Healthy cardiac muscle cells (cardiomyocytes) contain membrane invaginations, called t-tubules, that extend into their interior forming a complex network. In recent years, the malleable and dynamic nature of t-tubules has been highlighted, as well as their implications for cardiac contractility. These t-tubules form gradually during development (Figure A), critically ensuring synchronization of Ca2+ release and forceful cellular contraction. However, t-tubules can undergo dedifferentiation in diseases such as atrial fibrillation and heart failure. Despite their critical function, little is known about the molecular mechanism controlling t-tubule growth. What are the signals controlling this process?
To answer this question, Harmonie Perdreau-Dahl and colleagues have studied t-tubule assembly in the developing mouse heart and in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using confocal and Airyscan microscopy, she observed that in the postnatal mouse heart, the membrane-bending protein BIN1 (Bridging integrator 1) 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 increase in BIN1 mRNA and protein expression levels. Indeed, Harmonie observed that BIN1 overexpression was also able to induce tubulations in hiPSC-CMs. The resulting dyadic junctions between L-type Ca2+ channels and ryanodine receptors (Figure B), were found to effectively trigger Ca2+ release (Figure C).
Figure A. Confocal micrographs of isolated mouse cardiomyocytes labeled with di-8-ANNEPS. Scale bars, 10 µm (left) and 2 µm (right).
FigureB. Confocal micrographs of hiPSC-CMs transduced with AAV6-BIN1+13+17, and immunolabeled with pan-BIN1 (green) and α-actinin, CAV3 (caveolin-3), L-type voltage Ca2+ channel (LTCC) or RyR2 (ryanodine receptor 2) (red) antibodies. Scale bars, 20 µm and 5 µm (enlarged sections).
Figure C. Paired confocal images of t-tubules (di-8-ANEPPS staining, left) and Ca2+ fluorescence during spontaneous and triggered Ca2+ transients (centre and right, respectively, presented as F/F0), in hiPSC-CMs transduced with AAV6-BIN1+13+17.
Harmonie further observed that two partner proteins of BIN1 have opposing effects on t-tubule growth: high levels of the phosphoinositide 3’-phosphatase myotubularin (MTM1) critically support t-tubule maturation, consistent with a central role of lipid homeostasis, while low levels of dynamin-2 (DNM2) are required, in agreement with this protein’s role in membrane fission (Figures D and E). Thus, Harmonie’s work shows that the balanced expression of 3 proteins coordinates cardiac t-tubule growth.
Figure D. Overview of the relationship between t-tubule density and protein expression levels of BIN1, MTM1, and DNM2 in the mouse heart during postnatal development.
Figure E. Schematic illustration of t-tubule growth during cardiac development. jSR indicates junctional sarcoplasmic reticulum.
Why is this work important? Understanding how cardiomyocyte substructure is assembled has important implications for improving the differentiation of stem cells into cardiomyocytes, and developing new treatments for diseases such as heart failure where cells revert to an immature phenotype. Harmonie’s data indicate that the 3 proteins BIN1, MTM1, and DNM2 work in tandem to assemble the Ca2+ signalling apparatus, and could be concertedly targeted in cardiac bioengineering and disease.
