The Role And Mechanism Of BK Channel In Vascular Smooth Muscle Cells Differentiation Induced By Mechanical Strain

Cardiovascular diseases including hypertension and atherosclerosis result in vascular remodeling during progression of pathology, with phenotypic transformation and cell dysfunction. Mechanical factors play important role in vascular remodeling. Vascular smooth muscle cells(VSMCs) are main cell constituents in vascular media. VSMCs in mature vessels are highly differentiated cells whose principal functions are contraction and regulation of blood vessel tone-diameter under physiological cyclic mechanical strain. However, VSMCs are distinguished from other cells by their phenotypic transformation that could be changed by pathological increased strain. There are strong evidences that the differentiation of VSMCs, defined by phenotypic transformation, is induced by hypertension with increased vascular tone and pathological strain. Calcium is an important signal molecular which could be activated by many kinds of stimulation. Strain-induced VSMC dysfunction may be via calcium signaling. Then VSMC differentiation and vascular remodeling may be also regulated by ionic channels which are associated with calcium.Big conductance calcium and voltage-activated potassium(BK) channels are predominantly expressed in VSMCs and play important roles in modulating vascular tone to regulate blood flow, as stress axis–regulated exons(STREX) of BK channel α-subunit are necessary for mechano-sensitivity and diversity function. Contractile function of VSMCs is increased with intracellular calcium in hypertension, while the BK channel is activated to decrease intracellular calcium and vascular tone. Hence, intracellular calcium may be regulated by activation of BK channel in response to pathological strain, which result in VSMC dysfunction during hypertension. Taken together, the BK channel is very important to regulate vascular remodeling. However, roles and mechanisms of BK channels in pathological strain-induced VSMC differentiation remain to be elucidated, which may contribute to understand the pathogenesis of hypertension.In the present study, roles of the BK channel and mechano-sensitive alternative splicing of the BK channel in VSMC differentiation modulated by mechanical strain were demonstrated. VSMCs and HEK cells transfected with plasmids encoding whole BK channel(HEK-Slo) or the STREX-deleted BK channel(HEK-STREX delete) were subjected to 5% and 15% strain in vitro to mimic physiological and pathological mechanical situations, respectively. Effects of different strains on the expression and activity of BK channel, as well as on the differentiation markers, i.e., α-actin, calponin and SM22, and intracellular calcium levels, were also examined. Furthermore, a specific inducer of endoplasmic reticulum(ER) stress, xbp1-targeted siRNA transfection and BK channel overexpression were used to study the underlying mechanism of alternative splicing in response to the pathological strain.The results indicated that the expressions of VSMC differentiation markers and BK α-subunit were significantly decreased in 15%-strain group in comparison with 5%-strain group. However, the mRNA levels of STREX in the 15%-strain were significantly higher than those in the 5%-strain. Compared with HEK-STREX delete cells, the open probability(Po) of the BK channel in HEK-Slo cells were significantly upregulated with increased calcium. In the presence of same calcium, BK channel activation was significantly increased with voltage in HEK-Slo cells. Under same calcium and voltage condition, the activity of BK channel in 15%-strain was significantly increased. The frequency of calcium oscillation was also significantly increased in the 15%-strain compared with that of the 5%-strain. Protein expression of the specific ER stress marker, xbp1, was significantly increased following the application of 15%-strain compared with that of the 5%-strain. The ER stress repressed the expression of BK channel α-subunit and VSMC differentiation markers, but increased xbp1 and STREX alternative splicing. While the expression of BK α-subunit and VSMC differentiation markers were significantly increased by xbp1 siRNA transfection, the xbp1 and STREX alternative splicing was significantly decreased following xbp1 siRNA transfection. Expression levels of the VSMC differentiation markers, α-actin, calponin and SM22 were significantly higher in the BK channel-overexpressed group than that in the control group. Under calcium free DMEM condition, the expression of the BK α-subunit and VSMC differentiation markers were significantly decreased by the ER stress. Under calcium free DMEM condition, 15%-strain also significantly decreased the BK α-subunit and VSMC differentiation markers in comparison with the 5%-strain.Our results suggested that the pathological strain decreased the expression of the BK channel α-subunit but increased the activity via ER stress-induced alternative splicing of STREX, which subsequently modulated intracellular calcium and VSMC dedifferentiation. The mechanical response of BK channel and its effect on VSMC differentiation may be a potential mechanism of vascular remodeling in hypertension.