Heart failure is associated with increased risk of arrhythmia (1) and, at the cellular level, transverse-tubule (TT) network disruption (2), increased Na-Ca exchange (NCX) activity (1) and reduced caveolin-3 (Cav-3) expression (3). Cav-3 is a scaffolding protein which is implicated in TT formation (4) and in regulation of ion channels involved in excitation-contraction coupling, including the L-type Ca channel. However, little is known about whether and how altered Cav-3 expression affects TT structure, ryanodine receptor (RyR) and NCX activities. Ventricular myocytes were isolated from 3 month old Cav-3 knockout (KO) mice and wildtype (WT) C57Bl/6 control mice. Animal procedures were approved by the local ethics committee and conducted in accordance with UK legislation. Spontaneous contractile activity (SCA) was assessed at different [Na]o in intact and detubulated (DT) cells, to distinguish between TT and surface sarcolemmal NCX contribution (5). RyR activity was assessed by measuring spontaneous Ca spark frequency using confocal microscopy in cells loaded with the fluorescent Ca-indicator Fluo-4/AM. TT and RyR organisation were assessed using Alexa Fluor 488-conjugated wheat germ agglutinin (WGA), and RyRs with mouse α-RyR and Alexa Fluor 594 α-mouse IgGs and imaging using a confocal microscope. TT regularity (TTpower) was quantified using Fourier analysis and WGA/RyR colocalisation using Mander’s coefficients. Data are reported as mean±SEM of n cells. Statistical tests were performed as indicated and significance taken as p≤0.05. SCA was 23±3% greater in intact Cav-3 KO cells compared to WT controls (p<0.01, n=80, unpaired t-test), but did not change upon DT (ns for WT, n=80, and KO, n=80, 3-way Chi-Squared); thus SCA is unaltered by loss of TT NCX activity, but KO appears to have greater propensity for spontaneous activity. To determine whether a change in spontaneous RyR activity may be responsible, Ca sparks were measured. Total Ca spark frequency was reduced in KO (from 0.10±0.01 100 μm–2.s-1 in WT to 0.03±0.01 100 μm-2.s-1 in KO; p=0.0002, unpaired t-test), but activity was increased in the cell centre (p=0.02, Chi-Squared). To investigate whether changes in cell structure might also contribute to the changes seen in SCA, colocalisation analysis of WGA and RyR labeling was used, indicating that Cav-3 KO was associated with a ~30% decrease in RyR colocalisation with WGA (p=0.01, n=10, unpaired t-test) and vice versa (p=0.005, unpaired t-test). Fourier analysis of WGA staining revealed no changes to TT regularity in KO cells. These data suggest that while Cav-3 KO may not be associated with gross changes in TT morphology, it may be involved in regulating RyR and NCX, such that KO promotes SCA.