Muscle Nuclei Research Articles

Nuclei of aged myofibres undergo structural and functional changes suggesting impairment in RNA processing

Advancing adult age is associated with a progressive decrease in skeletal muscle mass, strength and quality known as sarcopenia. The mechanisms underlying age-related skeletal muscle wasting and weakness are manifold and still remain to be fully elucidated. Despite the increasing evidence that the progress of muscle diseases leading to muscle atrophy/dystrophy may be related to defective RNA processing, no data on the morpho-functional features of skeletal muscle nuclei in sarcopenia are available at present. In this view, we have investigated, by combining morphometry and immunocytochemistry at light and electron microscopy, the fine structure of myonuclei as well as the distribution and amount of RNA processing factors in skeletal myofibres of biceps brachii and quadriceps femoris from adult and old rats. Results demonstrate that the myonuclei of aged type II fibres show an increased amount of condensed chromatin and lower amounts of phosphorylated polymerase II and DNA/RNA hybrid molecules, clearly indicating a decrease in pre-mRNA transcription rate compared to adult animals. In addition, myonuclei of aged fibres show decreased amounts of nucleoplasmic splicing factors and an accumulation of cleavage factors, polyadenilated RNA and perichromatin granules, suggesting a reduction in the processing and transport rate of premRNA. During ageing, it seems therefore that in rat myonuclei the entire production chain of mRNA, from synthesis to cytoplasmic export, is less efficient. This failure likely contributes to the reduced responsiveness of muscle cells to anabolic stimuli in the elderly.

Reply: “Necklace” fibers and “trilaminar” fibers are different

We read with great attention Professor Michael Schroder’s letter on ‘‘necklace’’ fibers as a late clue to the interpretation of the forgotten ‘‘trilaminar’’ fibers. It is true that, at first glance, there is some resemblance between the ‘‘necklace’’ fibers we recently described in four cases of late-onset centronuclear myopathies [1], and the ‘‘trilaminar’’ fibers initially described by Ringel et al. [2] in a child with severe congenital muscle disorder and neonatal rigidity, and afterwards by Schroder himself with Schonberger [3, 4] in two brothers who also presented a very severe neuromuscular disorder and died at 3 and 9 months from bronchopneumonia. Both types of abnormal fibers exhibit a concentric pattern consisting of three zones (outer, intermediate and central). However, there are major and distinctive differences between these previous cases and those we reported, at the ultrastructural level. In the ‘‘necklace’’ fibers, the outer zone is formed by normally organized myofibrils, while in the trilaminar cases, this outer zone contains essentially sarcoplasmic components, similar to ‘‘sarcoplasmic masses’’ we described in various neuromuscular disorders, and in particular in myotonic dystrophy [5]. The intermediate zone in the ‘‘necklace fibers’’ is formed by small myofibrils with an oblique orientation and an increased density of mitochondria, sarcoplasmic reticulum (SR) profiles and glycogen granules, while in the ‘‘trilaminar’’ fibers there is an ‘‘increased density of myofibrillar structures’’. In the central zone, myofibrils and SR components have a normal organization in the ‘‘necklace’’ fibers, while in the ‘‘trilaminar’’ fibers, this central zone contains only a few remnants of myofibrils in Ringel’s case, and was ‘‘rather disorganized’’ in Schroder and Schonberger’s cases. These ultrastructural differences clearly explain the differences observed at the light microscopic level with histochemical and immunocytochemical techniques. Interestingly, the distribution of muscle nuclei also differs in these two conditions, the nuclei mainly being present in the intermediate and central zones in ‘‘necklace’’ fibers, while they are in their usual subsarcolemmal position in the ‘‘trilaminar’’ fibers. Furthermore, it should be emphasized how different the clinical presentations of the patients were in these two conditions, rather benign and of late onset in the MTM1 linked centronuclear myopathies, and very severe with early death in the three cases described as ‘‘trilaminar’’ myopathies. It is highly improbable that these differences in age and clinical severity might account for the histopathological differences. In the absence of any molecular genetic analysis of the ‘‘trilaminar’’ cases, we consider that we should establish a clear distinction between ‘‘necklace’’ and ‘‘trilaminar’’ muscle fibers. J. A. Bevilacqua Departamento de Neurologia y Neurocirugia, HCUCH, Santiago, Chile

