Calcineurin activation influences muscle phenotype in a muscle-specific fashion

Background  

The calcium activated protein phosphatase 2B, also known as calcineurin, has been implicated as a cell signaling molecule involved with transduction of physiological signals (free cytosolic Ca²⁺) into molecular signals that influence the expression of phenotype-specific genes in skeletal muscle. In the present study we address the role of calcineurin in mediating adaptations in myosin heavy chain (MHC) isoform expression and muscle mass using 3-month old wild-type (WT) and transgenic mice displaying high-level expression of a constitutively active form of calcineurin (MCK-CN* mice).     

Hybridization increases mitochondrial production of reactive oxygen species in sunfish

Mitochondrial dysfunction and oxidative stress have been suggested to be possible mechanisms underlying hybrid breakdown, as a result of mito‐nuclear incompatibilities in respiratory complexes of the electron transport system. However, it remains unclear whether hybridization increases the production of reactive oxygen species (ROS) by mitochondria. We used high‐resolution respirometry and fluorometry on isolated liver mitochondria to examine mitochondrial physiology and ROS emission in naturally occurring hybrids of pumpkinseed (Lepomis gibbosus) and bluegill (L. macrochirus). ROS emission was greater in hybrids than in both parent species when respiration was supported by complex I (but not complex II) substrates, and was associated with increases in lipid peroxidation. However, respiratory capacities for oxidative phosphorylation, phosphorylation efficiency, and O₂ kinetics in hybrids were intermediate between those in parental species. Flux control ratios of capacities for electron transport (measured in uncoupled mitochondria) relative to oxidative phosphorylation suggested that the limiting influence of the phosphorylation system is reduced in hybrids. This likely helped offset impairments in electron transport capacity and complex III activity, but contributed to augmenting ROS production. Therefore, hybridization can increase mitochondrial ROS production, in support of previous suggestions that mitochondrial dysfunction can induce oxidative stress and thus contribute to hybrid breakdown.     

Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function

Adiponectin (Ad) is linked to various disease states and mediates antidiabetic and anti-inflammatory effects. While it was originally thought that Ad expression was limited to adipocytes, we demonstrate here that Ad is expressed in mouse skeletal muscles and within differentiated L6 myotubes, as assessed by RT-PCR, Western blot, and immunohistochemical analyses. Serial muscle sections stained for fiber type, lipid content, and Ad revealed that muscle fibers with elevated intramyocellular Ad expression were consistently type IIA and IID fibers with detectably higher intramyocellular lipid (IMCL) content. To determine the effect of Ad on muscle phenotype and function, we used an Ad-null [knockout (KO)] mouse model. Body mass increased significantly in 24-wk-old KO mice [+5.5 +/- 3% relative to wild-type mice (WT)], with no change in muscle mass observed. IMCL content was significantly increased (+75.1 +/- 25%), whereas epididymal fat mass, although elevated, was not different in the KO mice compared with WT (+35.1 +/- 23%; P = 0.16). Fiber-type composition was unaltered, although type IIB fiber area was increased in KO mice (+25.5 +/- 6%). In situ muscle stimulation revealed lower peak tetanic forces in KO mice relative to WT (-47.5 +/- 6%), with no change in low-frequency fatigue rates. These data demonstrate that the absence of Ad expression causes contractile dysfunction and phenotypical changes in skeletal muscle. Furthermore, we demonstrate that Ad is expressed in skeletal muscle and that its intramyocellular localization is associated with elevated IMCL, particularly in type IIA/D fibers.     

The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype

Cells receive signals from the extracellular matrix through receptor-dependent interactions, but they are also influenced by the mechanical properties of the matrix. Although bulk properties of substrates have been shown to affect cell behavior, we show here that nanoscale properties of collagen fibrils also play a significant role in determining cell phenotype. Type I collagen fibrils assembled into thin films provide excellent viewing of cells interacting with individual fibrils. Cells can be observed to extensively manipulate the fibrils, and this behavior seems to result in an incompletely spread stellate morphology and a nonproliferative phenotype that is typical of these cells in collagen gels. We show here that thin films of collagen fibrils can be dehydrated, and when seeded on these dehydrated fibrils, smooth muscle cells spread and proliferate extensively. The dehydrated collagen fibrils appear to be similar to the fully hydrated collagen fibrils in topology and in presentation of β₁ integrin ligation sites, but they are mechanically stiffer. This decrease in compliance of dehydrated fibrils is seen by a failure of cell movement of dehydrated fibrils compared to their ability to rearrange fully hydrated fibrils and from direct measurements by nanoindentation and quantitative atomic force measurements. We suggest that increase in the nanoscale rigidity of collagen fibrils can cause these cells to assume a proliferative phenotype.     

