Research shows that exposure to space conditions causes muscle atrophy by actually altering gene expression, meaning that some genes will decrease or increase the amount of proteins they produce. Dr. Kandarian is examining the changes in gene expression to identify the molecular processes that regulate muscle atrophy.
Overview
Gene Expression Profiling of Unloaded Skeletal Muscle
Principal Investigator:
Susan C. Kandarian, Ph.D.
Organization:
Boston University
Technical Summary
Original aims:
The overall goal of this project is to elucidate signaling mechanisms that mediate the adaptation of mammalian skeletal muscle to mechanical unloading. In identifying cellular mechanisms we will be in a better position to develop effective countermeasures. Global gene expression profiling was a major tool used to address this goal. The specific aims were:
The overall goal of this project is to elucidate signaling mechanisms that mediate the adaptation of mammalian skeletal muscle to mechanical unloading. In identifying cellular mechanisms we will be in a better position to develop effective countermeasures. Global gene expression profiling was a major tool used to address this goal. The specific aims were:
- To conduct global gene expression analysis in mechanically unloaded mammalian skeletal muscle. Affymetrix RatU34A GeneChips were used to probe mRNA expression in rat soleus muscle after 1, 4, 7 and 14 days of hindlimb unloading.
- To identify candidate factors and pathways involved in the regulation of unloading induced muscle atrophy. Clustering algorithms were then used to elucidate sets of genes, with known or unknown functions, that are co-regulated based on temporal expression patterns. These approaches provided insight into possible gene associations and candidate players in the pathways that regulate the atrophy process, and thereby proposing them for further study. Quantitative analysis of candidate factors and pathways involved in regulating unloading induced atrophy.
Key findings of the project:
The results from the initial phases of the project have recently been published in their entirety. In brief, expression of 309 known genes was significantly changed by at least 2-fold. K-means clustering was used to divide these genes into co-regulated clusters based on the similarity of temporal expression patterns. This allowed the development of a timeline of the atrophy process with respect to the behavior of genes in a broad array of functional categories. Regulatory genes were often upregulated early, in either a transient or sustained manner, but they also populated clusters with later patterns of activation, suggesting different phases of molecular adaptations. Other early events were the activation of ubiquitination genes and downregulation of protein chaperones. In comparison, clusters representing slightly delayed activation patterns included genes involved in fast contraction, glycolysis, translational inhibition, oxidative stress, protein degradation, and amino acid catabolism. Downregulated genes exhibited fewer unique expression patterns and included structural and regulatory genes of the extracellular matrix and cytoskeleton, and genes that define a slow phenotype.
Other novel findings include the tight co-activation of proteasome subunit and ubiquitination genes, differential regulation of serine proteases and serine protease inhibitors, and the identification of transcriptional, signaling, growth and cell cycle genes that likely play a role in atrophy. The present work has uncovered temporal patterns of gene expression that highlight the molecular processes that underlie muscle atrophy and provide new avenues for study. Nedd4 project: Results from several laboratories have shown that the ubiquitin-proteasome system is responsible from the majority of muscle protein loss that occurs with disuse. Ubiquitin-protein ligases (E3s) are responsible for the targeting of specific proteins for degradation by the proteasome.
Our microarray data reconfirm that Atrogin1 and MuRF1 are upregulated during unloading, but we have also identified another upregulated ubiquitin-protein ligase not previously characterized with respect to muscle atrophy called Nedd4. Nedd4 has a pattern of activation very similar to that of Atrogin1/MAFbx. We have confirmed this pattern of activation at the mRNA and protein level. Nedd4 is known to ubiquitinate membrane proteins but its role in muscle protein turnover has not yet been defined. We are in the process investigating the role of Nedd4 in muscle using the tools described.
Impact of findings on objectives:
The data suggests several pathways that are at work during muscle atrophy. The results also indicate that there are several different temporal switches of regulatory genes that are activated during atrophy. The next step towards studying the function of these putative regulatory genes is to overexpress or inhibit their actions in muscle cell culture or in vivo. In order to study the role of the candidate genes we have identified from our microarray analysis of atrophying skeletal muscle, we are using conditional expression systems or transduction of candidate genes post-differentiation in cell culture and in vivo. The approaches we are using for the overexpression or inhibition of candidate genes are: a doxycycline conditional expression system and an adenoviral expression system. For in vivo experiments we are using direct plasmid injection with electroporation. The vectors encode one of the several genes we will study in either wild-type, constitutively active, or dominant-negative form. We will then test the effects of the overexpression of these genes in whole muscle or in muscle cell culture using standard biochemical and morphological assays.
Earth Applications
It is well known that exposure to microgravity, as well as muscular unloading on earth, leads to marked decreases in muscle size, functional capacity, and fatigue resistance. What is poorly understood about atrophy is how the protein loss is controlled, including the triggers and signaling mechanisms. Results from the microarray analysis of unloaded rat soleus muscles have not only given us insight into the processes and pathways involved in muscle atrophy but they have provided an excellent source of candidate genes to test for regulatory roles in the atrophy process. In identifying these genes we will be in a better position to develop more effective countermeasures to combat the deleterious changes in muscle function due to exposure to spaceflight.
This project's funding ended in 2004