Kenneth S. Campbell

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca2+ sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer

The mid/hindbrain (MHB) junction can act as an organizer to direct the development of the midbrain and anterior hindbrain. In mice, Otx2 is expressed in the forebrain and midbrain and Gbx2 is expressed in the anterior hindbrain, with a shared border at the level of the MHB organizer. Here we show that, in Gbx2-/- mutants, the earliest phenotype is a posterior expansion of the Otx2 domain during early somite stages. Furthermore, organizer genes are expressed at the shifted Otx2 border, but not in a normal spatial relationship. To test whether Gbx2 is sufficient to position the MHB organizer, we transiently expressed Gbx2 in the caudal Otx2 domain and found that the Otx2 caudal border was indeed shifted rostrally and a normal appearing organizer formed at this new Otx2 border. Transgenic embryos then showed an expanded hindbrain and a reduced midbrain at embryonic day 9.5–10. We propose that formation of a normal MHB organizer depends on a sharp Otx2 caudal border and that Gbx2 is required to position and sharpen this border.

CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function

MyoD, a master regulator of myogenesis, exhibits a circadian rhythm in its mRNA and protein levels, suggesting a possible role in the daily maintenance of muscle phenotype and function. We report that MyoD is a direct target of the circadian transcriptional activators CLOCK and BMAL1, which bind in a rhythmic manner to the core enhancer of the MyoD promoter. Skeletal muscle of ClockΔ19 and Bmal1−/− mutant mice exhibited ∼30% reductions in normalized maximal force. A similar reduction in force was observed at the single-fiber level. Electron microscopy (EM) showed that the myofilament architecture was disrupted in skeletal muscle of ClockΔ19, Bmal1−/−, and MyoD−/− mice. The alteration in myofilament organization was associated with decreased expression of actin, myosins, titin, and several MyoD target genes. EM analysis also demonstrated that muscle from both ClockΔ19 and Bmal1−/− mice had a 40% reduction in mitochondrial volume. The remaining mitochondria in these mutant mice displayed aberrant morphology and increased uncoupling of respiration. This mitochondrial pathology was not seen in muscle of MyoD−/− mice. We suggest that altered expression of both Pgc-1α and Pgc-1β in ClockΔ19 and Bmal1−/− mice may underlie this pathology. Taken together, our results demonstrate that disruption of CLOCK or BMAL1 leads to structural and functional alterations at the cellular level in skeletal muscle. The identification of MyoD as a clock-controlled gene provides a mechanism by which the circadian clock may generate a muscle-specific circadian transcriptome in an adaptive role for the daily maintenance of adult skeletal muscle.

Distinct growth hormone receptor signaling modes regulate skeletal muscle development and insulin sensitivity in mice

Skeletal muscle development, nutrient uptake, and nutrient utilization is largely coordinated by growth hormone (GH) and its downstream effectors, in particular, IGF-1. However, it is not clear which effects of GH on skeletal muscle are direct and which are secondary to GH-induced IGF-1 expression. Thus, we generated mice lacking either GH receptor (GHR) or IGF-1 receptor (IGF-1R) specifically in skeletal muscle. Both exhibited impaired skeletal muscle development characterized by reductions in myofiber number and area as well as accompanying deficiencies in functional performance. Defective skeletal muscle development, in both GHR and IGF-1R mutants, was attributable to diminished myoblast fusion and associated with compromised nuclear factor of activated T cells import and activity. Strikingly, mice lacking GHR developed metabolic features that were not observed in the IGF-1R mutants, including marked peripheral adiposity, insulin resistance, and glucose intolerance. Insulin resistance in GHR-deficient myotubes derived from reduced IR protein abundance and increased inhibitory phosphorylation of IRS-1 on Ser 1101. These results identify distinct signaling pathways through which GHR regulates skeletal muscle development and modulates nutrient metabolism.

Sp8 is crucial for limb outgrowth and neuropore closure

In this report we describe the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation.

