Ageing is associated with smaller, slower Ca transients in cardiac ventricular myocytes, which have been attributed to reduced Ca current and ryanodine receptor density and slowed Ca uptake by sarcoplasmic reticulum (1). However, the possible role of changes in the organisation of transverse (t-) tubules, invaginations of the ventricular cell membrane that are crucial to excitation-contraction coupling, is unknown. The role of caveolin-3 (Cav-3), a structural protein that has been implicated in the formation of t-tubules (2), in determining t-tubule structure with age, is also unknown. We therefore investigated t-tubule structure in ventricular myocytes isolated from wild type (WT) and cardiac-specific Cav-3 overexpressing (OE) mice at 21 days (d), 3 and 24 months (mo) of age. Animal procedures were approved by local ethics committee and conducted in accordance with UK legislation. Myocytes stained with di-8-ANEPPS were imaged on a confocal microscope. Image stacks were deconvolved and then, using a novel algorithm that does not rely on threshold (e.g. 3, 4), t-tubules were skeletonised (Fig. 1) and t-tubule density and orientation quantified. T-tubule regularity was determined using a discrete two-dimensional Fast Fourier Transform (FFT). Data are expressed as mean±SEM of n cells and analysed using a 2-way ANOVA or Mann-Whitney test as appropriate, with significance taken as p<0.05. FFT analysis of 21 d, 3 and 24 mo WT myocytes (n=30, 22, 15, respectively) revealed that t-tubule regularity changed with age (0.10 ± 0.01, 0.07 ± 0.01 and 0.13 ± 0.03, respectively, p=0.001) with no change in spatial frequency (~0.56 /μm). Age was also associated with a decrease in t-tubule density (0.77±0.04, 0.68±0.03 and 0.60±0.04 μm/μm2, p=0.0009) and fraction of longitudinal tubules (48±2, 44±1 and 43±3%, p=0.001), and an increase in half-distance to the nearest tubule (0.36±0.01, 0.40±0.02 and 0.43±0.03 μm, p=0.0006). Similar changes were observed in Cav-3 OE myocytes, in which there were no significant differences from WT for any variable in any age group. Preliminary analysis of WT 9 d myocytes showed that t-tubule regularity and density were reduced by 79% (p<0.0001) and 15% (p=0.0002), respectively, compared with 21 d WT myocytes. Thus ageing is associated with changes in t-tubule structure that may contribute to the altered Ca transient, but Cav-3 OE does not prevent these changes.