Importin 13 Regulates Neurotransmitter Release at theDrosophilaNeuromuscular Junction

In an unbiased genetic screen designed to isolate mutations that affect synaptic transmission, we have isolated homozygous lethal mutations in Drosophila importin 13 (imp13). Imp13 is expressed in and around nuclei of both neurons and muscles. At the larval neuromuscular junction (NMJ), imp13 affects muscle growth and formation of the subsynaptic reticulum without influencing any presynaptic structural features. In the absence of imp13, the probability of release of neurotransmitter and quantal content is increased, yet the abundance of the postsynaptic receptors and the amplitude of miniature excitatory junctional potentials are not affected. Interestingly, imp13 is required in the muscles to control presynaptic release. Thus, imp13 is a novel factor that affects neurotransmitter release at the fly NMJ. Its role in the context of synaptic homeostasis is discussed.

Activation of antioxidant defense during dehydration stress in the African clawed frog

The glutathione S-transferase (GST) and aldo-keto reductase (AKR) families of proteins are major groups of detoxifying enzymes that are known to be regulated by the NF-E2 related factor 2 (Nrf2) transcription factor via the antioxidant response element that is present in the promoter regions of GST and AKR genes. Expression of Nrf2, GST and AKR proteins was analyzed in the African clawed frog, Xenopus laevis, focusing on their responses to dehydration stress. Dehydration/rehydration cycles can generate oxidative stress and this could be ameliorated by enhancing antioxidant defenses. Dehydration to 28% of total body water lost triggered organ-specific changes in nrf2 mRNA expression (a 2-fold increase in liver), total Nrf2 protein (2–4-fold elevation in lung, heart, skin and liver), and a 4.3-fold increase in the content of Nrf2 in the nucleus in muscle. Protein levels of six GST and three AKR family members were assessed and showed organ-specific patterns of expression during dehydration. In particular, GSTP1 was strongly induced in liver, heart and skin, levels rising by 9-, 2.6- and 1.7-fold, respectively, whereas GSTM1 and M3 rose in skeletal muscle, kidney and skin. Selective expression of GSTK1, A3 and T1 also occurred. Dehydration also stimulated organ-specific increases in the levels of AKR family members (AKR1B4, AKR1A3, AFAR1) by 1.5–2-fold. The results show that metabolic responses to dehydration include activation of the Nrf2 transcription factor and selective up-regulation of genes under Nrf2 control.

Ribonuclear inclusions and MBNL1 nuclear sequestration do not affect myoblast differentiation but alter gene splicing in myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) is an autosomal dominant multisystemic disorder caused by a CCTG expansion in intron 1 of the zinc finger protein 9 gene on chromosome 3. Mutant transcripts are retained in muscle nuclei producing ribonuclear inclusions, which can bind specific RNA-binding proteins leading to a reduction in their activity. The nuclear sequestration of muscleblind-like proteins appears to be involved in splicing defects of genes directly related to the myotonic dystrophy phenotypes. Experimental evidence suggests that ribonuclear inclusions and muscleblind-like protein 1 (MBNL1) sequestration are strongly involved in DM2 pathogenesis. By using fluorescence in situ hybridization in combination with MBNL1-immunofluorescence, we have observed the presence of ribonuclear inclusions and MBNL1 nuclear sequestration at different time points of in vitro myoblast differentiation in each DM2 patient examined. Immunofluorescence and Western blot analysis of several markers of skeletal muscle differentiation reveal that the degree of differentiation of DM2 myoblasts is comparable to that observed in controls. Nevertheless the splicing pattern of the insulin receptor and MBNL1 transcripts, directly related to the DM2 phenotype, appears to be altered in in vitro differentiated DM2 myotubes. Our data seem indicate that the presence of ribonuclear inclusions and MBNL1 nuclear foci are involved in alteration of alternative splicing but do not impair DM2 myogenic differentiation.

