Defining the role of Xin and associated proteins in skeletal muscle health

Abstract

Xin is a F-actin cytoskeletal remodelling adaptor protein important for skeletal muscle health, including functionality and regeneration. Previous studies from our lab have proposed new roles for Xin based on Xin -/- studies in murine skeletal muscle and satellite cells (SCs). We discovered that Xin also maintained mitochondrial morphology and function and facilitated extracellular Ca2+ entry by stabilizing membrane protein channel components. However, these observations lacked a molecular basis, as many of Xin’s protein binding partners remain unknown. The main objective of this study was to utilize a Xin overexpression (OE) model to aid in discovering the function of Xin within skeletal muscle and identify new Xin binding partners. While Xin OE had a no discernible effect on myoblast morphological features and F-actin distribution, it significantly hindered differentiation into multi-nucleated myotubes. Time based differentiation assays from Xin OE myoblasts showed myotube lengths were decreased at day 5 (20%; P<.001) and further decreased at day 7 (60%; P<.0001). Xin OE cells displayed an abnormal increase mitochondrial enzyme content (~2.94 fold) and a reduction in myoblast motility (~45%, P <0.05), both which likely contributed to deficits in differentiation. Tracking Xin’s subcellular localization, Xin colocalized with mitochondria markers (~71 % Overlap; R2 0.68) and was present in mitochondria fractionations. Xin co-immunoprecipitated with mitochondrial-associated membrane (MAM) tether proteins: VDAC, Mitofusin 1, and Mitofusin 2. Furthermore, Xin interacted with extracellular calcium entry channels and related proteins such as: Orai1, Stim-1, Junctin, Triadin, and Homer. These results provide further evidence that Xin is a multi-functional protein which regulates mitochondria and cellular calcium entry by directly interacting with MAM tethers and calcium channels. Based on our Xin overexpression and knockout experiments, these molecular interactions ultimately play a role in myoblast differentiation and maintain contractile and metabolic function within skeletal muscle fibers.