Thyroid hormone maintains cardiac dyadic structures by regulating junctophilin-2 localization to ryanodine clusters

Abstract

Overt and subclinical hypothyroidism are associated with increased risk of cardiovascular disease and mortality. In pre-clinical studies, supplementation with triiodo-L-thyronine (T3) has been shown to regulate cardiomyocyte T-tubule (TT) organization, and thereby maintain ryanodine receptor-2 and L-type Ca 2+ channel (RyR-LTCC) clusters in dyadic structures necessary for efficient excitation-contraction (EC) coupling. We hypothesized that the TT-sarcoplasmic reticulum (SR)-spanning protein Junctophilin-2 (Jph) is regulated by T3. Thyroid hormone (TH) deficiency was induced in 12 adult female Sprague-Dawley rats by oral propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU treatment or with T3 (10 μg/kg/day) supplementation for 2 weeks (PTU+T3). Untreated rats served as euthyroid (EU) controls. Thyroid status was confirmed by plasma T3 and T4 levels. Left ventricular cardiomyocytes (CM) were isolated by collagenase digestion for single molecule imaging of Jph2, RyR2 and LTCC proteins using 2D and 3D STORM (stochastic optical reconstruction microscopy; Oxford Nanoimager). DBSCAN algorithms for cluster analysis (Web-based programs developed by LumeVR) measured cluster size and number, nearest neighbor cluster distances, and protein co-localizations. Calcium sparks in live CMs loaded with Fluo-4 were captured by line-scanning laser microscopy (Zeiss 980) to assess spontaneous SR Ca 2+ leak. STORM imaging of normal CMs showed that ~60% of RyR clusters consisted of 10-50 RyR localizations (locs) with 100-200 Jph molecules co-localized within 210 nm radius of the cluster centroid. Although Jph locs per cluster increased proportionately with increased cluster size, PTU cells had markedly less Jph co-localized with RyR at all cluster sizes compared to EU and PTU+T3 cells. The reduction of associated Jph was most evident in super-clusters (>250 RyR locs). 3D image analysis showed nearest neighbor distances between RyR clusters that were significantly increased in the PTU CMs vs EU (749±146 nm vs 640±140 nm), which were normalized with T3-treatment (616±60 nm). Numbers of RyR clusters without associated Jph locs were significantly higher in PTU cells, and reduced with T3. RyR Ca 2+ leak as measured by spontaneous Ca 2+ sparks was increased in PTU CMs, but decreased significantly to control values in T3-treated cells. This study supports a regulatory role of T3 in maintaining TT-SR dyad structures, in part by increasing Jph co-localization with RyR clusters, thus promoting efficient interaction of RyR with LTCC channels for effective EC-coupling and cardiac output. Study funded by NIH R15HL15406 and by resources of the NYIT Imaging Center. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.