The transverse (t-) tubules of cardiac ventricular myocytes show marked changes in structure during heart failure (HF), which lead to decreased synchrony of Ca release and Ca transient amplitude. We have previously shown that stimulation of L-type Ca current (ICa) by basal protein kinase A (PKA) activity occurs predominantly in the t-tubules and is facilitated by caveolin-3 (Cav-3). However, the effect of HF on such regulation is unknown. We have, therefore, investigated Cav-3 and PKA mediated regulation of ICa in a rat model of HF. Myocardial infarction was induced in male Wistar rats (~250g) by ligation of the left anterior descending coronary artery (LAD) under surgical anaesthesia (Ketamine 75 mg/kg, Medetomidine 0.5 mg/kg, i.p.) with appropriate analgesia (Buprenorphine 0.05 mg/kg, s.c.). Left ventricular myocytes were isolated from ligated (HF) or sham animals 16 weeks after surgery, following removal of the heart under Pentobarbitone (140 mg/kg i.p.) anaesthesia. All animal procedures were approved by the University of Bristol ethics committee and conducted in accordance with UK legislation. ICa was recorded using whole-cell patch-clamp at room temperature. Incubation with TAT-tagged C3SD peptide was used to disrupt normal Cav-3 protein binding (MacDougall et al., 2012); non-incubated cells were used as controls (Con). Cells stained with di-8-ANEPPS were imaged using confocal microscopy. T-tubule regularity was quantified using FFT analysis by plotting the amplitude of the first harmonic as a function of total intensity (TT power). Data are expressed as mean±SEM (n). Statistical significance at p<0.05 was determined using Student’s t-test or analysis of variance with the appropriate Bonferroni post hoc test. Animals that underwent LAD ligation had significantly increased heart:body weight (Sham 3.3±0.2 (9) vs HF 4.6±0.2 (7) g/kg, p<0.001) and lung:body weight (Sham 3.2±0.1 (9) vs HF 3.7±0.1 (7) g/kg, p<0.05). HF myocytes had increased cell capacitance (Sham 285±12 (31) vs HF 348±18 (21) pF, p<0.01 and reduced TT power (Sham 70.6±9.8 (48) vs HF 35.4±4.7 (58) a.u., p<0.002). Under basal conditions ICa density was not different between Sham and HF (Sham -6.1±0.2 (31), HF -5.8±0.3 (21) pA/pF, ns). However, after inhibition of basal PKA activity using H-89 (20 µM) ICa density was lower in HF (Sham -3.0±0.1 (18), HF -2.3±0.1 (14) pA/pF, p<0.05). ICa was reduced by C3SD in sham cells (Sham -6.5±0.4 (16), Sham+C3SD -4.9±0.3 (16) pA/pF, p<0.01) but was not affected by C3SD in HF cells (HF -5.6±0.4 (14), HF+C3SD -5.8±0.5 (15) pA/pF, ns). These data suggest that in HF, under basal conditions, there is increased stimulation of ICa by basal PKA activity but that the normal coupling of PKA activity to ICa by Cav-3 is disrupted.