The canonical Wnt/β-catenin signaling pathway upregulates carbonic anhydrase 2 via transcription factor 7-like 2 to promote cardiomyopathy in type 2 diabetic mice

Diabetic cardiomyopathy (DCM) is a major complication of diabetes mellitus. Excessive activation of the canonical Wnt/β-catenin pathway contributes to the development of cardiomyopathy in type 1 diabetes mellitus (T1DM). Transcription factor 7-like 2 (TCF7L2) is the main β-catenin partner of the TCF family in adult human hearts. In this study, we investigated the role of Wnt/β-catenin in the development of cardiomyopathy in type 2 diabetes mellitus (T2DM) by using streptozotocin (STZ)/high-fat diet (HFD)-induced diabetic mice and high glucose-stimulated neonatal rat cardiomyocytes (NRCMs) as in-vivo and in-vitro models of T2DM, respectively. Compared with the control mice, the T2DM mice exhibited increased myocardial β-catenin and TCF7L2 expression that was concentrated in the nucleus. Treatment of diabetic mice with the β-catenin/TCF7L2 bipartite inhibitor iCRT14 prevented myocardial remodeling and improved cardiac dysfunction. iCRT14 also prevented high glucose-induced hypertrophy in NRCMs, while the β-catenin stabilizer SKL2001 worsened hypertrophy. Immunoprecipitation experiments confirmed the formation of the β-catenin/TCF7L2 bipartite in the control and T2DM mouse cardiomyocytes. Moreover, carbonic anhydrase 2 (CA2) was upregulated in T2DM cardiomyocytes in vitro and in vivo. TCF7L2 overexpression upregulated CA2, while iCRT14 treatment or TCF7L2 knockdown downregulated CA2. Chromatin immunoprecipitation (ChIP) experiments revealed an increased interaction between β-catenin/TCF7L2 and the transcription initiation region of CA2 in the heart tissue of T2DM mice. CA2 knockdown ameliorated NRCM hypertrophy induced by high glucose and SKL2001. A luciferase reporter assay confirmed that CA2 is directly regulated by the β-catenin/TCF7L2 bipartite. Collectively, these results indicate that the canonical Wnt/β-catenin pathway upregulates CA2 via TCF7L2 to promote cardiomyopathy in T2DM. This research sheds new light on the pathogenesis of DCM and presents new potential therapeutic targets for this disease.糖尿病心肌病（Diabetic cardiomyopathy, DCM）是糖尿病（diabetes mellitus）的主要并发症。经典Wnt/β-连环蛋白通路（canonical Wnt/β-catenin pathway）的过度激活参与了1型糖尿病（type 1 diabetes mellitus, T1DM）患者心肌病的发生发展。转录因子7类似物2（Transcription factor 7-like 2, TCF7L2）是成人心脏中TCF家族的主要β-连环蛋白结合伴侣。本研究以链脲佐菌素（streptozotocin, STZ）/高脂饮食（high-fat diet, HFD）诱导的糖尿病小鼠作为2型糖尿病（type 2 diabetes mellitus, T2DM）的体内模型，以高糖刺激的新生大鼠心肌细胞（neonatal rat cardiomyocytes, NRCMs）作为体外模型，探究了经典Wnt/β-连环蛋白通路在2型糖尿病心肌病发生发展中的作用。与对照组小鼠相比，2型糖尿病小鼠的心肌组织中β-连环蛋白与TCF7L2的表达水平升高，且二者主要富集于细胞核内。给予糖尿病小鼠β-连环蛋白/TCF7L2二元抑制剂iCRT14处理，可阻止心肌重构并改善心功能障碍。iCRT14同样可抑制高糖诱导的新生大鼠心肌细胞肥大，而β-连环蛋白稳定剂SKL2001则会加重该肥大表型。免疫共沉淀实验证实，对照组与2型糖尿病小鼠的心肌细胞中均存在β-连环蛋白/TCF7L2二元复合物的形成。此外，碳酸酐酶2（carbonic anhydrase 2, CA2）在体外与体内的2型糖尿病心肌细胞中均呈现上调表达。TCF7L2过表达可上调CA2的表达，而iCRT14处理或TCF7L2敲低则会下调CA2的表达。染色质免疫共沉淀（Chromatin immunoprecipitation, ChIP）实验显示，2型糖尿病小鼠心脏组织中，β-连环蛋白/TCF7L2与CA2的转录起始区域的结合水平升高。敲低CA2可改善高糖与SKL2001诱导的新生大鼠心肌细胞肥大。荧光素酶报告基因实验证实，CA2直接受β-连环蛋白/TCF7L2二元复合物的调控。综上，本研究结果表明，经典Wnt/β-连环蛋白通路通过TCF7L2上调CA2的表达，进而促进2型糖尿病心肌病的发生发展。该研究为糖尿病心肌病的发病机制提供了新的见解，并为该疾病提出了全新的潜在治疗靶点。