Skeletal muscle regeneration is attenuated with repression of the cytoskeletal protein, Xin

Xin, an actin binding protein, has been implicated in the regulation of satellite cell function and is hypothesized to be essential for skeletal muscle regeneration following injury. To investigate the role of Xin during skeletal muscle regeneration, we inhibited Xin expression using a Xin shRNA adenoviral (XinAd) injection into the right tibialis anterior (TA) of 6 week old male mice, while the left TA served as a control receiving an injection of the virus lacking Xin shRNA. Injury was induced in both TAs 4 days later via cardiotoxin injection. At 5 days post‐injury, muscle fiber area was significantly smaller (Control: 1007 ± 62 μm2 ; Xin Ad: 785± 73 μm2) and the number of centrally located nuclei per muscle fiber was significantly increased (134 ± 9%) in XinAd regenerating muscle compared to control. These deficits in fiber area (Control: 3136±278 μm2;Xin Ad: 2273 ±167 μm2) and centrally located nuclei (137 ± 9% of control) in the XinAd muscles persisted to 14 days regeneration suggesting that the regenerating muscle injected with XinAd is less mature at 5 and 14 days of regeneration. A significant increase in apoptosis (TUNEL) at 5 and 14 days of regeneration (126±6%; 141±12% of control) was observed in the XinAd TA compared to control. These data support the hypothesis that Xin is important for muscle regeneration and future work will investigate the roles of Xin on satellite cell activation and maturation.

Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy

The present study provides evidence that abnormal patterns of global histone modification are present in the skeletal muscle nuclei of mdx mice and Duchenne muscular dystrophy (DMD) patients. A combination of specific histone H3 modifications, including Ser-10 phosphorylation, acetylation of Lys 9 and 14, and Lys 79 methylation, were found enriched in muscle biopsies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. In this context, chromatin immunoprecipitation experiments showed an enrichment of these modifications at the loci of genes involved in proliferation or inflammation, suggesting a regulatory effect on gene expression. Remarkably, the reexpression of dystrophin induced by gentamicin treatment or the administration of nitric oxide (NO) donors reversed the abnormal pattern of H3 histone modifications. These findings suggest an unanticipated link between the dystrophin-activated NO signaling and the remodeling of chromatin. In this context, the regulation of class IIa histone deacetylases (HDACs) 4 and 5 was found altered as a consequence of the reduced NO-dependent protein phosphatase 2A activity, indicating that both NO and class IIa HDACs are important for satellite cell differentiation and gene expression in mdx mice. In conclusion, this work provides the first evidence of a role for NO as an epigenetic regulator in DMD.

Prevention of oculopharyngeal muscular dystrophy by muscular expression of Llama single-chain intrabodies in vivo

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disorder characterized by progressive weakening of specific muscles. It is caused by short expansions of the N-terminal polyalanine tract in the poly(A) binding protein nuclear 1 (PABPN1), and it belongs to the group of protein aggregation diseases, such as Huntington's, Parkinson's and Alzheimer diseases. Mutant PABPN1 forms nuclear aggregates in diseased muscles in both patients and animal models. Intrabodies are antibodies that are modified to be expressed intracellularly and target specific antigens in subcellular locations. They are commonly generated by artificially linking the variable domains of antibody heavy and light chains. However, natural single-chain antibodies are produced in Camelids and, when engineered, combined the advantages of being single-chain, small sized and very stable. Here, we determine the in vivo efficiency of Llama intrabodies against PABPN1, using the established Drosophila model of OPMD. Among six anti-PABPN1 intrabodies expressed in muscle nuclei, we identify one as a strong suppressor of OPMD muscle degeneration in Drosophila, leading to nearly complete rescue. Expression of this intrabody affects PABPN1 aggregation and restores muscle gene expression. This approach promotes the identification of intrabodies with high therapeutic value and highlights the potential of natural single-chain intrabodies in treating protein aggregation diseases.

Mouse model of testosterone-induced muscle fiber hypertrophy: involvement of p38 mitogen-activated protein kinase-mediated Notch signaling

