Transverse tubules (t-tubules) regulate cardiac excitation-contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear. To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA-induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient. AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.