Popeye domain containing (Popdc) genes encode a novel class of cAMP-binding proteins. Functional analysis of Popdc1 and -2 genes in mice indicate a potential role in the chronotropic response to β-adrenergic signalling, with single null mutants displaying a stress-induced bradycardia.1 The overlapping phenotype in both mutants is age-dependent, suggesting the presence of genetic redundancy. Here we report our preliminary analysis of Popdc1;Popdc2 double knockout (P12dKO) mice and the functional interactions of Popdc1 and -2 at the protein level. Popdc1;Popdc2 double heterozygous (P12dHET), P12dKO, and wild type (WT) age-matched control male mice were subjected to telemetric ECG analysis at rest and during physical stress (6min swim, 15min recovery). In accordance with our single null mutant analysis, no significant difference was observed in the heart rates of 3 month old mice at rest (WT 514±34bpm, n=7; P12dHET 547±36bpm, n=5, two-tailed t-test p=0.14; P12dKO 552±36bpm, n=3, two-tailed t-test p=0.20). However, in contrast to the single null mutants where the stress-induced bradycardia was not observed until 5.5 months of age, the 3 month old P12dKO mice had a blunted response to physical stress (WT 39% increase in heart rate, n=7; P12dHET 32%, n=5, two tailed t-test p=0.35; P12dKO 18%, n=3, two-tailed t-test p=0.01). A blunted increase in heart rate in response to physical stress is consistently seen in P12dKO older mice (>9 months), although not exacerbated compared to P1KO mice (WT 719±14bpm, n=5; P1KO 593±39bpm, n=5; P12dKO 665±24bpm, n=6, WT vs P12dKO two-tailed t-test p<0.01), which suggests that during early postnatal development the two Popdc genes can functionally compensate each other and act in the same functional context since the loss of either gene is causing a pacemaker deficiency and phenotypic severity does not increase if both Popdc genes are missing. Immunohistochemistry revealed that both Popdc1 and -2 proteins are expressed throughout the heart, with enhanced expression in the cardiac conduction system. At the single cell level both proteins are located at the plasma membrane, intercalated disks and in T-tubules. To identify new interaction partners for Popdc proteins, we performed co-immunoprecipitation followed by mass spectrometry. By this screen Ankyrin-G and -B and the Na+/Ca2+ exchanger NCX1 were identified and subsequently confirmed by Western Blot as interacting proteins. Furthermore, we were able to demonstrate an interaction of Popdc1 and 2, suggesting that members of the Popdc family are interacting with each other and thereby acting in the same functional context. This may explain the overlapping phenotypes of Popdc1 and -2 mutants and the earlier manifestation of the bradycardia in P12dKO mice. Further analysis at the protein, cell, and organismic level, will help to define the role of Popdc proteins in cardiac stress adaptation.