ESCRT-Mediated Machinery Directly Drives Cardiac T-tubule Formation.

Abstract

Transverse tubules (T-tubules) are invaginations of the plasma membrane crucial for excitation-contraction coupling. Disruptions in T-tubule organization are frequently observed in heart diseases and are associated with impaired contractile function and malignant arrhythmias. In mammalian cells, the ESCRT (endosomal sorting complex required for transport) proteins mediate a fundamental mechanism for membrane deformation. This study aimed to elucidate the roles of key ESCRT proteins, including Chmp4b (charged multivesicular body protein 4b) and Tsg101 (tumor susceptibility gene 101), in the formation and maintenance of T-tubules. Myocardial-specific gene deletion was achieved using <i>Chmp4b</i>^<i>F/F</i>, <i>Tsg101</i>^<i>F/F</i>, and <i>Rosa26</i>^{<i>Cas9-GFP</i>} mouse strains in conjunction with adeno-associated virus 9-mediated gene editing. The polymerization state of Chmp4b was assessed through the introduction of point mutations combined with glycerol-gradient fractionation. Direct interaction between Chmp4b and membrane phospholipids was examined using genetically encoded biosensors, lipid strip binding, and liposome tubulation assays. An inducible <i>Chmp4b</i> knockout model was utilized to determine its role in T-tubule maintenance during adulthood. Chmp4b expression levels were analyzed in a heart failure mouse model and in human patients with dilated cardiomyopathy. Chmp4b gradually localizes to the dyad during postnatal development, with its deletion causing a complete loss of T-tubules and defects in cardiac structure and contractile function. Chmp4b polymerizes and binds to PtdIns(4,5)P2 (Phosphatidylinositol 4,5-bisphosphate) as well as other negatively charged membrane lipids, driving plasma membrane invagination in a process that depends on the ESCRT-I component Tsg101. In mature cardiomyocytes, Chmp4b remains anchored to the T-tubule membranes to maintain their structure, while Tsg101 detaches and becomes dispensable for T-tubule organization. Chmp4b expression was significantly reduced in heart samples from dilated cardiomyopathy patients and in a mouse model of heart failure. These results uncover an ESCRT-mediated membrane deformation machinery that is essential for shaping cardiomyocyte structure in physiological and disease conditions.