As a prerequisite for studies using mutant mice, we established a mouse model for investigating the molecular mechanisms by which testosterone (T) promotes muscle growth. Groups of six adult male mice (C57BL/6) received one of the following treatments: 1) vehicle (sterile distilled water; normal control) and 2) GnRH antagonist with empty (sham control) or 2 cm T- filled implant. Mice were killed 2, 6, and 8 weeks after treatment. T treatment for 8 weeks resulted in a significant (P<0.001) increase in fiber area of gastrocnemius muscles. T-induced fiber-hypertrophy was accompanied by up-regulation of the Notch ligand Delta 1 and activation of Notch signaling, as evidenced by increase in activated forms of Notch 1 and Notch 2. Consistent with this, we also observed an increase in the number of proliferating cell nuclear antigen (PCNA)-positive nuclei in muscles of T-treated mice, indicating that activation of Notch signaling enhanced cell proliferation. T supplementation not only triggered p38 mitogen-activated protein kinase (MAPK) activation but also concurrently inhibited c-Jun NH(2)-terminal kinase (JNK) activation within 2 weeks of treatment. Concomitant administration of SB203580, a p38 MAPK inhibitor, effectively blocked T-induced activation of Notch signaling and significantly (P<0.001) suppressed PCNA levels. Together, our results indicate that T induces muscle fiber hypertrophy through activation of Notch signaling and the inactivation of JNK together with the activation of p38 MAPK may be critical for T-induced activation of Notch signaling and, as a consequence, muscle fiber hypertrophy.

Effect of Dipsaci Radix on Hind Limb Muscle Atrophy of Sciatic Nerve Transected Rats

It was reported that Dipsaci radix (DR) has a reinforcement effect on the bone-muscle dysfunction in the oriental medical classics and the experimental animal studies. The muscle atrophy was induced by unilateral transection of the sciatic nerve of the rats. Water-extract of DR was used as treatment once a day for 12 days. The muscle weights of the hind limb, atrophic changes, glycogen contents, compositions and cross-section areas of muscle fiber types in soleus and medial gastrocnemius were investigated. Muscle fiber type was classified to type-I and type-II with MHCf immunohistochemistry. Furthermore, Bax and Bcl-2 expressions were observed with immunohistochemiatry. DR treatment significantly increased muscle weights of soleus, medial gastrocnemius, lateral gastrocnemius, and posterior tibialis of the damaged hind limb. DR treatment reduced apoptotic muscle nuclei and hyaline-degenerated muscle fibers in soleus and medial gastrocnemius of the damaged hind limb. DR treatment also significantly increased glycogen contents in medial gastrocnemius of the damaged hind limb. DR treatment significantly attenuated the slow-to-fast shift in soleus of the damaged hind limb but not in medial gastrocnemius. DR treatment significantly increased cross-section areas of type-I and type-II fibers in soleus and medial gastrocnemius of the damaged hind limb. In soleus and medial gastrocnemius, DR treatment significantly reduced Bax positive muscle nuclei in the damaged hind limb. These results suggest that DR treatment has an anti-atrophic effect and an anti-apoptotic effect against myonuclear apoptosis induced by the peripheral nerve damage.

Regulation of muscle growth in neonates

This review reports recent findings on the multiple factors that regulate skeletal muscle growth in neonates. Skeletal muscle is the fastest growing protein mass in neonates. The high rate of neonatal muscle growth is due to accelerated rates of protein synthesis accompanied by the rapid accumulation of muscle nuclei. Feeding profoundly stimulates muscle protein synthesis in neonates and the response decreases with age. The feeding-induced stimulation of muscle protein synthesis is modulated by enhanced sensitivity to the postprandial rise in insulin and amino acids. Insulin and amino acid signaling components have been identified that are involved in the feeding-induced stimulation of protein synthesis in neonatal muscle. The enhanced activation of these signaling components in skeletal muscle of the neonate contributes to the high rate of muscle protein synthesis and rapid gain in muscle protein mass in neonates. Recent findings suggest that the immature muscle has a heightened capacity to activate signaling cascades that promote translation initiation in response to the postprandial rise in insulin and amino acids thereby enabling their efficient utilization for muscle growth. This capacity is further supported by enhanced satellite cell proliferation, but how these two processes are linked remains to be established.

Altered distribution of lamin and emerin in muscle nuclei of sIBM patients

Sporadic inclusion body myositis (sIBM) is a chronic acquired inflammatory myopathy. The cause of sIBM remains unknown and its pathogenesis is controversial. There is a hypothesis [Karpati and Carpenter 1993] that the rimmed vacuoles result from nuclear breakdown, and IBM filaments are formed from components of the nuclear matrix. For nuclear membrane protein detection, six IBM patients were studied using immunohistochemical and immunochemical techniques. It was demonstrated that in the interior of 10-15% myonuclei emerin and lamin A/C inclusions appeared constantly. This finding indicated an abberant localization of lamin A/C epitopes, the presence of presumptive lamin A (67 KDu) and emerin as in the affected nuclei. We support the suggestion that truncated, changed lamin A protein takes part in nuclear disintegration and rimmed vacuole formation.

