BPAG1 isoform-b: Complex distribution pattern in striated and heart muscle and association with plectin and α-actinin

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

A hypoxia complement differentiates the muscle response to endurance exercise

Metabolic stress is believed to constitute an important signal for training-induced adjustments of gene expression and oxidative capacity in skeletal muscle. We hypothesized that the effects of endurance training on expression of muscle-relevant transcripts and ultrastructure would be specifically modified by a hypoxia complement during exercise due to enhanced glycolytic strain. Endurance training of untrained male subjects in conditions of hypoxia increased subsarcolemmal mitochondrial density in the recruited vastus lateralis muscle and power output in hypoxia more than training in normoxia, i.e. 169 versus 91% and 10 versus 6%, respectively, and tended to differentially elevate sarcoplasmic volume density (42 versus 20%, P = 0.07). The hypoxia-specific ultrastructural adjustments with training corresponded to differential regulation of the muscle transcriptome by single and repeated exercise between both oxygenation conditions. Fine-tuning by exercise in hypoxia comprised gene ontologies connected to energy provision by glycolysis and fat metabolism in mitochondria, remodelling of capillaries and the extracellular matrix, and cell cycle regulation, but not fibre structure. In the untrained state, the transcriptome response during the first 24 h of recovery from a single exercise bout correlated positively with changes in arterial oxygen saturation during exercise and negatively with blood lactate. This correspondence was inverted in the trained state. The observations highlight that the expression response of myocellular energy pathways to endurance work is graded with regard to metabolic stress and the training state. The exposed mechanistic relationship implies that the altitude specificity of improvements in aerobic performance with a 'living low-training high' regime has a myocellular basis.

Remodeling of calcium handling in skeletal muscle through PGC-1?: impact on force, fatigability, and fiber type

Regular endurance exercise remodels skeletal muscle, largely through the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α promotes fiber type switching and resistance to fatigue. Intracellular calcium levels might play a role in both adaptive phenomena, yet a role for PGC-1α in the adaptation of calcium handling in skeletal muscle remains unknown. Using mice with transgenic overexpression of PGC-1α, we now investigated the effect of PGC-1α on calcium handling in skeletal muscle. We demonstrate that PGC-1α induces a quantitative reduction in calcium release from the sarcoplasmic reticulum by diminishing the expression of calcium-releasing molecules. Concomitantly, maximal muscle force is reduced in vivo and ex vivo. In addition, PGC-1α overexpression delays calcium clearance from the myoplasm by interfering with multiple mechanisms involved in calcium removal, leading to higher myoplasmic calcium levels following contraction. During prolonged muscle activity, the delayed calcium clearance might facilitate force production in mice overexpressing PGC-1α. Our results reveal a novel role of PGC-1α in altering the contractile properties of skeletal muscle by modulating calcium handling. Importantly, our findings indicate PGC-1α to be both down- as well as upstream of calcium signaling in this tissue. Overall, our findings suggest that in the adaptation to chronic exercise, PGC-1α reduces maximal force, increases resistance to fatigue, and drives fiber type switching partly through remodeling of calcium transients, in addition to promoting slow-type myofibrillar protein expression and adequate energy supply.

Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of α-smooth muscle actin containing stress fibers

Rationale: Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether α-smooth muscle actin (α-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes. Objective: We investigated whether pharmacological ablation of α-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast–cardiomyocyte cross-talk. Methods and Results: Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of α-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes. Conclusions: The results suggest that α-SMA containing stress fibers importantly contribute to myofibroblast arrhythmogeneicity. After ablation of this cytoskeletal component, cells lose their arrhythmic effects on cardiomyocytes, even if heterocellular electrotonic coupling is sustained. The findings identify α-SMA containing stress fibers as a potential future target of antiarrhythmic therapy in hearts undergoing structural remodeling.

Cell Types as Natural Kinds

Talk of different types of cells is commonplace in the biological sciences. We know a great deal, for example, about human muscle cells by studying the same type of cells in mice. Information about cell type is apparently largely projectible across species boundaries. But what defines cell type? Do cells come pre-packaged into different natural kinds? Philosophical attention to these questions has been extremely limited [see e.g., Wilson (Species: New Interdisciplinary Essays, pp 187-207, 1999; Genes and the Agents of Life, 2005; Wilson et al. Philos Top 35(1/2): 189-215, 2007)]. On the face of it, the problems we face in individuating cellular kinds resemble those biologists and philosophers of biology encountered in thinking about species: there are apparently many different (and interconnected) bases on which we might legitimately classify cells. We could, for example, focus on their developmental history (a sort of analogue to a species' evolutionary history); or we might divide on the basis of certain structural features, functional role, location within larger systems, and so on. In this paper, I sketch an approach to cellular kinds inspired by Boyd's Homeostatic Property Cluster Theory, applying some lessons from this application back to general questions about the nature of natural kinds.

Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients

Several members of the human kallikrein-related peptidase family, including KLK6, are up-regulated in ovarian cancer. High KLK6 mRNA or protein expression, measured by quantitative polymerase chain reaction and enzyme-linked immunoassay, respectively, was previously found to be associated with a shortened overall and progression-free survival (OS and PFS, respectively). In the present study, we aimed at analyzing KLK6 protein expression in ovarian cancer tissue by immunohistochemistry. Using a newly developed monospecific polyclonal antibody, KLK6 immunoexpression was initially evaluated in normal tissues. We observed strong staining in the brain and moderate staining in the kidney, liver, and ovary, whereas the pancreas and the skeletal muscle were unreactive, which is in line with previously published results. Next, both tumor cell- and stromal cell-associated KLK6 immunoexpression were analyzed in tumor tissue specimens of 118 ovarian cancer patients. In multivariate Cox regression analysis, only stromal cell-associated expression, besides the established clinical parameters FIGO stage and residual tumor mass, was found to be statistically significant for OS and PFS [high vs. low KLK6 expression; hazard ratio (HR), 1.92; p=0.017; HR, 1.80; p=0.042, respectively]. These results indicate that KLK6 expressed by stromal cells may considerably contribute to the aggressiveness of ovarian cancer.

