Rnd3 Ameliorates Diabetic Cardiac Microvascular Injury via Facilitating Trim40-mediated Rock1 Ubiquitination

Diabetic microvascular dysfunction is evidenced by disrupted endothelial cell junctions and increased microvascular permeability. However, effective strategies against these injuries remain scarce. In this study, the type 2 diabetes mouse model was established by high-fat diet combined with streptozotocin injection in Rnd3 endothelial-specific transgenic and knockout mice. Echocardiography was employed to evaluate cardiac function. Microvascular corrosion casts, lanthanum nitrate perfusion, trans-endothelial electrical resistance, FITC-dextran permeability assay and laser speckle contrast imaging were performed to evaluate the integrity of endothelial cell junctions and microvascular function. RNA sequencing, mass spectrometry, co-immunoprecipitation, immunofluorescence and molecular docking were used to explore the downstream regulators of Rnd3. Evidence from gain/loss-of-function studies denoted a protective role for Rnd3 against microvascular dysfunction in diabetic heart. Endothelial-specific deletion of Rnd3 significantly exacerbated coronary microvascular barrier dysfunction under diabetic conditions, while Rnd3 overexpression effectively prevented these effects. Furthermore, Rnd3 overexpression also attenuated cardiac dysfunction in diabetic mice, as indicated by increased LVEF, LVFS, and E/A ratio. Rnd3 overexpression inhibited CMECs apoptosis and increased CMECs migration in response to HG-PA challenge. Rnd3 overexpression inhibited Rock1 activity and MLC phosphorylation in CMECs treated with HG-PA stimulus. Mechanically, Rnd3 recruited and interacted with the E3 ubiquitin ligase Trim40 which further facilitated the degradation of Rock1, thus inhibiting endothelial barrier hyperpermeability in HG-PA-stimulated CMECs. However, the cardioprotective effects of Rnd3 were largely abrogated by Trim40 deficiency in diabetic conditions. Collectively, Rnd3 alleviates microvascular hyperpermeability, maintains endothelial barrier integrity, and mitigates cardiac dysfunction by regulating the Rock1/MLC signaling pathway in the state of DCM.