Identification of sunfish species by muscle protein isoelectric focusing

Muscle protein phenotypes of nine recognized Lepomis species were developed by isoelectric focusing. Four classes were described based on putative parvalbumin patterns. All nine species could be distinguished by their protein phenotypes. Natural hybrids and their parentage were also identified, and the utility of this technique in studying natural sunfish hybridization is discussed.     

Understanding the tumor metabolic phenotype in the genomic era

Now, at the beginning of a new century, 80 years after Warburg's Nobel prize winning discoveries, we are beginning to make sense of the underlying causes of the well known metabolic phenotype of tumor cells. Building on decades of research to understand the interrelationships between respiration and glycolysis in cancer, the tumor metabolic phenotype can now begin to be understood in a genomic context. With the discovery of hypoxia inducible factor-1 (HIF-1), which is widely overexpressed across a broad range of cancers, modern molecular tools have allowed us to put together the pattern of events that might explain the metabolic differences between tumor and normal cells. HIF-1 controls cellular and systemic responses to oxygen availability and coordinates up-regulation of genes involved in many pathways concerned with tumour growth and metabolism including angiogenesis, glucose and energy metabolism, cellular proliferation, differentiation and viability, apoptosis, pH regulation and matrix metabolism. These findings begin to explain how glucose uptake and glycolysis could be up-regulated in cancer cells (through binding to a core DNA recognition sequence) in a co-ordinated and constitutive fashion that may also allow us to elucidate new targets for tumor therapy.     

Characterizing the metabolic phenotype: a phenotype phase plane analysis.

Genome-scale metabolic maps can be reconstructed from annotated genome sequence data, biochemical literature, bioinformatic analysis, and strain-specific information. Flux-balance analysis has been useful for qualitative and quantitative analysis of metabolic reconstructions. In the past, FBA has typically been performed in one growth condition at a time, thus giving a limited view of the metabolic capabilities of a metabolic network. We have broadened the use of FBA to map the optimal metabolic flux distribution onto a single plane, which is defined by the availability of two key substrates. A finite number of qualitatively distinct patterns of metabolic pathway utilization were identified in this plane, dividing it into discrete phases. The characteristics of these distinct phases are interpreted using ratios of shadow prices in the form of isoclines. The isoclines can be used to classify the state of the metabolic network. This methodology gives rise to a "phase plane" analysis of the metabolic genotype-phenotype relation relevant for a range of growth conditions. Phenotype phase planes (PhPPs) were generated for Escherichia coli growth on two carbon sources (acetate and glucose) at all levels of oxygenation, and the resulting optimal metabolic phenotypes were studied. Supplementary information can be downloaded from our website (http://epicurus.che.udel.edu).     

Osteogenic cell contact with biomaterials influences phenotype expression

Relationship between (1) osteoblast adhesion and spreading, and (2) phenotype expression was investigated. Cellular adhesion and spreading were estimated after short time (24 h), whereas proliferation and other osteoblast functions – after 7 days. Primary human osteogenic cells were seeded on the samples of titanium (T), surgical steel (S) and tissue culture polystyrene (PS), and incubated at 37 °C. After 24 h a number of samples were stained with crystal violet and Hoechst; the average single cell area (spreading) and adhering cell number was measured on each sample. The remaining cultures were supplemented with dexamethasone (10 nM) and -glycerophosphate (5 mM), and incubation was continued for 7 days. The cells on each sample were counted and the following tests were performed: XTT mitochondrial activity assay, total protein content, alkaline phosphatase activity (ALP), Sirius Red test for collagen, osteocalcin and calcium concentration. After 24 h significantly greater cell spreading (p < 0.05) and number (p < 0.05) were on T than on S. After 7 days significantly higher on T than on S were: ALP activity (p < 0.000001), collagen (p < 0.0015) and calcium concentration (p < 0.03). XTT results were bigger on S than on T. In control – XTT results were higher than on the metals; collagen and ALP were lower than on T, and calcium level was significantly lower than on T and S (p < 0.025). After 7 days there were no differences in cell number between T and S. Cell number (24 h) correlated with ALP activity (7 days) on steel (coefficient of correlation, CC = 0.866) and titanium (CC = 0.742). The spreading correlated on steel and on titanium with calcium concentration (CC = 0.645 on S, CC = 0.696 on T) and collagen level (CC = –0.638 on S, CC = –0.69 on T). Conclusions: Better conditions for osteoblast phenotype expression on T after 7 days of culture coincided with greater adhesion and spreading of cells after 24 h on T, as compared with S. The initial contact of cells with underlying surface may influence osteoblast functions and possibly, bone regeneration and implant osteointegration in vivo. Early cell spreading may be an indicator of further expression of osteoblast phenotype and may be important for application of osteogenic cells in reconstructive surgery.     

