Expression of SERCA2a is independent of innervation in regenerating soleus muscle

The speed of contraction of a skeletal muscle largely depends on the myosin heavy chain isoforms (MyHC), whereas the relaxation is initiated and maintained by the sarcoplasmic reticulum Ca²⁺-ATPases (SERCA). The expression of the slow muscle-type myosin heavy chain I (MyHCI) is entirely dependent on innervation, but, as we show here, innervation is not required for the expression of the slow-type sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) in regenerating soleus muscles of the rat, although it can play a modulator role. Remarkably, the SERCA2a level is even higher in denervated than in innervated regenerating soleus muscles on day 7 when innervation is expected to resume. Later, the level of SERCA2a protein declines in denervated regenerated muscles but it remains expressed, whereas the corresponding mRNA level is still increasing. SERCA1 (i.e., the fast muscle-type isoform) expression shows only minor changes in denervated regenerating soleus muscles compared with innervated regenerating controls. When the soleus nerve was transected instead of the sciatic nerve, SERCA2a and MyHCI expressions were found to be even more uncoupled because the MyHCI nearly completely disappeared, whereas the SERCA2a mRNA and protein levels decreased much less. The transfection of regenerating muscles with constitutively active mutants of the Ras oncogene, known to mimic the effect of innervation on the expression of MyHCI, did not affect SERCA2a expression. These results demonstrate that the regulation of SERCA2a expression is clearly distinct from that of the slow myosin in the regenerating soleus muscle and that SERCA2a expression is modulated by neuronal activity but is not entirely dependent on it. slow type sarcoplasmic reticulum Ca²⁺ pump; MyHCI; nerve influence     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.     

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.     

A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling

Here we describe a small family of proteins, termed MCIP1 and MCIP2 (for myocyte-enriched calcineurin interacting protein), that are expressed most abundantly in striated muscles and that form a physical complex with calcineurin A. MCIP1 is encoded by DSCR1, a gene located in the Down syndrome critical region. Expression of the MCIP family of proteins is up-regulated during muscle differentiation, and their forced overexpression inhibits calcineurin signaling to a muscle-specific target gene in a myocyte cell background. Binding of MCIP1 to calcineurin A requires sequence motifs that resemble calcineurin interacting domains found in NFAT proteins. The inhibitory action of MCIP1 involves a direct association with the catalytic domain of calcineurin, rather than interference with the function of downstream components of the calcineurin signaling pathway. The interaction between MCIP proteins and calcineurin may modulate calcineurin-dependent pathways that control hypertrophic growth and selective programs of gene expression in striated muscles.     

Mitotic activity and nucleic acid precursor incorporation in denervated and innervated limb stumps of axolotl larvae.

Mitotic activity and DNA and RNA precursor incorporation were compared in innervated regenerating limbs and in denervated, non-regenerating limbs on days 8 and 9 post-amputation. Innervated limbs had well-developed cone stage blastemas which showed high cellular mitotic indices and H3-thymidine labeling indices of 0.40-0.50 and H3-uridine labeling indices of 0.50-0.75. In contrast, denervated limbs showed dedifferentiated cells distally under thickened wound epithelia, but essentially no mitotic activity and no blastema formation. These dedifferentiated cells showed lower levels of H3-thymidine (0.10 index) and H3-uridine (0.50) incorporation than regenerating limbs. Labeling indices of wound epithelia are also compared.     

Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle

Monoclonal antibodies were prepared to stage-specific chicken pectoral muscle myosin heavy chain isoforms. From comparison of serial sections reacted with these antibodies, the myosin heavy chain isoform composition of individual myofibers was determined in denervated pectoral muscle and in regenerating myotubes that developed following cold injury of normal and denervated muscle. It was found that the neonatal myosin heavy chain reappeared in most myofibers following denervation of the pectoral muscle. Regenerating myotubes in both innervated and denervated muscle expressed all of the myosin heavy chain isoforms which have thus far been characterized in developing pectoral muscle. However, the neonatal and adult myosin heavy chains appeared more rapidly in regenerating myotubes compared to myofibers in developing muscle. While the initial expression of these isoforms in the regenerating areas was similar in innervated and denervated muscles, the neonatal myosin heavy chain did not disappear from noninnervated regenerating fibers. These results indicate that innervation is not required for the appearance of fast myosin heavy chain isoforms, but that the nerve plays some role in the repression of the neonatal myosin heavy chain.     