The protein l-isoaspartyl- O-methyltransferase functions in the Caenorhabditis elegans stress response

The efficient use of nutrients is important in development and aging. In this study, we asked if the protein repair methyltransferase has a related or additional role in energy metabolism and stress response in the nematode Caenorhabditis elegans. Worms lacking the pcm-1 gene encoding this enzyme exhibit reduced longevity as SDS-isolated dauer larvae and as arrested L1 larvae under starvation stress, while overexpression leads to increased adult longevity. These findings led us to question whether pcm-1 deficient C. elegans may have inappropriate metabolic responses to stress. We assayed dauer and dauer-like larvae for starvation survival and observed a two-fold reduction of median survival time for pcm-1 mutants compared to N2 wild-type worms. Under these conditions, pcm-1 deficient dauer larvae had reduced fat stores, suggesting that PCM-1 may have a role in the initiation of the correct metabolic responses to stress starvation. We show expression of the pcm-1 gene in neurons, body wall and reproductive tissues. Upon heat shock and dauer formation-inducing conditions, we observe additional pcm-1 expression in body wall muscle nuclei and actomyosin filaments and in hypodermal cells. These results suggest that this enzyme may be important in stress response pathways, including proper decision making for energy storage.

MYONUCLEI ARE NOT LOST DURING MUSCLE ATROPHY

We have all experienced the fact that skeletal muscles can change size. When we go to the gym and lift weights, our muscles get bigger. However, when we are lazy and stop working out, our muscles decrease in size. Such muscle shrinkage is termed disuse atrophy and, currently, the only way to build the muscle up again is to increase physical activity and nerve-evoked contractile activity. Muscles also deteriorate with numerous neuromuscular disorders that medical research is endeavoring to cure.A change in muscle size normally results from an alteration of the size of the individual muscle cells (otherwise known as muscle fibers), which contain numerous nuclei (called myonuclei). The classical belief is that muscle cell size is closely correlated to the number of myonuclei, and that a reduction of muscle fiber size is accompanied by a regulated reduction (i.e. apoptosis) of myonuclei. Thus, a loss of nuclei during disuse atrophy implies that the recovery of muscle strength requires the nuclei to be replenished by muscle satellite cells (the muscle's stem cells). Further, this notion dictates that potential intervention and treatment therapies for neuromuscular disorders should involve activation of the satellite cells or interference of the apoptotic pathways. However, it is likely that the classical view of muscle disuse atrophy needs to be revised, given a recent publication by Jo Bruusgaard and Kristian Gundersen in The Journal of Clinical Investigation.The team from the University of Oslo, Norway, decided to examine the phenomenon of muscle disuse atrophy using an in vivo time-lapse microscopy technique. First, the team stained myonuclei of both slow- and fast-twitch leg muscle fibers in live mice by transfecting the mice with a plasmid encoding green fluorescent protein (GFP) that localized to muscle nuclei. Subsequently, they inactivated the leg muscles of the mice by denervating the muscle by severing the sciatic nerve, blocking nerve impulses to the muscle with the voltage-gated sodium channel blocker tetrodotoxin, or mechanically unloading the muscle. Following these procedures, the team monitored changes in fiber size and number of myonuclei in the same muscle segment over several days and weeks.Astonishingly, despite a greater than 50% reduction in the cross-sectional area of the muscle fibers following the inactivation techniques, the team did not observe any loss of myonuclei. Even though the muscle fibers were inactivated, by denervation, nerve impulse block or mechanical unloading, the number of myonuclei in individual muscle fibres did not decrease. Thus the team discovered that, in contrast with the classical belief, muscle nuclei are not lost during disuse. Indeed, the team observed high numbers of nuclei undergoing regulated reduction by apoptosis in inactive muscles, but the apoptosis was confined to the nuclei in satellite cells and surrounding stromal cells, not those situated inside the muscle fiber.The team argues that their novel findings indicate that future therapeutic regimes for the treatment of muscle atrophy should focus on intracellular regulatory mechanisms related to protein degradation and synthesis, and not on the regeneration of myonuclei from stem cells. Nevertheless, the team cautions that their findings do not exclude the possibility that myonuclear apoptosis occurs after longer periods of disuse.

