Type 2 diabetes disrupts T-tubule and RyR2 organization in male but not in female rat ventricular muscle

Females with diabetes often experience more severe cardiovascular outcomes than males with diabetes. It is unclear whether these sex-specific outcomes are rooted in distinct cellular alterations of key cardiac excitation-contraction coupling (ECC) proteins, specifically transverse tubules (T-tubules), ryanodine receptors (RyR2), and filamentous actin (F-actin). This study investigated myocardial ECC structures in both sexes under type 2 diabetic conditions and measured cardiac functional outcomes at muscle level. Type 2 diabetes was induced in male and female Wistar rats using a high-fat diet and low-dose streptozotocin protocol. Left ventricular trabeculae were isolated and subjected to mechano-calorimetric experiments to quantify heat output and twitch force production. The same trabeculae were imaged using high-resolution stimulated emission depletion (STED) microscopy to assess structural organizations of the T-tubule, RyR2, and F-actin. Diabetic rats of both sexes developed hyperglycemia and glucose intolerance, without evidence of ventricular hypertrophy. Mechanoenergetic indices including activation heat, cross-bridge economy, and twitch-force kinetics were unaffected by diabetes in either sex. F-actin organization was not affected by diabetes. However, compared with their respective controls, diabetic males, but not diabetic females, exhibited structural differences in cardiac ECC proteins: reduced T-tubule area and skeleton length, and enlarged RyR2 clusters and calcium release units (CRUs) with increased RyR2 density. These results show that structural reorganization of cardiac RyR2 proteins occurred in diabetic males and not females, despite absence of ventricular hypertrophy and mechanoenergetic dysfunction in the muscles of these two groups.<b>NEW & NOTEWORTHY</b> This study reveals sex-specific cardiac structural alterations of subcellular excitation-contraction coupling proteins in type 2 diabetic rats. Specifically, diabetic males showed evidence of reduced T-tubule area, enlarged RyR2 cluster, and increased calcium release unit density, but without macroscopic functional impairment. In contrast, diabetic females showed no such cardiac structural alterations, suggesting potential resilience mechanisms. These findings highlight that subcellular structural alterations precede functional decline and may underlie sex differences in diabetic cardiovascular outcomes.