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Principal Investigator:
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Susan L. Hamilton, Ph.D.
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Organization:
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Baylor College of Medicine
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Project Ended:
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2000
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The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca
2+ concentration (Ca
2+i). In most cells the consequences of changes in Ca
2+i depend on the amplitude, frequency and duration of the Ca
2+ signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several Ca
2+ regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NF B and the pro-apoptotic protein BAD. Other Ca2+ sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.
Mechanisms by which hind limb-suspension could alter the concentration of resting Ca2+ - In the second year of NSBRI funding our goals were to determine both the cause and the functional consequences of the rise in resting Ca2+. Muscle inactivity increases the production of ROS (Am. J. Physiol. 28: E839-E844, 1993) and decreases nitric oxide (NO) production (Am.J. Physiol. 275: C260-C266, 1998). We demonstrated that, in muscle, there is a delicate balance between ROS, NO and calmodulin (CaM) regulation of Ca2+ leak via the skeletal muscle Ca2+ release channel (RYR1). Both oxidants and Ca2+ free CaM activate RYR1, but their interactions with the channel are mutually exclusive. When cytoplasmic Ca2+ rises, Ca2+CaM helps to shut the channel, but this effect can be blocked by channel oxidation and, conversely, both Ca2+CaM and Ca2+ free CaM partially protect RYR1 from oxidation. NO blocks oxidative activation of RYR1, has no effect on Ca2+CaM inhibition of the channel, but prevents the activation of the channel by Ca2+ free CaM. The net effect of conditions that increase ROS but decrease NO would be prolonged opening of RYR1 and increased cytoplasmic Ca2+. These findings have led to our current working hypothesis that the increase in resting Ca2+ in skeletal muscle during hind limb suspension arises, at least in part, from increased SR Ca2+ leak. Decreases in SR luminal Ca2+ could also activate store operated Ca2+ influx pathways. Mechanisms to regulate Ca2+i will be investigated in the coming year.
Effects of sustained increases in Ca2+i on muscle function - Increases in intracellular Ca2+ can alter a variety of signal transduction pathways and the actual pathways altered will depend on the amplitude, frequency and duration of the Ca2+ signal. Our studies show a sustained increased Ca2+i after about three days of hind-limb suspension. We are exploring the possibility that the increased Ca2+i activates pro-apoptotic pathways leading to loss of myonuclei and muscle remodeling. Activation of apoptotic pathways has previously been suggested to contribute to hind limb suspension induced muscle remodeling (Am. J. Physiology 273: C579-C587, 1997). In B cells low sustained increases in intracellular Ca2+ have been shown to activate NFAT while large Ca2+ transients activate NF B and JNK transcription factors (Nature 386:855-858, 1997). For the NFAT pathway, the increases in resting Ca2+ activate calcineurin and this dephosphorylates NFAT, allowing it to translocate to the nucleus. The transcriptional events activated by NFAT depend on the status of a number of other signal transduction pathways, but NFAT activation can increase the transcription of several pro-apoptotic genes. We have identified the presence of NFAT in skeletal muscle and have shown that cytoplasmic levels of NFAT decrease with hindlimb suspension. We have not, however, yet shown that this is due to translocation to the nucleus. We are currently assessing the nuclear translocation of NFAT with hind limb suspension.
In addition to its effects on NFAT, calcineurin can dephosphorylate the apoptotic protein BAD (Science 284:339-343, 1999), leading to its interaction with the anti-apoptotic proteins Bcl-2 and BClxl. We have detected BAD in skeltal muscle and are currently attempting to assess whether hind limb suspension alters in phosphorylation status. We have, however, shown that there significant increase in cytosolic BAD with 14 days of hind limb suspension. We are currently assessing if this is due to new protein or decreased breakdown. An anti-apoptotic protein that can interact with Bcl-2 is smn (Nature 390:413-417, 1997), the protein missing in spinal muscular atrophy. Our preliminary studies suggest a decrease in cytoplasmic smn with hind limb suspension. We also have preliminary evidence for a decrease in cytosolic IκB.
Skeletal muscle specific FKBP12 deficient mice as models for effects of microgravity - FKBP12 is an endogenous modulator of both RYR1 and calcineurin. FKBP12 inhibits calcineurin and stabilizes a closed state of RYR1. The absence of FKBP12 is likely to lead to increases in both resting Ca2+ and calcineurin activity. If calcineurin activation produces muscle atrophy, the knockout of FKBP12 may mimic the effects of hind limb suspension on skeletal muscle. An animal model of muscle atrophy would be extremely useful for drug intervention. We have previously prepared FKBP12 deficient mice. Skeletal muscle force production was markedly diminished but, unfortunately these animals die of cardiac hypertrophy. To avoid the cardiac complications we are in the process of creating a skeletal muscle specific knockout of FKBP12 using the Cre-loxP system. To generate the skeletal muscle restricted FKBP12-deficient mice, two transgenic mouse lines will be used, the skeletal muscle specific Cre mouse and the FKBP12-loxP mouse. In collaboration with Dr. Weinian Shou, we now have both the FKBP12-loxP targeted ES cell lines and the linear myogenin-Cre construct. We anticipate having the skeletal muscle specific FKBP12 knockout mice within the next six months. We will compare soleus muscle from these mice to that obtained from hind limb suspended mice to determine if the mechanisms of atrophy are related.
Summary of Progress in the Second Year
In the second year of NSBRI funding we have demonstrated that: 1) there is an early and sustained increase in intracellular Ca2+, 2) an increase in oxidants or a decrease in NO can increase resting Ca2+, 3) hind limb suspension alters NF B and possibly NFAT signaling in skeletal muscle, 4) NFκB mediates proteolytic signaling in muscle by upregulating components of the ubiquitin/proteasome pathway, and 5) hind limb suspension increases the amount of the pro-apoptotic protein, BAD and decreases anti-apoptotic protein, SMN. In addition to this, we are well on our way to producing skeletal muscle specific FKBP12 deficient mice. These will be used to test our hypothesis that increased cytoplasmic Ca2+ activates calcineurin, producing effects similar to those found with hind-limb suspension.
In summary, we propose that skeletal muscle adaptation to microgravity represents an increase in both calcineurin and ubiquitin/proteasome activity and a shift in the balance between pro-apoptotic and anti-apoptotic pathways. We propose to test this hypothesis during the third year. The demonstration of the activation of these pathways by hind-limb suspension will allow us then to design and test new countermeasures.
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