Mechanical stimuli play a major role in regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and quality of life. Although the link between mechanical signals and the control of muscle mass has been recognized for decades, the mechanisms involved in converting mechanical information into the molecular events that control this process are not known. Thus, the primary research interest of this lab is to determine how skeletal muscles sense mechanical information and convert this stimulus into the molecular events that regulate changes in mass (mechanotransduction). Our studies in the field of mechanotransduction and the regulation of skeletal muscle mass have led us to focus on a protein kinase called the mammalian target of rapamycin (mTOR). Signaling by mTOR is necessary for mechanically-induced growth of skeletal muscle, and we have recently determined that mechanical stimuli activate mTOR signaling through a unique mechanism involving phospholipase D and the lipid second messenger phosphatidic acid (PLD→PA→mTOR). Thus, our current efforts are now focused on further defining how mechanical stimuli activate the PLD→PA→mTOR pathway, and muscle growth. To accomplish this, we are using an integrative approach based on th e following three projects.

1. Live Cell Imaging of Mechanically-Induced Signaling Events

2. Defining the Mechanically-induced PLD→PA→mTOR Signaling Pathway

3. Identification of Molecules that Regulate Skeletal Muscle Growth Atrophy

 

 

1. Live Cell Imaging of mechanically induced signaling events

New technologies based on live cell imaging of fluorescent indicators are making it possible to accurately quantify and visualize the dynamic nature of molecular signaling in an intact cellular environment. In this project, a microscope-mounted cell stretch device (MMSD) is being used to apply live cell imaging technologies to the study of mechanotransduction in skeletal muscle. These technologies include FRET-based biosensors recently developed by colleagues in the Whittaker Institute of Biomedical Engineering at UCSD and GFP-tagged probes. For example, a GFP-tagged phosphatidic acid binding domain from the Raf-1 protein is being used to monitor the mechanically-induced spatiotemporal dynamics of phosphatidic acid accumulation in real-time. With these state-of-the-art technologies, we can test our current hypotheses about mechanotransduction within the context of a live cell. Furthermore, the observations made in these studies will inspire novel hypotheses about mechanotransduction in skeletal muscle . These hypotheses can then be tested with the in vitro, ex vivo and in vivo models employed in projects #2 and #3.

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2. Defining the Mechancially-Induced PLD→PA→mTOR Signaling Pathway

We have recently developed both in vitro and ex vivo models for studying the mechanisms involved in the mechanical activation of mTOR signaling. In this project, the in vitro model of mechanical stimulation is being combined with the use of pharmacological inhibitors and molecular interventions, such as siRNA and transfection of wild type / mutant proteins, to identify molecules that are necessary for the mechanical activation of the PLD→PA→mTOR pathway. The observations made in vitro will then be confirmed with our ex vivo model for mechanical stimulating muscle explants from wild type and / or transgenic mice. This project will extend our current knowledge about the mechanisms involved in the mechanical activation of the PLD→PA→mTOR pathway and act as a compass to guide us step-by-step through the elucidation of the upstream signaling pathway. The long-term goal of this project is to identify the mechanosensitive molecule(s) that convert the initial mechanical signal into the molecular events that activate mTOR signaling and skeletal muscle growth . Furthermore, these studies will identify molecules of interest for project #3.

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3. Identification of Molecules that Regulate Skeletal Muscle Growth Atrophy

We have established proficiency with the use of in vivo technologies for modifying gene expression such as transgenic mice and electroporation. Using these techniques, variants of the molecules identified as being necessary for the mechanical activation of the PLD→PA→mTOR pathway in project #2, will be knocked-out or over-expressed in skeletal muscle, and the resulting effect on muscle mass / fiber size will be determined. For example, our current hypothesis predicts that constitutive activation of the PLD→PA→mTOR pathway will promote skeletal muscle growth. Thus, plans are underway to over-express PLD1 and PLD2 (a constitutively-active isoform) and determine the effect of these manipulations on skeletal muscle fiber size. The experiments from this portion of the project will lead to the identification of molecules that are sufficient for growth. Furthermore, we will also use the above approach to determine the effect of over-expressing molecules, such as PLD, on preventing unloading-induced atrophy (disuse).

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Taken together, the projects in our lab form an integrative platform for defining how mechanical signals are converted into the molecular events that regulate skeletal muscle growth and atrophy. The long-term goal of these projects is to identify molecular targets for genetic or pharmacological therapies aimed at preventing skeletal muscle atrophy during conditions such as, chronic bedrest, immobilization, spaceflight, aging, cancer cachexia and muscular dystrophy.