophageal transit and gastric emptying of the placebo film-coated risedronate tablet when taken with 50 or 120 mL of water in subjects with Kyphosis. A total of 23 patients with radiologically documented osteoporosis participated in a single-center, open-label, crossover gamma scintigraphy study. The mean esophageal transit times were 15.6 s (50 mL) and 12.0 s (120 mL) and the mean gastric emptying half-times were 20.5 min (50 mL) and 14.3 min (120 mL). There was no relationship between the degree of Kyphosis measured from lateral standing radiographs and the esophageal transit time. This study demonstrated that even when taken with a minimal volume of water the esophageal transit and gastric emptying of the film-coated 35 mg weekly risedronate placebo tablet was similar in kyphotic subjects to previously obtained results from healthy control subjects.

Progression of kyphosis in mdx mice.

Spinal deformity in the form of kyphosis or kyphoscoliosis occurs in most patients with Duchenne muscular dystrophy (DMD), a fatal X-linked disorder caused by an absence of the subsarcolemmal protein dystrophin. Mdx mice, which also lack dystrophin, show thoracolumbar kyphosis that progresses with age. We hypothesize that paraspinal and respiratory muscle weakness and fibrosis are associated with the progression of spinal deformity in this mouse model, and similar to DMD patients there is evidence of altered thoracic conformation and area. We measured kyphosis in mdx and age-matched control mice by monthly radiographs and the application of a novel radiographic index, the kyphotic index, similar to that used in boys with DMD. Kyphotic index became significantly less in mdx at 9 mo of age (3.58 +/- 0.12 compared with 4.27 +/- 0.04 in the control strain; P < or = 0.01), indicating more severe kyphosis, and remained less from 10 to 17 mo of age. Thoracic area in 17-mo-old mdx was reduced by 14% compared with control mice (P < or = 0.05). Peak tetanic tension was significantly lower in mdx and fell 47% in old mdx latissimus dorsi muscles, 44% in intercostal strips, and 73% in diaphragm strips (P < or = 0.05). Fibrosis of these muscles and the longissimus dorsi, measured by hydroxyproline analysis and histological grading of picrosirius red-stained sections, was greater in mdx (P < 0.05). We conclude that kyphotic index is a useful measure in mdx and other kyphotic mouse strains, and assessment of paralumbar and accessory respiratory muscles enhance understanding of spinal deformity in muscular dystrophy.