Caveolin-3 (Cav-3) plays an important role in localizing L-type calcium current (ICa) to the t-tubules of cardiac ventricular myocytes, via localised protein kinase A (PKA)-dependent phosphorylation (1). Ageing is associated with a decline in cardiac function, and a decrease in ICa that appears to occur predominantly at the t-tubules (2). We have, therefore, investigated the role of Cav-3 and PKA in this decrease, using ventricular myocytes isolated from 3 month (3 mo) and 24 month (24 mo) old wild type (WT) and cardiac-specific Cav-3 overexpressing (Cav-3 OE) mice. Animal procedures were approved by local ethics committee and conducted in accordance with UK legislation. Ventricular myocytes were isolated by enzymatic digestion of hearts from male Cav-3 OE or WT littermate mice. ICa was recorded using the whole-cell patch-clamp technique in intact cells and following acute detubulation (DT, using formamide-induced osmotic shock (3)), at 22-25°C. ICa was elicited by a 500 ms step depolarisation from -40 to 0 mV following a 200 ms pre-pulse from -80 to -40 mV, at 0.2Hz and measured as the difference between peak current and that at the end of the pulse. ICa recorded from DT myocytes represents ICa at the surface membrane and the difference in ICa between intact and DT myocytes gives ICa at the t-tubule membrane. Data are expressed as mean±SEM (n cells). Student’s t-test or ANOVA were used for statistical analysis, with the Bonferroni post hoc test; the limit of statistical confidence was p<0.05. In WT mice, ICa density was ~ 28% (p<0.001) smaller in myocytes isolated from 24 mo old compared to 3 mo old mice. In contrast, ICa density was not significantly different in myocytes isolated from 24 mo and 3 mo old Cav-3 OE mice (3 mo, -4.8±0.3 (n=25); 24 mo, -5.1±0.3 (n=28), pA/pF), nor was ICa density significantly different in DT myocytes from 24 mo and 3 mo old Cav-3 OE mice; thus, calculated ICa density in the t-tubules was not significantly different at the two ages in Cav-3 OE mice (3 mo, -9.1±0.4; 24 mo, -9.3±0.3, pA/pF). In the presence of the PKA inhibitor H-89, ICa density was smaller in myocytes from 24 mo compared to 3 mo WT mice (3 mo, -2.5±0.2 (n=12); 24 mo, -1.6±0.1 (n=11), pA/pF, p<0.01) but decreased to the same level following DT. However, in intact cells from Cav-3 OE mice in the presence of H-89, ICa density was similar (3 mo, -1.7±0.2 (n=10); 24 mo, -1.6±0.1 (n=17), pA/pF, ns) and decreased to the same level following DT. These data suggest that Cav-3 OE may be protective against the decrease in t-tubular ICa density observed in aged WT mice. The effect of age, and the protective effect of Cav-3 OE, persist in the presence of H-89 and may therefore reflect, in part, t-tubular calcium channel density.