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF(164) levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Immediate and delayed transplantation of mesenchymal stem cells improve muscle force after skeletal muscle injury in rats

Mesenchymal stem cell (MSC) therapy is a promising approach for regaining muscle function after trauma. Prior to clinical application, the ideal time of transplantation has to be determined. We investigated the effects of immediate and delayed transplantation. Sprague-Dawley rats received a crush trauma to the left soleus muscle. Treatment groups were transplanted locally with 2 × 10(6) autologous MSCs, either immediately or 7 days after trauma. Saline was used as sham therapy. Contraction force tests and histological analyses were performed 4 weeks after injury. GFP-labelled MSCs were followed after transplantation. The traumatized soleus muscles of the sham group displayed a reduction of twitch forces to 36 ± 17% and of tetanic forces to 29 ± 11% of the non-injured right control side, respectively. Delayed MSC transplantation resulted in a significant improvement of contraction maxima in both stimulation modes (twitch, p = 0.011; tetany, p = 0.014). Immediate transplantation showed a significant increase in twitch forces to 59 ± 17% (p = 0.043). There was no significant difference in contraction forces between muscles treated by immediate and delayed cell transplantation. We were able to identify MSCs in the interstitium of the injured muscles up to 4 weeks after transplantation. Despite the fundamental differences of the local environment, which MSCs encounter after transplantation, similar results could be obtained with respect to functional muscle regeneration. We believe that transplanted MSCs residing in the interstitial compartment evolve their regenerative capabilities through paracrine pathways. Our data suggest a large time window of the therapeutical measures.

Leiomyomatoid angiomatous neuroendocrine tumor (LANT) of the pituitary: a distinctive biphasic neoplasm with primitive secretory phenotype and smooth muscle-rich stroma

We describe a hitherto undocumented variant of dimorphic pituitary neoplasm composed of an admixture of neurosecretory cells and profuse leiomyomatous stroma around intratumoral vessels. Radiologically perceived as a macroadenoma of 3.8 cm in diameter, this pituitary mass developed in an otherwise healthy 43-year-old female. At the term of a yearlong history of amenorrhea and progressive bitemporal visual loss, subtotal resection was performed via transsphenoidal microsurgery. Discounting mild hyperprolactinemia, there was no evidence of excess hormone production. Histologically, solid sheets, nests and cords of epithelial-looking, yet cytokeratin-negative cells were seen growing in a richly vascularized stroma of spindle cells. While strong immunoreactivity for NCAM, Synaptophysin and Chromogranin-A was detected in the former, the latter showed both morphological and immunophenotypic hallmarks of smooth muscle, being positive for vimentin, muscle actin and smooth muscle actin. Architectural patterns varied from monomorphous stroma-dominant zones through biphasic neuroendocrine-leiomyomatous areas, to pseudopapillary fronds along vascular cores. Only endothelia were labeled with CD34. Staining for S100 protein and GFAP, characteristics of sustentacular cells, as well as bcl-2 and c-kit was absent. Except for alpha-subunit, anterior pituitary hormones tested negative in tumor cells, as did a panel of peripheral endocrine markers, including serotonin, somatostatin, calcitonin, parathormone and vasoactive intestinal polypeptide. Mitotic activity was absent and the MIB-1 labeling index low (1-2%). While assignment of this lesion to any established neoplastic entity is not forthcoming, we propose it is being considered as a low-grade neuroendocrine tumor possibly related to null cell adenoma.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Primary perivascular epithelioid cell tumor of the liver not related to hepatic ligaments: hepatic PEComa as an emerging entity

Primary perivascular epithelioid cell tumor (PEComa) of the liver is a very rare example of an emerging family of hepatic PEC tumors. Only few cases have been described so far. We report the case of a large but benign hepatic PEComa in a 53-year-old man without signs of tuberous sclerosis. In contrast to recently described PEC-derived liver tumors in children and young adults, this neoplasm was not related to the hepatic ligaments but had developed deeply within the liver substance. The neoplastic cells displayed the complete phenotype typical for PEComas, i.e. reactivity for several melanoma markers and for smooth muscle actin. The unique relationship of myoid tumor cells to the adventitia of blood vessels prompted us, in comparison with published findings obtained with angiomyolipomas, to comment on the possible origin of the still enigmatic perivascular epithelioid cells.

Langerhans cell histiocytosis as differential diagnosis of a mediastinal tumor

We describe the case of a 55-year-old man who presented with parasternal swelling. The chest CT scan showed a large tumor of the chest wall infiltrating the subcutaneous tissue. To assume histologic diagnosis an open biopsy was performed. Between the myofibrils a coarse, white tumor with infiltrative growth was noted. Histopathologic examination revealed expanded atrophic skeletal muscle that was infiltrated by histiocytic cells. Numerous eosinophilic granulocytes and lymphocytes CD20 and CD3 positive could be detected and immunohistochemical staining was also positive for S-100 proteins and CD1a. Histologic findings were characteristic of Langerhans cell histiocytosis (LCH). To the best of our knowledge a LCH originating from the mediastinum in an adult as presented has not been previously described.

Medroxyprogesterone abrogates the inhibitory effects of estradiol on vascular smooth muscle cells by preventing estradiol metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.