Aberrant splicing of Ca_V1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during...

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The Ca_V1.2 calcium channel is one primary pathway for Ca²⁺ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular Ca_V1.2 functions under hyperglycemic condition remains unknown. Here, Sprague-Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K⁺-induced arterial constriction and Ca_V1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K⁺-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and Ca_V1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, Ca_V1.2 channels exhibit hyperpolarized current-voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of Ca_V1.2 channels in VSMCs and MAs. Notably, GS increases K⁺-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates Ca_V1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.