Soft and flexible poly(ethylene glycol) nanotubes for local drug delivery.

Nanotubes are emerging as promising materials for healthcare applications but the selection of clinically relevant starting materials for their synthesis remains largely unexplored. Here we present, for the first time, the synthesis of poly(ethylene glycol) (PEG) based nanotubes via the photopolymerization of poly(ethylene glycol) diacrylate and other diacrylate derivatives within the pores of anodized aluminum oxide templates. Template-assisted synthesis allowed the manufacture of a diverse set of polymeric nanotubes with tunable physical characteristics including diameter (∼200-400 nm) and stiffness (405-902 kPa). PEG nanotubes were subjected to cytotoxicty assessment in cell lines and primary stem cells and showed excellent cytocompatability (IC50 > 120 µg ml-1). Nanotubes were readily drug loaded but released the majority of the drug over 5 days. Direct administration of drug loaded nanotubes to human orthotopic breast tumors substantially reduced tumor growth and metastasis and outperformed i.v. administration at the equivalent dose. Overall, this nanotube templating platform is emerging as a facile route for the manufacture of poly(ethylene glycol) nanotubes.

Thermospheric Nitric Oxide Response to Shock-led Storms.

We present a multi-year superposed epoch study of the Sounding of the Atmosphere using Broadband Emission Radiometry nitric oxide (NO) emission data. NO is a trace constituent in the thermosphere that acts as cooling agent via infrared (IR) emissions. The NO cooling competes with storm time thermospheric heating resulting in a thermostat effect. Our study of nearly 200 events reveals that shock-led interplanetary coronal mass ejections (ICMEs) are prone to early and excessive thermospheric NO production and IR emissions. Excess NO emissions can arrest thermospheric expansion by cooling the thermosphere during intense storms. The strongest events curtail the interval of neutral density increase and produce a phenomenon known as thermospheric 'overcooling'. We use Defense Meteorological Satellite Program particle precipitation data to show that interplanetary shocks and their ICME drivers can more than double the fluxes of precipitating particles that are known to trigger the production of thermospheric NO. Coincident increases in Joule heating likely amplify the effect. In turn, NO emissions more than double. We discuss the roles and features of shock/sheath structures that allow the thermosphere to temper the effects of extreme storm time energy input and explore the implication these structures may have on mesospheric NO. Shock-driven thermospheric NO IR cooling likely plays an important role in satellite drag forecasting challenges during extreme events.

Expression and functional implications of troponin T isoforms in soleus muscle fibers of rat after unloading.

The expression pattern of troponin T (TnT) isoforms was studied in rat soleus muscle fibers in control and after hindlimb unloading (HU) conditions. To determine the functional consequence of TnT expression, the fibers were also examined for their calcium activation characteristics. With regard to TnT expression, four populations of fibers were distinguished in control muscle. Slow fibers expressing only slow isoforms of TnT (TnT1s, 2s, 3s ) were predominant (54%). Hybrid slow fibers (16%) differed from slow fibers by the additional expression of two TnTf isoforms. Hybrid fast fibers (22%) expressed slow and fast isoforms of TnT while fast fibers (8%) expressed only fast TnT isoforms. The expression of the other regulatory protein isoforms was checked for each population. The contractile experiments revealed steeper slopes of the tension/pCa relationship from hybrid slow fibers expressing fast TnT in a completely slow molecular environment. The expression of TnTs in hybrid fast fibers did not modulate the intrinsic co-operativity. After HU, the fast population was increased and reached 55%. The slow population decreased to 41% and a very small amount of hybrid slow fibers remained (4%). These data demonstrated the implication of TnT isoforms in the calcium activation properties and, more particularly, in the modulation of co-operativity within the myofibrillar lattice. Regulation of TnT expression appeared as a very fast and complete process compared to moderate changes of TnC and TnI.

Mitochondrial enzyme defects in normal and low-frequency-stimulated muscles of young and aging rats.