Nitric oxide and vascular remodeling modulate hepatic arterial vascular resistance in the isolated perfused cirrhotic rat liver

Hepatic arterial resistance is modulated by the hepatic arterioles but the role of NO and vascular remodeling in hepatic arterial resistance in cirrhosis is unknown. Cirrhosis was induced by CCl(4) or BDL. Using a bivascular liver perfusion dose-responses curves to methoxamine were obtained from the hepatic artery in absence and presence of L-NMMA. Lumen-diameter, wall thickness and number of smooth muscle nuclei were quantitated in the arteries using image analysis. Hepatic arterial resistance and the response to methoxamine were lower in cirrhosis compared to controls (p< or = 0.04) and lower in BDL compared to CCl(4) (p< or = 0.01). L-NMMA increased the response to methoxamine in CCl(4) (p=0.002) and BDL (p=0.05) but corrected the response only in CCl(4) (p=n.s. vs. control). Wall thickness and the number of smooth muscle nuclei were significantly smaller in cirrhosis compared to controls (p<0.05) and the number of nuclei was also lower in BDL compared to CCl(4) (p=0.005). NO is the main modulator of hepatic arterial resistance in CCl(4) but not in BDL. Intrahepatic arterial remodeling is present in both cirrhotic models but is greater in BDL. This indicates a larger role of structural changes in the control of hepatic arterial resistance in BDL.

A putative role of ribonuclear inclusions and MBNL1 in the impairment of gallbladder smooth muscle contractility with cholelithiasis in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of unstable trinucleotide (CTG) repeats at 3′ untranslated region of the DMPK gene on chromosome 19q13.3. Mutant transcripts are retained in muscle nuclei as ribonuclear inclusions and interact with RNA-binding proteins, such as muscleblind-like protein 1 (MBNL1), leading to a reduction in their activity. The reduced MBNL1 activity has been associated to skeletal and cardiac muscle dysfunction. However, other organs and systems may be involved. It has been reported that 25–50% of DM1 patients have abdominal symptoms due to cholelithiasis or gallstones. Since impaired gallbladder motility plays an important role in gallstones formation, we have analyzed by FISH combined with MBNL1-immunofluorescence, the gallbladder obtained from a woman affected by DM1 who required a cholecystectomy at the age of 30. Gallbladders obtained from two no-DM1 subjects have been used as controls. Ribonuclear inclusions and MBNL1 foci accumulate and colocalize in nuclei of DM1 gallbladder smooth muscle cells. On the contrary, no ribonuclear inclusions are detectable in cell nuclei of control gallbladders and MBNL1 is uniformly distributed in smooth muscle cell nuclei. These results suggest that nuclear accumulation of MBNL1 and ribonuclear inclusions may have a direct adverse effect on gallbladder smooth muscle contractility and thus contribute to gallstones formation in DM1 patients.

Nuclear domains during muscle atrophy: nuclei lost or paradigm lost?

According to the current paradigm, muscle nuclei serve a certain cytoplasmic domain. To preserve the domain size, it is believed that nuclei are injected from satellite cells fusing to fibres undergoing hypertrophy, and lost by apoptosis during atrophy. Based on single fibre observations in and ex vivo we suggest that nuclear domains are not as constant as is often indicated. Moreover, recent time lapse in vivo imaging of single fibres suggests that at least for the first few weeks, atrophy is not accompanied by any loss of nuclei. Apoptosis is abundant in muscle tissue during atrophy conditions, but in our opinion it has not been unequivocally demonstrated that such nuclei are myonuclei. As we see it, the preponderance of current evidence suggests that disuse atrophy is not accompanied by loss of nuclei, at least not for the first 2 months. Moreover, it has not been proven that myonuclear apoptosis does occur in permanent fibres undergoing atrophy; it seems more likely that it is confined to stromal cells and satellite cells. If muscle atrophy is not related to loss of nuclei, design of intervention therapies should focus on protein metabolism rather than regeneration from stem cells.

