Stimulation of t-tubular ICa in cardiac ventricular myocytes by both basal and β2-adrenoceptor-stimulated protein kinase A (PKA) activity is dependent on caveolin-3 (Cav-3) binding (Bryant et al., 2014). It is unknown whether Cav-3-dependent stimulation of ICa is altered in heart failure (HF). We have therefore investigated the regulation of ICa by Cav-3 and PKA-dependent mechanisms in HF.Animal procedures were approved by local ethics committee and conducted in accordance with UK legislation. Coronary artery ligation (CAL) was performed in male Wistar rats (200-250g) under anaesthesia (ketamine 75 mg/kg, medetomidine 0.5 mg/kg, i.p.) with appropriate analgesia (buprenorphine 0.05 mg/kg s.c.). 17 weeks post-surgery, hearts were excised from ligated (CAL) or sham-operated (Sham) animals under pentobarbitone (140 mg/kg i.p.) anaesthesia and left ventricular myocytes isolated. ICa was recorded using whole-cell patch-clamp in intact cells and following acute detubulation (22-25°C). PKA was inhibited using 20 μM H-89; β2-adrenoceptors were stimulated using the β2-agonist zinterol (3 μM) in combination with 10 μM atenolol. Cav-3 binding was disrupted using the peptide C3SD as described previously (Bryant et al., 2014). Data are expressed as mean±SEM (n cells). Statistical analyses were performed using Student’s t-test or ANOVA with the appropriate Bonferroni post hoc test; ** p<0.01, *** p<0.001.Under basal conditions ICa density was not different between Sham and CAL myocytes (Fig 1A); however, after inhibition of PKA ICa density was smaller in CAL than in Sham myocytes (Fig 1B), suggesting that PKA stimulation helps maintain ICa density in CAL myocytes.Measurement of ICa density in intact and detubulated myocytes showed that the difference in ICa density between t-tubular (TT) and surface sarcolemmal (SS) membrane observed in Sham cells was reduced in CAL myocytes (Fig 1A). H-89 abolished this difference in ICa density in CAL but not Sham myocytes (Fig 1B). C3SD decreased basal ICa in Sham (con -6.5±0.4 (16) vs C3SD -4.9±0.3 (16) pA/pF, p<0.001) but not in CAL myocytes (con -5.6±0.4 (14) vs C3SD -5.8±0.5 (14) pA/pF, ns) suggesting loss of Cav-3-dependent stimulation of ICa in CAL.Zinterol increased ICa in intact but not detubulated Sham myocytes (by 46±8% (7), p<0.01 and 2±5% (7), ns, respectively), but increased ICa in both intact and detubulated CAL myocytes (by 39±12% (5), p<0.01 and 37±2% (4), p<0.01). These data suggest loss of Cav-3 dependent localization of PKA activity at the t-tubules in HF, accompanying redistribution of β2-adrenoceptors to the SS (Nikolaev et al., 2010).