The age-related increase in cytochrome c-oxidase-deficient (COX(-)) muscle fibers has been suggested to be positively correlated with mitochondrial content (Müller-Höcker, Brain Pathol. 1992; 2:149-158). As a way to test this relationship, tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway rats were exposed to chronic low-frequency stimulation (CLFS) for 50 days, an experimental protocol known to induce marked increases in mitochondrial content. CLFS produced elevated activity levels of COX and succinate dehydrogenase (SDH) in most fibers of young and aging rats. Some fibers low or deficient in COX and a few fibers low or deficient both in COX and SDH (COX(-)/SDH(-)) were detected in unstimulated muscles of young and, more frequently, aging rats. According to their myosin complement, these fibers were immunohistochemically identified as type I fibers. CLFS increased their number in young muscles, but reduced it in aging muscles. Stimulated aging muscles contained some very small, most likely newly formed COX(+) and SDH(+) type I fibers. Thus, the fraction of COX(-) fibers was reduced in aging muscle by enhanced contractile activity.

Fiber-type transitions and satellite cell activation in low-frequency-stimulated muscles of young and aging rats.

We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both young and aging BN rats. These changes were accompanied by a twofold increase in the satellite cell content in both the young and aging groups; satellite cell content reached a level that was significantly higher in the young group (p <.04). The total muscle precursor cell content (i.e., satellite cells plus progeny), however, did not differ between groups, because there was a greater number of satellite cell progeny passing through the proliferative and differentiative compartments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging BN rats possess an unaltered capacity to contribute to the adaptive response.

Transitions of muscle fiber phenotypic profiles.

Skeletal muscle is a complex, versatile tissue composed of a large variety of functionally diverse fiber types. The overall properties of a muscle largely result from a combination of the individual properties of its different fiber types and their proportions. Skeletal muscle fiber types, which can be delineated according to various parameters, for example, myofibrillar protein isoforms, metabolic enzyme profiles, and structural and contractile properties, are not fixed units but are capable of responding to altered functional demands and a variety of signals by changing their phenotypic profiles. This brief review summarizes our current understanding of the delineation of fiber types, modulations of their phenotypic profiles as induced under various conditions, and potential mechanisms involved in these transitions.

Deficiency in parvalbumin increases fatigue resistance in fast-twitch muscle and upregulates mitochondria.

The soluble Ca2+-binding protein parvalbumin (PV) is expressed at high levels in fast-twitch muscles of mice. Deficiency of PV in knockout mice (PV -/-) slows down the speed of twitch relaxation, while maximum force generated during tetanic contraction is unaltered. We observed that PV-deficient fast-twitch muscles were significantly more resistant to fatigue than were the wild type. Thus components involved in Ca2+ homeostasis during the contraction-relaxation cycle were analyzed. No upregulation of another cytosolic Ca2+-binding protein was found. Mitochondria are thought to play a physiological role during muscle relaxation and were thus analyzed. The fractional volume of mitochondria in the fast-twitch muscle extensor digitorum longus (EDL) was almost doubled in PV -/- mice, and this was reflected in an increase of cytochrome c oxidase. A faster removal of intracellular Ca2+ concentration ([Ca2+]i) 200-700 ms after fast-twitch muscle stimulation observed in PV -/- muscles supports the role for mitochondria in late [Ca2+]i removal. The present results also show a significant increase of the density of capillaries in EDL muscles of PV -/- mice. Thus alterations in the dynamics of Ca2+ transients detected in fast-twitch muscles of PV -/- mice might be linked to the increase in mitochondria volume and capillary density, which contribute to the greater fatigue resistance of these muscles.

Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle.

The present study investigates the role of two major proteolytic systems in transforming rabbit and rat muscles. The fast-to-slow transformation of rabbit muscle by chronic low-frequency stimulation (CLFS) induces fast-to-slow transitions of intact, mature fibers and replacement of degenerating fibers by newly formed slow fibers. Ubiquitination, an indicator of the ATP-dependent proteasome system, and calpain activity were measured in homogenates of control and stimulated extensor digitorum longus muscles. Calpain activity increased similarly (approximately 2-fold) in stimulated rat and rabbit muscles. CLFS had no effect on protein ubiquitination in rat muscle but led to elevations in ubiquitin protein conjugates in rabbit muscle. Immunohistochemistry was used to study the distribution of micro-calpain and m-calpain and of ubiquitinated proteins in myosin heavy chain-based fiber types. The findings suggest that both proteolytic systems are involved in fiber transformation and replacement. Transforming mature fibers displayed increases in micro-calpain and accumulation of ubiquitin protein conjugates. The majority of these fibers were identified as type IIA. Enhanced ubiquitination was also observed in degenerating and necrotic fibers. Such fibers additionally displayed elevated m-calpain levels. Conversely, p94, the skeletal muscle-specific calpain, decayed rapidly after stimulation onset and was hardly detectable after 4 days of CLFS.

