The transverse (t)-tubule system plays an essential role in healthy and diseased heart muscle, particularly in Ca2+-induced Ca2+ release (CICR), and its structural disruption is an early event in heart failure. Both mechanical overload and unloading alter t-tubule structure, but the mechanisms mediating the normally tight regulation of the t-tubules in response to load variation are poorly understood. Telethonin (Tcap) is a stretch-sensitive Z-disc protein that binds to proteins in the t-tubule membrane. To assess its role in regulating t-tubule structure and function, we used Tcap knockout (KO) mice and investigated cardiomyocyte t-tubule and cell structure and CICR over time and following mechanical overload. In cardiomyocytes from 3-month-old KO (3mKO), there were isolated t-tubule defects and Ca2+ transient dysynchrony without whole heart and cellular dysfunction. Ca2+ spark frequency more than doubled in 3mKO. At 8 months of age (8mKO), cardiomyocytes showed progressive loss of t-tubules and remodelling of the cell surface, with prolonged and dysynchronous Ca2+ transients. Ca2+ spark frequency was elevated and the L-type Ca2+ channel was depressed at 8 months only. After mechanical overload obtained by aortic banding constriction, the Ca2+ transient was prolonged in both wild type and KO. Mechanical overload increased the Ca2+ spark frequency in KO alone, where there was also significantly more t-tubule loss, with a greater deterioration in t-tubule regularity. In conjunction, Tcap KO showed severe loss of cell surface ultrastructure. These data suggest that Tcap is a critical, loadsensitive regulator of t-tubule structure and function. © The Author 2012. Published by Oxford University Press. All rights reserved.