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Aims: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the A T I receptor. Methods and results: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS and results of the isolated complexes showed that Atrap interacts with the cardiac Ca2+-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca2+ uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca2+]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap--/-- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap--/-- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap--/-- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap--/-- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap--/-- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap--/-- (725 ± 48 μL/s) compared with WT mice (1065 ± 122 μL/s; P = 0.01; n = 15). Conclusion: We identified Atrap as a novel regulatory protein of the cardiac Ca2+-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation. [ABSTRACT FROM AUTHOR]