We have observed a persistent increase in the expression and activity of the immunoproteasome (iProt) in post‐ischemic heart (post‐MI). The objective of this study was to identify the pathway and mediator(s) responsible for iProt upregulation. Using RT‐PCR, ifng, tnfa, and ifnb1 mRNAs were assayed. There were no changes in either ifng or tnfa but ifnb1 was elevated ~ 17‐fold (P<0.01) in the heart 4 days post‐MI. Exposure of a mixture of rat neonatal cardiomyocytes/fibroblasts (CM/CFs) to IFN‐β from 1U/ml up to100u/ml for 24h resulted in a variable response with an 85% (P<0.05) increase in iProt at 100 u/ml as measured using the ProCISE assay. Since the CM/CF preparation typically contains about 20% cardiac fibroblasts, (confirmed by vimentin staining), isolated CFs were exposed to IFN‐β for 24hs which resulted in a robust concentration‐dependent increase in iProt that was almost 3‐fold (P<0.01) at 100 u/ml. An enriched CM (eCM) preparation in which CFs were limited to ≤5% was prepared and these had the lowest iProt response to 10u/ml IFN‐β (eCM, +27% ± 5%; CF, +104 ± 15%; CM/CF, +52 ± 18%). To determine a potential source of IFN‐β in the post‐MI heart, CFs, CM/CFs and eCMs were exposed to 1% O2 for 22h to simulate ischemia. This resulted in +2 ±1%, +31 ± 8% (P<0.05), −16 ± 5% changes in iProt, respectively, suggesting that the CMs were the source of the IFN‐β. RT‐PCR analysis revealed that ifnb1 transcription in the control and in hypoxic eCMs was increased 2.75‐fold (P<0.01) and 3.3‐fold (P<0.01) respectively when compared to hypoxic CM/CFs. To probe a potential function of IFN‐β, CM/CFs were pretreated with 100 u/ml IFN‐β 24h prior to exposure to hypoxia (1% O2) for 22h. Cardiomyocyte injury was determined using protein array to measure release of cardiac injury markers, cTNI, cTNT and FABP3, into the media. All of the markers were significantly increased at the end of the hypoxic challenge in cells that were not pre‐treated with IFN‐β as compared to controls. In the absence of hypoxia, IFN‐β had no effect on the levels of these markers. However, when IFN‐β pre‐treated CM/CFs were exposed to hypoxia there was a significant reduction (P<0.05) in all three markers as compared to hypoxic CM/CFs alone. These results suggest that in response to hypoxia CMs release IFN‐β which then stimulates CF to upregulate an iProt that serves to protect CMs. Overall, these studies suggest the existence of a previously unrecognized signaling pathway in the heart involving CM IFN‐β signaling to the cardiac fibroblast.Support or Funding InformationThis study was supported in part by an AHA ‐ Founders Affiliate Grant in Aid and a grant from Onyx Pharmaceuticals, Inc., an Amgen subsidiary.