Bin1, A Bar Domain Protein, Is Necessary For The Maintenance Of T-tubule Structure And Intracellular Ca2+ Homeostasis In Skeletal Muscle

Abstract

Efficient intracellular Ca2+ homeostasis in skeletal muscle requires establishment and maintenance of an intact triad junctional complex. Bin1, an amphiphysin family protein contains a conserved BAR domain that facilitates membrane curvature. Bin1 also contains a unique phosphatidylinositol-4,5-bisphosphate (PIP2) binding domain that is required for transverse-tubule membrane biogenesis during C2C12 myogenic cell differentiation. The lethality associated with Bin1 knockout limits the opportunities to study the function of Bin1 in adult skeletal muscle. Using in vivo electroporation to deliver shRNA against Bin1, we knockdown Bin1 expression in the FDB muscle of adult mice. We find that transient loss of Bin1 alters the properties of Ca2+ sparks induced by osmotic stress. Specifically, we observe that the mean frequency of Ca2+ sparks per minute is reduced in fibers that exhibit a high level of Bin1 knockdown (33.1 ± 5.7), compared to control fibers (138.1 ± 21.7). Kinetic analysis of individual Ca2+ sparks shows that spark amplitude (ΔF/F0) is reduced in the Bin1 knockdown fibers (0.67 ± 0.02) when compared to control (0.85 ± 0.01). Bin1 knockdown also alters the full duration at half maximal (FDHM) of Ca2+ sparks. A two-exponential decay function fit of the FDHM histograms indicates that, in the control fiber, the corresponding time constants for Ca2+ sparks (t01) are 38.0 ± 2.8 ms and 281.3 ± 40.0 ms for Ca2+ bursts (t02). The time constant for Ca2+ bursts (t02) are significantly reduced in the high Bin1 knockdown muscle fiber (140.9 ± 41.2 ms). Electron microscopy reveals Bin1 knockdown fibers exhibit vacuolation and swelling of t-tubule structures. Thus, alteration of triad junction structure can potentially affect the resting cytosolic Ca2+ levels and internal Ca2+ stores in Bin1 knockdown fibers.