Calhex231 Alleviates High Glucose-Induced Myocardial Fibrosis via Inhibiting Itch-Ubiquitin Proteasome Pathway in Vitro

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, and features myocardial fibrosis as its main pathological feature. Calcium sensing receptor (CaSR) is a G protein-coupled receptor, which involves in myocardial fibrosis by regulation of calcium homeostasis. Calhex₂₃₁, the CaSR inhibitor, is not clear whether it regulates myocardial fibrosis in DCM. In the present study, type 1 diabetic (T1D) rats and primary neonatal rat cardiac fibroblasts were used to observe the role of Calhex₂₃₁. In vivo experiments showed that in the T1D group, contractile dysfunction and the deposition of collagen I and III were obvious after 12 weeks. In vitro experiments, we found that high glucose (HG) could increase the expression of CaSR, α-smooth muscle actin (α-SMA), Transforming Growth Factor-β₁ (TGF-β₁) collagen I/III, matrix metalloproteinase-2 (MMP-2), MMP9, along with cardiac fibroblast migration and proliferation. We further demonstrated that CaSR activation increased intracellular Ca²⁺ concentration and upregulated the expression of Itch (atrophin-1 interacting protein 4), which resulted in increasing the ubiquitination levels of Smad7 and upregulating the expression of p-Smad2, p-Smad3. However, treatment with Calhex₂₃₁ clearly inhibited the above-mentioned changes. Collectively these results suggest that Calhex₂₃₁ could inhibit Itch-ubiquitin Proteasome and TGF-β₁/Smads pathways, and then depress the proliferation of cardiac fibroblasts, along with the reduction deposition of collagen, alleviate glucose-induced myocardial fibrosis. Our findings indicate an important new mechanism for myocardial fibrosis, and suggest Calhex₂₃₁ would be a new therapeutic agent for the treatment of DCM.