Abstract Body: Tissue remodeling that occurs in myocardial infarction (MI)-induced heart failure includes ultra-structural changes in the transverse (T)-tubule (TT) network and in EC-coupling ion channels, ryanodine receptor-2 (RYR) and L-type calcium channels (LTCC). We hypothesized that low myocardial triiodothyronine (T3) content in failing cardiomyocytes would result in reduced Junctophilin-2 (JPH), a bridging protein that approximates the RYR and LTCC channels, thus leading to impaired calcium-induced calcium release. Single molecule localization microscopy (STORM) was used to image RYR clusters and co-localization of LTCC and JPH. MI was produced in adult female Sprague-Dawley rats by permanent ligation of the LAD coronary artery, while Sham animals underwent surgery without ligation. HF was confirmed at 4 mo. post-surgery by echocardiography. Left ventricular myocytes were isolated enzymatically and plated into 8-well chamber slides. Cardiomyocytes (CM) were fixed, permeabilized and stained with fluorescent-tagged primary antibodies: RyR-Dylight550 and JPH-CF647, while secondary anti-Ca_V1.2 IgG -Dylight488 was used to identify LTCC. STORM images were captured on ONI (Oxford NanoImager Inc, UK) microscope, and DBSCAN algorithm (analysis platform developed by LumeVR, UK) was used to determine RYR cluster size and number, nearest neighbor distances between clusters, and co-localizations of JPH and LTCC within clusters. Spontaneous Ca²⁺ sparks were imaged in live cardiomyocytes loaded with a Ca²⁺-indicator dye, and captured by line-scanning laser confocal microscopy. The T-tubule structures were stained with membrane dye (di-8-ANEPPS), and confocal images were analyzed for TT integrity. MI-induced failing hearts (EF<35%) had significantly low T3 content as measured by HPLC-MS. Increased number, amplitude and duration of Ca²⁺ sparks in CM implied significant ion leakage from the SR. Ultrastructure imaging indicated T-tubule disruption. STORM images showed significantly reduced RYR cluster numbers and size with altered co-localization of LTCC and JPH, leading to reduced CM contractility.