Muscle LIM protein is upregulated in fast skeletal muscle during transition toward slower phenotypes.

Muscle LIM protein (MLP) is constitutively expressed in slow, but undetectable in fast, muscles of the rat. Here we show that MLP was upregulated at both the mRNA and protein levels under experimental conditions leading to transitions from fast to slower phenotypes. Chronic low-frequency stimulation and mechanical overloading by synergist removal both induced fast-to-slow shifts in myosin heavy chain (MHC) isoforms and expression of MLP in fast muscles. High amounts of MLP mRNA and protein were also present in fast muscles of the myotonic, hyperactive ADR mouse. Hypothyroidism evoked shifts in myosin composition toward slower isoforms and increased the MLP protein content of soleus (SOL) muscle but failed to induce MLP in fast muscles. Unweighting by hindlimb suspension elicited slow-to-fast transitions in MHC expression without altering MLP levels in SOL muscle. Hyperthyroidism shifted the MHC pattern toward faster isoforms but did not affect MLP content in SOL muscle. We conclude that alterations in MLP expression are associated with transitions from fast to slower phenotypes but not with slow-to-fast muscle fiber transitions.

Historical Perspectives: plasticity of mammalian skeletal muscle.

More than 40 years ago, the nerve cross-union experiment of Buller, Eccles, and Eccles provided compelling evidence for the essential role of innervation in determining the properties of mammalian skeletal muscle fibers. Moreover, this experiment revealed that terminally differentiated muscle fibers are not inalterable but are highly versatile entities capable of changing their phenotype from fast to slow or slow to fast. With the use of various experimental models, numerous studies have since confirmed and extended the notion of muscle plasticity. Together, these studies demonstrated that motoneuron-specific impulse patterns, neuromuscular activity, and mechanical loading play important roles in both the maintenance and transition of muscle fiber phenotypes. Depending on the type, intensity, and duration of changes in any of these factors, muscle fibers adjust their phenotype to meet the altered functional demands. Fiber-type transitions resulting from multiple qualitative and quantitative changes in gene expression occur sequentially in a regular order within a spectrum of pure and hybrid fiber types.

Low-frequency stimulation of fast muscle affects the abundance of Ca(2+)-ATPase but not its oligomeric status.

After chronic, low-frequency stimulation, a rapid decline in Ca(2+) pump activity is observed during the early stages of skeletal muscle transformation. However, this variation in enzymatic activity does not coincide with a drastic reduction in the amount of sarcoplasmic reticulum Ca(2+)-ATPases. To investigate whether changes in subunit interactions within Ca(2+) pump complexes contribute to this phenomena, we performed a chemical cross-linking analysis of 4 days continuously, and 4 days discontinuously, electrostimulated fast muscle fibers. The abundance of the slow and fast Ca(2+)-ATPase isoforms sarco(endo)plasmic reticulum Ca(2+)- ATPase types 1 and 2 was affected during the fast-to-slow transition process, demonstrating that, even after short-term stimulation, distinct changes in the isoform expression pattern of muscle proteins occur. However, the oligomeric status of both ion pump species did not change. Hence, chemical modifications of critical enzyme domains must be responsible for the rapid stimulation-induced activity changes, not variations in protein-protein interactions within Ca(2+)-ATPase units. Oligomerization appears to be of central importance to the proper physiological functioning of the Ca(2+)-ATPase and does not undergo changes during skeletal muscle conditioning.

Adaptive potentials of skeletal muscle in young and aging rats.

We compared responses of the fast extensor digitorum longus (EDL) and tibialis anterior (TA) muscles in young (15-week) and aging (101-week) male Brown Norwegian rats to 50 days of chronic low-frequency stimulation (CLFS, 10 Hz, 10 hours/day). After 50 days of CLFS, the EDL muscles of the young (22-week) and aging (108-week) rats displayed similar increases in type IIA fibers, relative concentration of myosin heavy chain MHCIIa, elevations in mitochondrial citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities, and similar decreases in glycolytic enzyme activities (glyceraldehydephosphate dehydrogenase, lactate dehydrogenase). TA muscle in young rats contained a few cytochrome c oxidase negative (COX-) type I fibers. Their number was approximately 2-fold elevated by CLFS. Conversely, aging muscle, which contained a slightly higher amount of COX- fibers than young TA muscle, responded to CLFS with a significant decrease in COX- fibers. The appearance of small COX-positive type I fibers in stimulated aging muscle indicated that regenerating type I fibers "diluted" the COX-deficient fiber population.

