Nanobar Array Assay Revealed Complementary Roles of BIN1 Splice Isoforms in Cardiac T-Tubule Morphogenesis

Abstract

Bridging integrator-1 (BIN1) is a family of banana-shaped molecules implicated in cell membrane tubulation. To understand the curvature sensitivity and functional roles of BIN1 splicing isoforms, we engineered vertical nanobars on a cell culture substrate to create high and low curvatures. When expressed individually, BIN1 isoforms with phosphoinositide-binding motifs (pBIN1) appeared preferentially at high-curvature nanobar ends, agreeing well with their membrane tubulation in cardiomyocytes. In contrast, the ubiquitous BIN1 isoform without phosphoinositide-binding motif (uBIN1) exhibited no affinity to membranes around nanobars but accumulated along Z-lines in cardiomyocytes. Importantly, in pBIN1-uBIN1 coexpression, pBIN1 recruited uBIN1 to high-curvature membranes at nanobar ends, and uBIN1 attached the otherwise messy pBIN1 tubules to Z-lines. The complementary cooperation of BIN1 isoforms (comboBIN1) represents a novel mechanism of T-tubule formation along Z-lines in cardiomyocytes. Dysregulation of BIN1 splicing, e.g., during myocardial infarction, underlied T-tubule disorganization, and correction of uBIN1/pBIN1 stoichiometry rescued T-tubule morphology in heart disease.

The role of junctophilin proteins in cellular function

“…Similar to JPH2, neonatal cardiomyocytes exhibit a very low expression of BIN1 isoforms ( 147 ). Immunoblotting based on an antibody against the phosphoinositide-binding motif has recently demonstrated the location of pBIN1 both in cardiac and skeletal muscle ( 151 ). While pBIN1 and uBIN1 are differentially localized in neonatal cardiomyocytes, overexpression of both fluorophore-tagged isoforms in cultured neonatal cardiomyocytes has resulted in induction of tubulation and the cooperative binding of both proteins to membrane tubules at Z-disks ( 151 ).…”

“…Immunoblotting based on an antibody against the phosphoinositide-binding motif has recently demonstrated the location of pBIN1 both in cardiac and skeletal muscle ( 151 ). While pBIN1 and uBIN1 are differentially localized in neonatal cardiomyocytes, overexpression of both fluorophore-tagged isoforms in cultured neonatal cardiomyocytes has resulted in induction of tubulation and the cooperative binding of both proteins to membrane tubules at Z-disks ( 151 ). Distinct BIN1 isoforms may contribute to this T-tubule membrane folding and together with JPH2 support the postnatal tubulation spring and tubular Cav1.2 enrichment linked through JPH2 binding to RyR2 Ca 2+ release sites.…”

The Physiology and Pathophysiology of T-Tubules in the Heart

“…However, understanding of these roles has been complicated by the fact that BIN1 is expressed in several tissue- and species-specific isoforms. Early work suggested that the presence of exon 11 is required to induce tubulogenesis ( Lee et al, 2002 ; Kojima et al, 2004 ), and exon 11-containing BIN1 isoforms expressed in rat, sheep, and human myocardium have indeed been observed to induce t-tubule formation ( Caldwell et al, 2014 ; De La Mata et al, 2019 ; Lawless et al, 2019 ; Li L. L. et al, 2020 ). However, other studies have reported that exon 11 is dispensable for t-tubule development ( Prokic et al, 2020 ), and shown that the mouse heart expresses four BIN1 splice variants which do not contain this motif (BIN1, BIN1+13, BIN1+17, and BIN1+13+17) ( Forbes and Sperelakis, 1976 ; Hong et al, 2014 ).…”

Phosphatidylinositol-4,5-Bisphosphate Binding to Amphiphysin-II Modulates T-Tubule Remodeling: Implications for Heart Failure

“…Thus, the current study suggests a novel mechanism that allows cBIN1 and PIP2 to play a crucial role in TT biogenesis. It is also important to note that recent work has also identified the presence of exon 11-containing isoform 8 in rat ( Li et al, 2020 ) but not apparently in mouse ( Hong et al, 2014 ) myocardium. Furthermore, Guo et al (2021) have shown that at least two different exon 11-containing isoforms 8 and 13 (i.e., Bin1+11 and Bin1+11+17, respectively) are also present in human heart.…”