Regulation of Junctophilin‐2 Expression by Triiodothyronine in Adult Rat Cardiomyocytes

Abstract

Introduction Cardiac junctophilin‐2 (Jph2) plays a central role in myocardial contractility by anchoring transverse tubules (TT) to the junctional sarcoplasmic reticulum (jSR), essential for excitation‐contraction (EC) coupling and calcium signaling. Decreases in the expression levels of Jph2 have been observed in a variety of pathological conditions including hypertrophic cardiomyopathy, arrhythmias, and progression of disease in failing hearts. Studies have shown that T3 regulates SR Ca2+ ATPase (SERCa2), phospholamban (PLN), L‐type Ca2+ channels and ryanodine receptors (RyR2), that are responsible for regulating intracellular Ca2+ and maintaining normal systolic and diastolic function. We previously reported that T3 improves cardiac contractile function after induction of myocardial infarction (MI) by maintaining TT network integrity and by regulating expression of myofibrillar and Ca2+ handling proteins. We hypothesize that T3 regulates Jph2 expression and its localization within the jSR‐TT complex to maintain functional TT structures and thus normal Ca2+ transients and contractility. Methods Thyroid deficiency was induced in female Sprague Dawley rats (~220g) by ingestion of 0.025% PTU (propylthiouracil) in drinking water. Control (EU) rats had access to untreated water. After 8 weeks, a subgroup of PTU treated rats received T3 (10ug/kg/d) in PTU‐containing water for an additional 2 weeks. RNA was extracted from left ventricular (LV) tissue to measure Jph2, SERCa2, PLN, LTCC and RyR2 mRNA by RT‐qPCR. RNAseq analysis interrogated 15,000 genes. LV myocytes were isolated by collagenase digestion and cultured with and without T3 (10−7 M). Cells were harvested at 24, 48, and 72 hrs for RT‐qPCR analysis of Jph2, SERCa2, and Bridging Integrator‐1 (Bin1). Isolated myocytes from all study groups were immunolabeled with RyR2, Cav1.2 and Jph2 for nanoimaging using STORM. Results RT‐PCR analysis showed significant upregulation of Jph2 expression compared to thyroid deficiency (1.404±0.73 vs 0.322±0.17 fold change). RNAseq revealed 567 differentially expressed genes with 377 genes up‐regulated and 190 down‐regulated in T3‐treated hearts compared to TH deficient hearts. Results showed up‐regulation of Jph2 (p<0.001) and SERCa2 by >1.5‐fold (p<0.00001). Cultured EU and PTU cardiomyocytes similarly showed significantly (p<0.05) increased Jph2 expression in T3 treatment vs non‐treated cells at 48 and 72 hr time points. STORM imaging showed increased clustering of LTCC (Cav1.2) within the RyR/Cav1.2 clusters of TT‐jSR complexes in the T3‐treated vs TH deficient cardiomyocytes (0.53±0.13 vs 0.36±0.03). Conclusions T3 regulates expression of Jph2 which in turn is a critical regulator of the jSR‐TT organization in cardiomyocytes allowing for optimal calcium transients and contractile function.