Early Stage Morphogenesis of Transverse Tubules in Rat Cardiomyocytes: The Role of pBIN1

Transverse tubules (TTs) are tubular invaginations of myocyte membrane forming junctions with sarcoplasmic reticulum and are essential for excitation-contraction coupling. Although it is known that TTs begin to develop 2 weeks after birth in rodent cardiomyocytes, the spatial profile and molecular mechanisms of TT morphogenesis are not clear. Understanding the molecular mechanism of TT morphogenesis may provide potential solutions for TT loss in pathogenic conditions such as hypertrophy and heart failure.

To understand early stage morphogenesis of cardiac TTs, we utilized a scanning electron microscope equipped with a focused ion beam to reconstruct a 3-dimensional spatial profile of developing TT network in cardiomyocytes from 2-week-old rats. We created tamoxifen-inducible cardiac-specific knockout rats to explore the role of exons 11 and 13 of the BIN1 (bridging integrator 1) gene.

We found that TTs began to develop as intracellular membrane hubs around Z-discs, from which pseudopod-like tubules budded in a relatively random way toward different directions without necessarily connecting to the cell surface. A tubule network forms when membrane branches from adjacent hubs are interconnected. Cardiac-specific knockout of BIN1 exon 13 (13KO) suppressed TT microfolds. In contrast, cardiac-specific knockout of BIN1 exon 11 (11KO), which encodes the PIBM (phosphoinositide-binding motif), suppressed the formation of budding tubules, resulting in a sparse tubule network with swollen membrane hubs. Due to the underdeveloped TT network, TT-sarcoplasmic reticulum couplon density/size and excitation-contraction coupling gain in 11KO cardiomyocytes were decreased, similar to those occurring in failing heart cells.

TTs start to develop as budding tubules branching from membrane hubs around Z-discs. This process depends at least partially on the tubulation function of pBIN1 (BIN1 isoforms with PIBM [Bin1+11 and Bin1+11+17]), which is constitutively expressed in rat and human cardiomyocytes. Defective TT morphogenesis due to altered BIN1 splicing in cardiomyocytes may have potential implications in heart diseases.