BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes

Abstract

Background: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca 2+ homeostasis. The membrane bending and scaffolding protein BIN1 (amphiphysin-2) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3′-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. Methods: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca 2+ release was recorded using Fluo-4. Results: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca 2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca 2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. Conclusions: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Dynamics of membrane tubulation coupled with fission by a two-component module revealed on polymer cushioned bilayer islands

“…High Dyn2 concentrations overcome this inhibition and renders the BIN1 tubule susceptible to fission. BIN1 and Dyn2 levels are mutually regulated during muscle development 16 . Perhaps, at early stages during development where Dyn2 levels are relatively high, fission of emergent BIN tubules acts as a quality control mechanism.…”

“…Mutations in BIN1 and Dyn2 are linked to autosomal dominant and recessive forms of centronuclear myopathies (CNMs), which are a group of congenital hereditary disorders characterized by disorganized T-tubules leading to uncoordinated force generation and muscle hypotrophy 15 . The relative levels of BIN1 and Dyn2 are tightly regulated during development, with an increase in BIN1 and a decrease in Dyn2 levels correlating with increased T-tubule densities in muscle cells 16 . Increased expression of Dyn2 inhibits T-tubule growth during development and overexpression of Dyn2 in myoblasts leads to the fragmentation of BIN1 tubules 17,18 .…”

Shaping transverse-tubules: central mechanisms that play a role in the cytosol zoning for muscle contraction

“…Mice with skeletal muscle-specific deletion of BIN1 display a CNM phenotype with T-tubule abnormalities ( 16 ) . Furthermore, the expression level of BIN1 reaches its peak when the organized T-tubule is formed in mouse hearts ( 17 ) . Collectively, BIN1 is an evolutionarily conserved central player for T-tubule organization.…”

“…Mice lacking exon11 do not exhibit any muscle defects, whereas humans and dogs with mutations in the splice acceptor site of exon11 present with a CNM phenotype associated with T-tubule abnormalities ( 20 , 21 ) . In-cell tubulation assays also provided inconsistent results ( 17 , 22 ) , suggesting that the contribution of the PI-binding motif of exon11 for T-tubule organization would vary among species, tissues, and cell lines.…”

“…This result suggests that the interactors with the SH3 domain play a critical role in T-tubule organization. Overexpressed BIN1-induced tubulation in cells depends on the expression of other genes associated with CNM and with other muscle diseases, including Cav3, MTM1, and DNM2 ( 17 , 22 , 23 ) . Therefore, these players should function coordinately with BIN1 in T-tubule organization.…”