Myosin and SERCA isoform expression in denervated slow-twitch muscle of euthyroid and hyperthyroid rabbits.

Changes in the isoform patterns of myosin heavy chains (MHC) and Ca2+-ATPase (SERCA) were studied in long-term (72 days) denervated slow-twitch soleus muscles of euthyroid and hyperthyroid rabbits. MHC isoforms were separated electrophoretically. SERCA isoforms were assessed by electrophoretic separation of the trypsin-digested and phosphorylated fragments. Denervation led to pronounced slow-to-fast transitions with increases in the fast MHC and fast SERCA1a isoforms. Hyperthyroidism had no significant effect on the denervation-induced changes in SERCA isoforms, but led to slight increases in the relative concentrations of the fast MHC isoforms. A high correlation existed between the relative concentrations of slow myosin and SERCA2a in both innervated euthyroid and hyperthyroid soleus muscles. This correlation, however, seems to be less tight in denervated soleus muscles.

Inactivation of sarcoplasmic reticulum Ca(2+)-atpase in low-frequency stimulated rat muscle.

Continuous low-frequency stimulation (CLFS) by implanted electrodes for 12-24 h led to a significant (approximately 30%) decrease in the activity of sarcoplasmic reticulum Ca(2+)-ATPase in fast-twitch extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of intact rats. The decline in catalytic activity after 24 h of CLFS was accompanied by an approximately twofold increase in dinitrophenylhydrazine-reactive carbonyl groups of the enzyme. It also correlated with an immunochemically determined 30% decrease in Ca2(+)-ATPase protein. Recovery studies after 12 h of CLFS revealed a relatively slow (48-72 h) re-establishment of normal catalytic activity. These findings suggest that the 30% decline of Ca(2+)-ATPase activity in low-frequency stimulated rat muscle led to an irreversible modification by protein oxidation. The decrease in Ca(2+)-ATPase protein most likely resulted from the degradation of inactive Ca(2+)-ATPase molecules. The relatively slow recovery of Ca(2+)-ATPase activity suggests that de novo synthesis of the enzyme may be necessary to re-attain normal activity.

Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat.

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the beta(2)-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb ("fast-to-faster transitions"). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.

Myosin isoforms, muscle fiber types, and transitions.

Skeletal muscle is an extremely heterogeneous tissue composed of a variety of fast and slow fiber types and subtypes. Moreover, muscle fibers are versatile entities capable of adjusting their phenotypic properties in response to altered functional demands. Major differences between muscle fiber types relate to their myosin complement, i.e., isoforms of myosin light and heavy chains. Myosin heavy chain (MHC) isoforms appear to represent the most appropriate markers for fiber type delineation. On this basis, pure fiber types are characterized by the expression of a single MHC isoform, whereas hybrid fiber type express two or more MHC isoforms. Hybrid fibers bridge the gap between the pure fiber types. The fiber population of skeletal muscles, thus, encompasses a continuum of pure and hybrid fiber types. Under certain conditions, changes can be induced in MHC isoform expression heading in the direction of either fast-to-slow or slow-to-fast. Increased neuromuscular activity, mechanical loading, and hypothyroidism are conditions that induce fast-to-slow transitions, whereas reduced neuromuscular activity, mechanical unloading, and hyperthyroidism cause transitions in the slow-to-fast direction.

Tetanus relaxation of fast skeletal muscles of the mouse made parvalbumin deficient by gene inactivation.

