Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

Abstract

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

Phosphatidylinositol 3-kinase inhibition restores Ca 2+ release defects and prolongs survival in myotubularin-deficient mice

“…S6). This interpretation also fits with the proposed role of MTM1 and associated protein amphiphysin-2 in membrane tubulation (15) and with the fact that disrupted continuity of Ca 2+ transients was observed in muscle fibers downexpressing amphiphysin 2 (16).…”

Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation

“…Bin1-KO mice with Dnm2 haploinsufficiency have normal muscle ultrastructure and T-tubules. As BAR proteins can remodel membrane in vitro and as Bin1 downregulation with shRNA in adult muscle led to swollen T-tubules (3,26,27), the sarcomere and triad structures were investigated. In skeletal muscles from E18.5 WT mice, DNM2 and α-actinin were detected predominantly in the transverse orientation, consistent with Z-line orientation (Supplemental Figure 5B).…”

Ca2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives

“…Milder forms of CNM have been associated with dominant and recessive mutations in BIN1 and DNM2, encoding the functionally related proteins dynamin 2 and amphiphysin 2, respectively [132][133][134][135]. Dominant mutations in DNM2 are associated with defective tubular membrane structures [134,135] most likely because they alter the function of amphiphysin 2 [134].…”

“…Amphiphysin 2 is ubiquitously expressed but the skeletal muscle isoform contains a PI binding domain and is localized to the T-tubules where it plays an important role in T-tubule biogenesis [136]. Acute down regulation of Bin1 by siRNA in mouse flexor digitorum brevis muscle fibers recapitulates T-tubule disruption as seen in the muscles of affected patients [132]. Furthermore, murine muscles knocked down for Bin1 show profound alterations in Ca 2+ homeostasis, including depletion of intracellular Ca 2+ stores, reduced membrane current in response to depolarizing pulses leading to a reduced Ca 2+ transient and fewer sparks.…”