Diminished Rbfox1 increases vascular constriction by dynamically regulating alternative splicing of CaV1.2 calcium channel in hypertension

Abstract

Calcium influx from depolarized CaV1.2 calcium channels triggers the contraction of vascular smooth muscle cells (VSMCs), which is important for maintaining vascular myogenic tone and blood pressure. The function of CaV1.2 channel can be subtly modulated by alternative splicing (AS), and its aberrant splicing involves in the pathogenesis of multiple cardiovascular diseases. The RNA binding protein Rbfox1 is reported to regulate the AS events of CaV1.2 channel in the neuronal development, but its potential roles in vascular CaV1.2 channels and vasoconstriction remain undefined. Here, we detect Rbfox1 is expressed in rat vascular smooth muscles. Moreover, the protein level of Rbfox1 is dramatically decreased in the hypertensive small arteries from spontaneously hypertensive rats in comparison to normotensive ones from Wistar-Kyoto rats. In VSMCs, Rbfox1 could dynamically regulate the AS of CaV1.2 exons 9* and 33. By whole-cell patch clamp, we identify knockdown of Rbfox1 induces the hyperpolarization of CaV1.2 current-voltage relationship curve in VSMCs. Furthermore, siRNA-mediated knockdown of Rbfox1 increases the K+-induced constriction of rat mesenteric arteries. In summary, our results indicate Rbfox1 modulates vascular constriction by dynamically regulating CaV1.2 alternative exons 9* and 33. Therefore, our work elucidates the underlying mechanisms for CaV1.2 channels regulation and provides a potential therapeutic target for hypertension.

Dysregulated Rbfox2 produces aberrant splicing of CaV1.2 calcium channel in diabetes-induced cardiac hypertrophy

“…Ca V 1.2 alternative exon 9* is known to be regulated by splicing factors RBM20 [ 23 ] and Rbfox proteins [ 16 , 25 ]. However, the expression of RBM20 in HFD/STZ-treated hearts remained unchanged (Figure S2 ), implying that RBM20 may not involve in the AS regulation in diabetic heart.…”

“…Mechanistically, RBM20 bound to introns surrounding exon 9*, promoted the inclusion of exon 9* in Ca V 1.2 channel [ 23 ]. Whereas, Rbfox proteins bound to UGCAUG elements of Cacna1c pre-mRNAs to repress Ca V 1.2 alternative exon 9* in neural development [ 24 ] and vascular smooth muscle [ 16 , 25 ]. Previously, we discovered that Rbfox2 regulates the expression of Ca V 1.2 alternative exon 9* in vascular smooth muscles by taking a key role in the pathogenesis of hypertension [ 16 ].…”

RBPMS promotes contractile smooth muscle splicing and alters phenotypic behaviour of human embryonic stem cell derived vascular smooth muscle cells

“…However, in smooth muscle rich tissues such as the aorta and bladder, where RBFOX2 is predominantly expressed (GTEX), the role of the RBFOX family is not well understood. We only have limited knowledge of the splicing activity of RBFOX2 in vasculature from its role in the endothelium in mediating the response to low blood flow [38] and its regulation of the splicing of the Calcium channel CaV1.2 in VSMCs implicated in the pathology of hypertension alongside RBFOX1 [39, 67].…”

Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia

“…The Myograph system (620M, DMT, Denmark) was used to evaluate vasoconstriction by measuring the isometric tension [33]. Briefly, second-order mesenteric arteries (MAs), were isolated in K + -free Hanks' solution (containing in mmol/L: 1.26 CaCl 2 , 0.41 MgSO 4 -7H 2 O, 137.93 NaCl, 0.34 Na 2 HPO 4 , 0.49 MgCl 2 -6H 2 O, 4.17 NaHCO 3 , 5.56 d-Glucose).…”

“…Through bioinformatic analysis, it has been discovered that CACNA1C gene contains multiple UGC AUG elements, which can be bound by Rbfox1/2 to regulate its AS events during neuronal development [32]. Previously, we found that Rbfox1/2 dynamically regulates Ca V 1.2 exons 9* and 33 in VSMCs, playing important roles in vasoconstriction of hypertensive arteries [28,33]. However, the roles of Rbfox proteins in modulating the key function of vascular Ca V 1.2 channels and arterial constriction during diabetic hyperglycemia remain unidentified.…”