Ecological and morphological differentiation of pumpkinseed sunfish in lakes without bluegill sunfish

Summary Over the last three decades, sunfish of the familyCentrarchidae have become recognized as a model system in which the ecological consequences of species interactions can be observed and tested. The evolutionary consequences of species interactions in sunfish have received less attention. Bluegill (Lepomis machrochirus) and pumpkinseed (Lepomis gibbosus) sunfish are two common and well-studied species that occupy separate ecological niches. Adult bluegill are generalists that feed in the open water on zooplankton during much of the year, while adult pumpkinseeds specialize on crushing hard-bodied prey such as snails. These species coexist over much of their geographical ranges, but bluegill are historically absent from several large drainage basins in the northeastern US. Here we show that pumpkinseeds from an Adirondack lake without bluegills have differentiated into two morphological forms, one of which is planktivorous. Differentiation is independent of sex and occurs over a broad range of sizes. Thus, the ecological diversity that exists between the bluegill and pumpkinseeds in sympatry has been replaced by a comparable degree of diversity within pumpkinseeds in allopatry.     

Circadian rhythms in the green sunfish retina

We investigated the occurrence of circadian rhythms in retinomotor movements and retinal sensitivity in the green sunfish, Lepomis cyanellus. When green sunfish were kept in constant darkness, cone photoreceptors exhibited circadian retinomotor movements; rod photoreceptors and retinal pigment epithelium (RPE) pigment granules did not. Cones elongated during subjective night and contracted during subjective day. These results corroborate those of Burnside and Ackland (1984. Investigative Ophthalmology and Visual Science. 25:539-545). Electroretinograms (ERGs) recorded in constant darkness in response to dim flashes (lambda = 640 nm) exhibited a greater amplitude during subjective night than during subjective day. The nighttime increase in the ERG amplitude corresponded to a 3-10-fold increase in retinal sensitivity. The rhythmic changes in the ERG amplitude continued in constant darkness with a period of approximately 24 h, which indicates that the rhythm is generated by a circadian oscillator. The spectral sensitivity of the ERG recorded in constant darkness suggests that cones contribute to retinal responses during both day and night. Thus, the elongation of cone myoids during the night does not abolish the response of the cones. To examine the role of retinal efferents in generating retinal circadian rhythms, we cut the optic nerve. This procedure did not abolish the rhythms of retinomotor movement or of the ERG amplitude, but it did reduce the magnitude of the nighttime phases of both rhythms. Our results suggest that more than one endogenous oscillator regulates the retinal circadian rhythms in green sunfish. Circadian signals controlling the rhythms may be either generated within the eye or transferred to the eye via a humoral pathway.     

Studying metabolic regulation in human muscle

Phosphorus magnetic resonance spectroscopy, despite some limitations, is a valuable non-invasive window on muscle metabolism in vivo, particularly oxidative ATP synthesis. A number of experiments have shown this to be dominated by closed-loop feedback mechanisms: a well-known model posits regulation by ADP, but there are others, difficult to distinguish experimentally. Moreover the contribution of open-loop control mechanisms ('feed forward' or 'parallel activation') in vivo remains controversial. Progress will require more precise data, better integrated with other measurements (e.g. muscle oxygenation), and improvement of the conceptual tools appropriate to such studies, where data are limited and steady-state assumptions frequently inapplicable.     

Emergence of the mature myosin phenotype in the rat diaphragm muscle

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.