Protein-tyrosine Phosphatase 1B Expression Is Induced by Inflammation in Vivo

Protein-tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin and leptin sensitivity. PTP1B overexpression in adipose tissue and skeletal muscle of humans and rodents may contribute to insulin resistance and obesity. The mechanisms mediating PTP1B overexpression in obese and diabetic states have been unclear. We find that adipose tissue inflammation and the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) regulate PTP1B expression in vivo. High fat feeding of mice increased PTP1B expression 1.5- to 7-fold in adipose tissue, liver, skeletal muscle, and arcuate nucleus of hypothalamus. PTP1B overexpression in high fat-fed mice coincided with increased adipose tissue expression of the macrophage marker CD68 and TNFalpha, which is implicated in causing obesity-induced insulin resistance. TNFalpha increased PTP1B mRNA and protein levels by 2- to 5-fold in a dose- and time-dependent manner in adipocyte and hepatocyte cell lines. TNFalpha administration in mice increased PTP1B mRNA 1.4- to 4-fold in adipose tissue, liver, skeletal muscle, and hypothalamic arcuate nucleus and PTP1B protein 2-fold in liver. Actinomycin D treatment blocked, and high dose salicylate treatment inhibited by 80%, TNFalpha-induced PTP1B expression in adipocyte cell lines, suggesting TNFalpha may induce PTP1B transcription via nuclear factor kappaB (NFkappaB) activation. Chromatin immunoprecipitation from adipocyte cell lines and liver of mice demonstrated TNFalpha-induced recruitment of NFkappaB subunit p65 to the PTP1B promoter in vitro and in vivo. In mice with diet-induced obesity, TNFalpha deficiency also partly blocked PTP1B overexpression in adipose tissue. Our data suggest that PTP1B overexpression in multiple tissues in obesity is regulated by inflammation and that PTP1B may be a target of anti-inflammatory therapies.

Age-related reductions in expression of serum response factor and myocardin-related transcription factor A in mouse skeletal muscles

The molecular signaling pathways linking the atrophy of skeletal muscle during aging have not been identified. Using reverse transcription (RT)-PCR, Western blotting, and immunofluorescence microscopy, we investigated whether the amounts of RhoA, RhoGDI, SRF, MRTF-A, and MyoD in the triceps brachii and quadriceps muscles change with aging in mice. Young adult (3 mo) and aged (24 mo) C57BL/6J mice were used. Senescent mice possessed many fibers with central nuclei in the quadriceps muscle. Western blotting using a homogenate of whole muscle or the cytosolic fraction clearly showed that the amount of SRF protein was significantly decreased in the aged skeletal muscles. Immunofluorescence labeling indicated more SRF-positive muscle fibers in young mice. Both young and old mice possessed SRF immunoreactivity in some satellite cells expressing Pax7. MRTF-A and STARS mRNA levels significantly declined with aging in the triceps brachii and quadriceps muscles. The amount of MRTF-A protein was markedly reduced in the nuclear fraction of aged muscle of mice. The amounts of RhoA and RhoGDI in the crude homogenate or the cytosolic and membrane fractions were greater in the aged muscle. Senescent mice possessed significantly higher levels of MyoD protein in the cytosol and nucleus. Decreased SRF and MRTF expression may induce the atrophy of skeletal muscle with aging.

In vivo time-lapse microscopy reveals no loss of murine myonuclei during weeks of muscle atrophy

Numerous studies have suggested that muscle atrophy is accompanied by apoptotic loss of myonuclei and therefore recovery would require replenishment by muscle stem cells. We used in vivo time-lapse microscopy to observe the loss and replenishment of myonuclei in murine muscle fibers following induced muscle atrophy. To our surprise, imaging of single fibers for up to 28 days did not support the concept of nuclear loss during atrophy. Muscles were inactivated by denervation, nerve impulse block, or mechanical unloading. Nuclei were stained in vivo either acutely by intracellular injection of fluorescent oligonucleotides or in time-lapse studies after transfection with a plasmid encoding GFP with a nuclear localization signal. We observed no loss of myonuclei in fast- or slow-twitch muscle fibers despite a greater than 50% reduction in fiber cross-sectional area. TUNEL labeling of fragmented DNA on histological sections revealed high levels of apoptotic nuclei in inactive muscles. However, when costained for laminin and dystrophin, virtually none of the TUNEL-positive nuclei could be classified as myonuclei; apoptosis was confined to stromal and satellite cells. We conclude that disuse atrophy is not a degenerative process, but is rather a change in the balance between protein synthesis and proteolysis in a permanent cell syncytium.