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March 2007

Novel Trafficking Pathway Found for Heart and Muscle Protein

Live-cell imaging provides new insights into mechanisms that regulate heart disease

Results: In a paper that appears in the American Journal of Physiology: Cell Physiology, scientists at Pacific Northwest National Laboratory describe a new cellular trafficking, or processing, pathway for an important protein that regulates heart and skeletal muscle. Phospholamban (PLB) is one of the main molecular targets of beta-blocker drugs for heart disease. The paper also introduces the use of fluorescent tags (tetracysteine-FlAsH fluorophore tags) for live-cell imaging in muscle cells (myocytes). These dual-purpose tags can also be used to identify protein-protein complexes.
Kinetic resolution of cellular trafficking
PNNL scientists obtained kinetic resolution of cellular trafficking through one- (A) and two- (B) color pulse-chase experiment that measures cellular localization of newly synthesized proteins. A) Tagged PLB (upper row) or sarco/endoplasmic reticulum calcium ATPase1a (lower row) expressing myoblasts were directly imaged after labeling. B) Resolution of newly synthesized proteins. Overlay (right panel in B) shows co-localization of newly synthesized (green) and existing (red) proteins. Enlarged View

Methods: Using a combination of new and established imaging tools, the PNNL team measured changes in abundance, cellular location and turnover of proteins in living myoblasts (immature muscle cells) understand how coordinated changes in the abundance and intracellular trafficking of PLB and Ca-ATPase contribute to muscle maturation. The Ca-ATPase is a membrane transporter that couples adenosine triphosphate (ATP) hydrolysis to calcium ion transport into intracellular stores; that is, the sarcoplasmic reticulum (SR), which mediates muscle relaxation, the slowest step in contraction. The association of PLB with the Ca-ATPase modulates contractile force on a beat-to-beat basis in response to adrenalin, an important regulator of cardiac muscle function.

Why it matters: Changes in the abundance of PLB and the SR Ca-ATPase occurring in heart disease, aging and other diseases suggest the importance of understanding the processes regulating the formation of PLB-ATPase interactions in the myocyte. This research team found that the early expression of PLB during muscle development is accompanied by a unique trafficking pattern within the cell that occurs outside the compartment in which Ca-ATPase is subsequently expressed. Thus, upon expression of the Ca-ATPase, its functional interaction with PLB requires re-programming of the cell and PLB's localization in the myocyte.

These findings suggest an important role for PLB during muscle differentiation that is distinct from its previously recognized role in regulating Ca-ATPase.

Next Steps: Future measurements should focus on understanding 1) the role of PLB's trafficking pattern in the immature myocyte prior to Ca-ATPase expression, and 2) how this relates to maintaining cardiac function.

Funding: The research was conducted under the Biomolecular Systems Initiative at PNNL. Portions of the work were also supported by the National Institutes of Health.

Source: Stenoien DL, TV Knyushko, MP Londono, LK Opresko, MU Mayer, ST Brady, TC Squier and DJ Bigelow. 2007. "Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation." American Journal of Physiology: Cell Physiology 292:C2084-C2094.

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