Abstract：
OBJECTIVE: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs.
RESULTS: Whole-cell voltage-clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance, and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae, and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF.
CONCLUSIONS: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs.
RESULTS: Whole-cell voltage-clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance, and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae, and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF.
CONCLUSIONS: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs.
摘要：
目标：坎图综合征（CS），具有复杂心血管表型的多系统疾病，由ATP敏感性钾（KATP）通道的Kir6.1/SUR2亚基中的GoF变体引起，其特点是全身血管阻力低，以及曲折，扩张的血管,脉搏波速度降低。因此，CS血管功能障碍是多因素的，同时具有肌强直和超弹性成分。为了剖析这种复杂性是否在血管平滑肌细胞（VSMC）内由细胞自主产生，或者作为对病理生理环境的二次反应，我们评估了人类诱导多能干细胞来源的VSMC（hiPSC-VSMC）的电特性和基因表达，从对照和CS患者来源的HiPSC分化，以及在本机鼠标控制和CSVSMC中。
结果：从野生型（WT）和Kir6.1[V65M]（CS）小鼠分离的主动脉和肠系膜动脉VSMC的全细胞电压钳显示电压门控K（Kv）或Ca2电流没有明显差异。Kv和Ca2+电流在从对照分化的验证的hiPSC-VSMC和CS患者来源的hiPSC之间也没有差异。虽然对照hiPSC-VSMC中的吡那地尔敏感的KATP电流与WT小鼠VSMC中的一致，它们在CShiPSC-VSMC中相当大。在电流钳位条件下，CShiPSC-VSMC也是超极化的，与基础钾电导增加一致，并为CS的音调降低和血管阻力降低提供了解释。在分离的CS小鼠主动脉中观察到顺应性增加，并与弹性蛋白mRNA表达增加有关。这与CShiPSC-VSMC中弹性蛋白mRNA的高水平一致，表明CS血管病变的超弹性成分是血管KATPGoF的细胞自主结果。
结论：结果表明，hiPSC-VSMC重申了与初级VSMC相同的主要离子电流的表达，验证使用这些细胞来研究血管疾病。源自CS患者细胞的hiPSC-VSMC的结果表明，CS血管病变的肌强直和超弹性成分都是由VSMC内KATP过度活动驱动的细胞自主现象。
结果：从野生型（WT）和Kir6.1[V65M]（CS）小鼠分离的主动脉和肠系膜动脉VSMC的全细胞电压钳显示电压门控K（Kv）或Ca2电流没有明显差异。Kv和Ca2+电流在从对照分化的验证的hiPSC-VSMC和CS患者来源的hiPSC之间也没有差异。虽然对照hiPSC-VSMC中的吡那地尔敏感的KATP电流与WT小鼠VSMC中的一致，它们在CShiPSC-VSMC中相当大。在电流钳位条件下，CShiPSC-VSMC也是超极化的，与基础钾电导增加一致，并为CS的音调降低和血管阻力降低提供了解释。在分离的CS小鼠主动脉中观察到顺应性增加，并与弹性蛋白mRNA表达增加有关。这与CShiPSC-VSMC中弹性蛋白mRNA的高水平一致，表明CS血管病变的超弹性成分是血管KATPGoF的细胞自主结果。
结论：结果表明，hiPSC-VSMC重申了与初级VSMC相同的主要离子电流的表达，验证使用这些细胞来研究血管疾病。源自CS患者细胞的hiPSC-VSMC的结果表明，CS血管病变的肌强直和超弹性成分都是由VSMC内KATP过度活动驱动的细胞自主现象。