A cross-bridge mechanism can explain the thixotropic short-range elastic component of relaxed frog skeletal muscle

1 The passive tension and sarcomere length of relaxed frog skeletal muscle fibres were measured in response to imposed length stretches. The tension response to a constant‐velocity stretch exhibited a clear discontinuity. Tension rose more rapidly during the initial ∼ 0.4 %L0 of the stretch than during the latter stages (where L0 is the resting length of the fibre). This initial tension response is attributed to the short‐range elastic component (SREC). 2 The use of paired triangular stretches revealed that the maximum tension produced during the SREC response of the second stretch was significantly reduced by the first stretch. This history‐dependent behaviour of the SREC reflects thixotropic stiffness changes that have been previously described in relaxed muscle. 3 The biphasic nature of the SREC tension response to movement was most apparent during the first imposed length change after a period at a fixed length, irrespective of the direction of movement. 4 If a relaxed muscle was subjected to an imposed triangular length change so that the muscle was initially stretched and subsequently shortened back to its original fibre length, the resting tension at the end of the stretch was reduced relative to its initial pre‐stretch value. Following the end of the stretch, tension slowly increased towards its initial value but the tension recovery was not accompanied by a contemporaneous increase in sarcomere length. This finding suggests that the resting tension of a relaxed muscle fibre is not entirely due to passive elasticity. The results are compatible with the suggestion that a portion of the resting tension ‐ the filamentary resting tension (FRT) ‐ is produced by a low level of active force generation. 5 If a second identical stretch was imposed on the muscle at a time when the resting tension was reduced by the previous stretch, the maximal tension produced during the second stretch was the same as that produced during the first, despite the second stretch commencing from a lower initial resting tension. 6 In experiments using paired triangular length changes, an inter‐stretch interval of zero did not produce a substantially greater thixotropic reduction in the second stretch elastic limit force than an inter‐stretch interval in the range 0.5‐1 s. 7 A theoretical model was developed in which the SREC and FRT arise as manifestations of a small number of slowly cycling cross‐bridges linking the actin and myosin filaments of a relaxed skeletal muscle. The predictions of the model are compatible with many of the experimental observations. If the SREC and FRT are indeed due to cross‐bridge activity, the model suggests that the cross‐bridge attachment rate must increase during interfilamentary movement. A mechanism (based on misregistration between the actin binding sites and the myosin cross‐bridges) by which this might arise is presented.

TNF-α acts via TNFR1 and muscle-derived oxidants to depress myofibrillar force in murine skeletal muscle

Tumor necrosis factor-alpha (TNF) diminishes specific force of skeletal muscle. To address the mechanism of this response, we tested the hypothesis that TNF acts via the type 1 (TNFR1) receptor subtype to increase oxidant activity and thereby depress myofibrillar function. Experiments showed that a single intraperitoneal dose of TNF (100 microg/kg) increased cytosolic oxidant activity (P < 0.05) and depressed maximal force of male ICR mouse diaphragm by approximately 25% within 1 h, a deficit that persisted for 48 h. Pretreating animals with the antioxidant Trolox (10 mg/kg) lessened oxidant activity (P < 0.05) and abolished contractile losses in TNF-treated muscle (P < 0.05). Genetic TNFR1 deficiency prevented the rise in oxidant activity and fall in force stimulated by TNF; type 2 TNF receptor deficiency did not. TNF effects on muscle function were evident at the myofibrillar level. Chemically permeabilized muscle fibers from TNF-treated animals had lower maximal Ca2+-activated force (P < 0.02) with no change in Ca2+ sensitivity or shortening velocity. We conclude that TNF acts via TNFR1 to stimulate oxidant activity and depress specific force. TNF effects on force are caused, at least in part, by decrements in function of calcium-activated myofibrillar proteins.

Cdc42 deficiency causes Sonic hedgehog-independent holoprosencephaly.

