The ER/SR Ca2+ sensor, stromal interaction molecule 1(STIM1) is a major regulator of store-operated Ca2+entry(SOCE), in non-excitable cells. In the adult rodent heart STIM1 has been shown to be essential in the progression of pathological hypertrophy(2); however, the physiological role of STIM1 in the heart is not well understood. We have shown that mice lacking cardiomyocyte STIM1 (crSTIM1-KO) develop ER stress, mitochondrial abnormalities, and dilated cardiomyopathy(1). However, the specific signaling pathways regulated by STIM1 in the heart remain unknown. Therefore, we used a discovery based kinomics approach to identify kinases that were differentially regulated by STIM1. 12-week male control and crSTIM1-KO mice were injected with saline or phenylephrine(PE, 15mg/Kg; s.c), that activates SOCE in cardiomyocytes. 15min following treatment mice were sacrificed and hearts processed for kinomic analysis. 2μg of whole cell lysates were prepared and loaded on to a Ser/Thr kinome chip. Primary analysis was performed in BioNavigator 6.0 (PamGene) and subsequent analysis using the Kinexus PhosphoNET database and GeneGo MetaCore. Downstream confirmation was performed using standard immunoblotting techniques. In parallel experiments standard electrocardiogram analysis was performed +/- PE under 2% isoflurane anesthesia. Pathway analysis of the kinomic array revealed significantly lower Protein Kinase C (PKC) and PKG signaling in the hearts of the KO in comparison to control hearts at baseline. Immunoblotting confirmed that activation of Ca2+-dependent PKCα at pThr497(1.00 ± 0.03 vs 0.75 ± 0.06, p<0.05) and one if its downstream targets MARCKS at pSer158/162 were lower in crSTIM1-KO hearts. Similar reductions in KO hearts were found for several additional kinases such as MEK1/2 at pSer217/221, AMPK at pThr172(1.00 ± 0.04 vs 0.57 ± 0.09, p<0.05) and PDK1 at pSer241(1.00 ± 0.06 vs 0.75 ± 0.03, p<0.05). PKC and PKG regulate cardiac contractility via the regulation of voltage-dependent ion channels. Additional analysis of the kinomic data identified several genes including those encoding the L-type Ca2+ channel, Cav1.2, the delayed rectifier potassium channels K+ channels, Kv1.2 and Kv1.6, all as potential target downstream peptide sequences of the identified kinases. In support of the potential impact of these changes on cardiac contractility, electrocardiogram analysis revealed that KO have significantly lower HR(461.7 ± 0.73 vs 428.7 ± 1.98 bpm, p<0.05) and prolonged QT interval(23.2 ±0.00 vs 47.6 ± 0.00 ms, p<0.05) in comparison to control mice. In conclusion we have shown for the first time that activation of Ca2+-dependent kinases, such as PKC and PKG, are regulated by STIM1 in the adult mouse heart. This has important implications in understanding how STIM1 contributes to the regulation of normal cardiac physiology.