Background: Cardiovascular pathologies are a major cause of death in people with diabetes. Impaired excitation-contraction (EC) coupling, involving ryanodine receptor (RyR2), is a hallmark of both type 1/2 diabetes1. We have previously reported an interaction between caveolin-3 (Cav3) and RyR2; however, the functional consequences of this interaction are unknown. Cav3 is a negative regulator of nitric oxide (NO) production via NO synthase (NOS). Significantly, post-translational modification of RyRs, S-nitrosylation, the addition of nitroso group, has been linked to ‘leaky’ channels. We hypothesize that the formation of a Cav3-RyR2 complex has a cardioprotective role regulating NOS activity within cardiomyocytes by maintaining the nitroso-redox state of the receptor a relationship that is perturbed in obesity and diabetes. Methods: Male Sprague Dawley rats were randomly split into two groups and fed high-fat diet (HFD) (45% fat) ad libitum and control group (10%) for 16 weeks and glucose, insulin and weight were recorded over time. Echocardiography was conducted prior to sacrificing the animals under constant 1.5% Isoflurane and 50% oxygen. All procedures were performed in accordance with Scientific Procedures Act 1986. Protein expression levels analyzed using Western blot. Data were expressed as mean ± SEM and analyzed using unpaired t-test. Recombinant SPRY domain of RyR2 and full-length Cav3 were expressed and purified and microscale thermophoresis (MST) used for interaction studies. Results: The pre-diabetic (obese) heart exhibits early signs of cardiac dysfunction, with trends towards increased LV mass, fractional shortening and E/A ratio. We have determined a down-regulation of Cav3, nNOS and eNOS in the pre-diabetic heart but no change to RyR2 levels. Although Cav3 expression levels are also down-regulated in STZ hearts nNOS (p=0.008) and eNOS (p=0.011) are up-regulated. Bioinformatics analyses identified multiple putative caveolin binding motifs (CBMs) within the RyR1 and RyR2 primary sequences. Analysis of a 3-D structure of skeletal RyR13 allowed us to identify one of the CBMs within a SPRY domain. Sequence alignment with RyR2 showed this region is conserved. Our preliminary data indicate a putative interaction between recombinant Cav3 and a recombinant SPRY domain using MST. Conclusion: While both pre-diabetic (obese) and type 1 diabetic models show a down-regulation of Cav3, and no change to RyR2, we determined that the eNOS/nNOS profiles were significantly different. Functional studies are underway to investigate the implications of an imbalance between nNOS/eNOS and Cav3 in terms of RyR2 nitrosylation and calcium release. Biophysical and structural studies are on-going to investigate the putative SPRY-Cav3 interaction, to further understand the implications of Cav3 depletion in the diseased heart.