The telencephalic neuroepithelium (NE) of mammalian brain has an apical-basal polarity that is marked by the positioning of neural progenitors and adherens junctions on the apical/ventricular surface and the ascending of radial glia/progenitor fibers toward the pial/basal surface. The signaling pathway that establishes this apical-basal polarity of NE is not completely understood, but the Rho-family GTPase Cdc42 may play a critical role because it controls cadherin-based intercellular junctions and cell polarity in many species. Here, we tested this hypothesis by a conditional gene-targeting strategy by using the Foxg1-Cre line to delete Cdc42 in the telencephalic neural progenitors in mouse embryos. We found that Cdc42-deletion abolishes the apical localization of PAR6, aPKC, E-cadherin, β-catenin, and Numb proteins in the NE, and severely impairs the extension of nestin-positive radial fibers. Consequently, neural progenitors were scattered throughout the entire depth of the NE, and the Cdc42-deficient telencephalon failed to bulge or separate into two cerebral hemispheres, resulting in holoprosencephaly. However, neither the midline expression of Sonic hedgehog nor the dorso-ventral patterning of the telencephalon was affected by Cdc42-deletion. Taken together, these results indicate that Cdc42 has an essential role in establishing the apical-basal polarity of the telencephalic NE, which is needed for the expansion and bifurcation of cerebral hemispheres.

Rac1 Controls the Formation of Midline Commissures and the Competency of Tangential Migration in Ventral Telencephalic Neurons

Previous studies using dominant-mutant constructs have implicated Rac1 GTPase in neuritogenesis and neuronal migration. However, overexpression of dominant mutants generally blocks Rho–GTPase activity; thus, it may not reveal the specific or physiological functions of Rac1. To address this issue, we have applied a conditional gene-targeting strategy, using Foxg1–Cre mice to delete Rac1 in the ventricular zone (VZ) of telencephalon and Dlx5/6–Cre–IRES (internal ribosomal entry site)–EGFP (enhanced green fluorescent protein) (Dlx5/6–CIE) in the subventricular zone (SVZ) of ventral telencephalon, respectively. Surprisingly, the deletion of Rac1 in VZ progenitors did not prevent axonal outgrowth of telencephalic neurons. However, the anterior commissure was absent, and the corpus callosal as well as hippocampal commissural axons failed to cross the midline in Rac1/Foxg1–Cre knock-out embryos. The thalamocortical and corticothalamic axons also showed defasciculation or projection defects. These results suggest that Rac1 controls axon guidance rather than neuritogenesis. In addition, although Rac1/Foxg1–Cre knock-out embryos showed delayed radial migration of cortical projection neurons and severe impairment of tangential migration by the ventral telencephalon-derived interneurons, deletion of Rac1 in the SVZ by Dlx5/6–CIE mice produced no discernible defects in tangential migration. These contrasting effects of Rac1 deletion on tangential migration suggest that Rac1 is dispensable for cellular motility per se during neuronal migration. Together, these results underscore the challenge of deciphering the biological functions of Rac1, and Rho–GTPases in general, during mammalian brain development. Moreover, they indicate that Rac1 has a critical role in axon guidance and in acquisition of migratory competency during differentiation of the progenitors for the ventral telencephalon-derived interneurons.

Titin isoform changes in rat myocardium during development

Developmental changes in the alternative splicing patterns of titin were observed in rat cardiac muscle. Titin from 16-day fetal hearts consisted of a single 3710 kDa band on SDS agarose gels, and it disappeared by 10 days after birth. The major adult N2B isoform (2990 kDa) first appeared in 18-day fetal hearts and its proportion in the ventricle increased to approximately 85% from 20 days of age and older. Changes in three other intermediate-sized N2BA isoform bands also occurred during this same time period. The cDNA sequences of fetal cardiac, adult ventricle, and adult soleus were different in the PEVK and alternatively spliced middle Ig domain. Extensive heterogeneity in splice patterns was found in the N2BA PEVK region. The extra length of the fetal titin isoforms appeared to be due to both a greater number of middle Ig domains expressed plus the inclusion of more PEVK exons. Passive tension measurements on myocyte-sized fragments indicated a significantly lower tension in neonate versus adult ventricles at sarcomere lengths greater than 2.1 microm, consistent with the protein and cDNA sequence results. The time course of the titin isoform switching was similar to that occurring with myosin and troponin I during development.