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.
Highlights
Bin1 is a member of Amphiphysin family of proteins that contains a canonical NH2-terminal BAR (Bin/Amphiphysin/Rvs) protein domain which induces membrane bending, a Src homology 3 (SH3) domain at the carboxy-terminus and a variable region at the center of the protein [1,2]
While these studies indicate a role for Bin1 in establishing the muscle cell ultrastructure and excitation-contraction (EC) coupling, limited progress has been made in the study of the role of Bin1 in Ca2+ homeostasis in adult skeletal muscle due to the lethality associated with systemic ablation of Bin1 in mice
Studies in C2C12 cells and isolated muscle tissue showed that Bin1 is important for membrane fusion and t-tubule development [4,5,7]
Summary
Bin is a member of Amphiphysin family of proteins that contains a canonical NH2-terminal BAR (Bin/Amphiphysin/Rvs) protein domain which induces membrane bending, a Src homology 3 (SH3) domain at the carboxy-terminus and a variable region at the center of the protein [1,2]. Knock-in mice overexpressing SH3 domain of Bin suggests that the SH3 domain is associated with actin, myosin filaments and pro-myogenic kinase CDK 5 proteins that are important for sarcomeric assembly and organization [9]. Studies in cultured cardiomyocytes suggested that Bin is important for proper localization of DHPR (or CaV 1.2), a family of L-type Ca2+ channels, to the t-tubule membrane [10]. While these studies indicate a role for Bin in establishing the muscle cell ultrastructure and excitation-contraction (EC) coupling, limited progress has been made in the study of the role of Bin in Ca2+ homeostasis in adult skeletal muscle due to the lethality associated with systemic ablation of Bin in mice. Bin1(2/2) mice die shortly after birth due to hypertrophic dilated cardiomyopathy without apparent defects in vesicle trafficking, suggesting one role for Bin during cardiac development [11]
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