Developmentally regulated expression of cytochrome-c oxidase isoforms in regenerating rat skeletal muscle

The developmentalexpression of tissue-specific isoforms ofcytochrome-c oxidase(COX) subunit VIII [heart (COX VIII-H) and liver (COXVIII-L)] and the influence of innervation were examined inregenerating fast [extensor digitorum longus (EDL)] andslow (soleus) muscles. In adult muscles, COX VIII-H was the predominant isoform. The COX VIII-L mRNA was expressed 3 days after induction ofregeneration, and it progressively decreased after 7, 10, 14, and 30 days of regeneration in both muscles. In contrast, the expression ofCOX VIII-H mRNA accumulated as myogenesis proceeded to the myotubestage between 7 and 10 days of regeneration and progressively increasedto near control levels by 30 days. The influence of innervation on theexpression of COX VIII and -actin isoforms wasexamined in control, innervated, and denervatedregenerating muscles at 3 and 10 days. The relative expression of COXVIII-L mRNA in denervated regenerating EDL muscles was significantly greater, while that of COX VIII-H was significantlyless than in innervated regenerating EDL muscles after 10 days ofregeneration. Similarly, cardiac -actin mRNA levelswere elevated in denervated regenerating EDL muscles after 10 days ofregeneration. In conclusion, motor innervation influences thetransition from the COX VIII-L to COX VIII-H isoform during myogenesisin regenerating muscles.

     

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.     

Long-term control of renal blood flow: what is the role of the renal nerves?

We have developed a system for long-term continuous monitoring of cardiovascular parameters in rabbits living in their home cage to assess what role renal sympathetic nerve activity (RSNA) has in regulating renal blood flow (RBF) in daily life. Blood pressure, heart rate, locomotor activity, RSNA, and RBF were recorded continuously for 4 wk. Beginning 4-5 days after surgery a circadian rhythm, dependent on feeding time, was observed. When averaged over all days RBF to the innervated and denervated kidneys was not significantly different. However, control of RBF around these mean levels was dependent on the presence of the renal sympathetic nerves. In particular we observed episodic elevations in heart rate and other parameters associated with activity. In the denervated kidney, during these episodic elevations, the increase in renal resistance was closely related to the increase in arterial pressure. In the innervated kidney the renal resistance response was significantly more variable, indicating an interaction of the sympathetic nervous system. These results indicate that whereas overall levels of RSNA do not set the mean level of RBF the renal vasculature is sensitive to episodic increases in sympathetic nerve activity.     

Effect of moderate acute exercise on expression of mRNA involved in the calcineurin signaling pathway in human skeletal muscle

Calcineurin, a calcium-regulated protein phosphatase, activates gene expression specific to slow muscle fibers by dephosphorylating a family of the nuclear factor of activated T cells (NFAT), which cooperates with myocyte enhancer factor-2 (MEF2) and AP-1. However, it remains unknown how acute exercise influences this signaling pathway and leads to the development of slow muscle fibers. In the present study, we investigated the effect of moderate acute exercise on mRNA expression of genes in the calcineurin signaling pathway in human skeletal muscle. Five healthy volunteers underwent 1 h bicycle ergometer at 50%VO2max, and vastus lateralis muscle biopsies were collected before and after exercise. Four hours after exercise, alterations in mRNA expression of NFAT 1-3 were observed with a wide variety among subjects, while c-fos mRNA was significantly induced in all subjects. By contrast, the expression of calcineurin, MEF2, and myocyte-enriched calcineurin-interacting protein 1 (MCIP1) remained unchanged. These results suggest that even moderate acute exercise may change mRNA expression of genes in the calcineurin-signaling pathway.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Role of adrenergic innervation in experimental renal hypertension

Renal hypertension was induced by ligation of the aorta between renal arteries in rats sympathectomized with 6-hydroxydopamine. In the early phase, equally severe hypertension developed in the denervated group as compared to innervated controls. Later, blood pressure was lower in the denervated rats. Initially, increases in plasma renin were seen in both groups; the levels, however, were markedly lower in the denervated rats. Later, the renin levels were similar and not different from baseline. It is concluded that adrenergic neural activity is not essential in the development of renal hypertension; the maintenance of the chronic state, however, depends in part on adrenergic innervation.