The effects of tetanus duration on the relaxation rate of extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles were studied in normal (wild-type, WT) and parvalbumin-deficient (PVKO) mice, at 20 C. In EDL of PVKO, the relaxation rate was low and unaffected by tetanus duration (< 3.2 s). In contrast, the relaxation rate of WT muscles decreased when tetanus duration increased from 0.2 to 3.2 s. In WT muscles, fast relaxation recovered as the rest interval increased. Specific effect of parvalbumin was asserted by calculating the difference in relaxation rate between WT and PVKO muscles. For EDL, the rate constant of relaxation slowing was 1.10 s-1 of tetanization; the rate constant of relaxation recovery was 0.05 s-1 of rest. In FDB, the effects of tetanus duration on WT and PVKO muscles were qualitatively similar to those observed in EDL. Relaxation slowing as tetanus duration increases, reflects the progressive saturation of parvalbumin by Ca2+, while recovery as rest interval increases reflects the return to Ca2+-free parvalbumin. At all tetanus durations, relaxation rate still remained slightly faster in WT muscles. This suggested that parvalbumin facilitates calcium traffic from myofibrils to the SR. No difference was found between WT and PVKO muscles for: (i) the expression of the fast isoforms of myosin heavy chains, (ii) the force-velocity relationship and maximal shortening velocity and (iii) the Ca2+-activated ATPase activity from isolated preparations of the sarcoplasmic reticulum (SR).

Upregulation of myosin heavy chain MHClalpha in rat muscles after unweighting and clenbuterol treatment.

Myosin heavy chain (MHC) mRNA isoforms were quantified in soleus (SOL) and gastrocnemius (GAS) muscles from rats exposed to 14 days of either hindlimb unweighting (HU), clenbuterol treatment (CB), or HU combined with CB treatment (HU-CB). All conditions induced in SOL a shift from slow to faster MHC mRNA isoforms and an upregulation of MHClalpha. Increases were highest with CB, lowest with HU-CB, and coincided mainly with elevations in MHClla mRNA isoforms. The changes in MHC mRNA levels in GAS muscle corresponded to fast-to-faster transitions. Elevations in MHClalpha mRNA were smaller than in SOL and seemed to occur in parallel with decreases in MHClbeta. Taken together, our results suggested that MHClalpha is expressed in transforming rat slow and fast muscles, most likely as an intermediate step between MHClbeta and MHClla.

Calpain activity in fast, slow, transforming, and regenerating skeletal muscles of rat.

Fiber-type transitions in adult skeletal muscle induced by chronic low-frequency stimulation (CLFS) encompass coordinated exchanges of myofibrillar protein isoforms. CLFS-induced elevations in cytosolic Ca(2+) could activate proteases, especially calpains, the major Ca(2+)-regulated cytosolic proteases. Calpain activity determined by a fluorogenic substrate in the presence of unaltered endogenous calpastatin activities increased twofold in low-frequency-stimulated extensor digitorum longus (EDL) muscle, reaching a level intermediate between normal fast- and slow-twitch muscles. micro- and m-calpains were delineated by a calpain-specific zymographical assay that assessed total activities independent of calpastatin and distinguished between native and processed calpains. Contrary to normal EDL, structure-bound, namely myofibrillar and microsomal calpains, were abundant in soleus muscle. However, the fast-to-slow conversion of EDL was accompanied by an early translocation of cytosolic micro-calpain, suggesting that myofibrillar and microsomal micro-calpain was responsible for the twofold increase in activity and thus involved in controlled proteolysis during fiber transformation. This is in contrast to muscle regeneration where m-calpain translocation predominated. Taken together, we suggest that translocation is an important step in the control of calpain activity in skeletal muscle in vivo.

Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat.

Satellite cell proliferation was assessed in low-frequency-stimulated hypothyroid rat fast-twitch muscle by 5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining of labeled muscle nuclei, and by staining for proliferating cell nuclear antigen (PCNA). BrdU labeling and PCNA staining were highly correlated and increased approximately fourfold at 5 days of stimulation, decayed thereafter, but remained elevated over control in 10- and 20-day stimulated muscles. Myogenin mRNA was approximately 4-fold elevated at 5 days and 1.5-fold at 10 days. Staining for myogenin protein yielded results similar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequency stimulation. By 20 days, myogenin staining was observed in transforming fast fibers that coexpressed embryonic and adult myosin heavy chain isoforms. In the slower fiber populations (i.e., IIA and I), myogenin-positive transforming fibers that coexpressed embryonic myosin heavy chain, appeared already at 5 days. Thus the satellite cell progeny on slower fibers seemed to proliferate less and to fuse earlier to their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.