CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
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DOI：
10.1161/hypertensionaha.120.14772
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发表时间：
2020-08
期刊：
影响因子：
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通讯作者：
Gupte SA
中科院分区：
文献类型：
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作者：
Kitagawa A;Kizub I;Jacob C;Michael K;D'Alessandro A;Reisz JA;Grzybowski M;Geurts AM;Rocic P;Gupte R;Miano JM;Gupte SA
流行病学研究表明，地中海地区具有功能丧失、非同义单核苷酸多态性（SNP；S188F）、葡萄糖-6-磷酸脱氢酶（G6pd）的个体不易患血管疾病。然而，这种关联尚未得到实验证明。在这里，我们着手确定地中海突变是否能预防血管疾病，并发现潜在的保护机制。我们使用 CRISPR-Cas9 基因组编辑生成了具有地中海 SNP (G6PDS188F) 的大鼠模型。在突变纯合子大鼠中，G6PD 活性（而非表达）降低至野生型 (WT) 同窝小鼠的 20%。此外，无偏代谢组学分析显示，与 WT 大鼠相比，G6PDS188F 大鼠动脉中的磷酸戊糖途径 (PPP) 和与 PPP 相关的其他辅助代谢途径减少 (P<0.05)。有趣的是，与 WT 大鼠相比，G6PDS188F 突变体因高脂肪饮食和 L-NG-硝基精氨酸甲酯 (L-NAME) 抑制一氧化氮合酶而引起的大动脉僵硬度和高血压较少。静脉注射电压门控 L 型 Ca2+ 通道激动剂（甲基 2,6-二甲基-5-硝基-4-[2-(三氟甲基)苯基]-1,4-二氢吡啶-3-羧酸酯；Bay K8644）会导致 WT 大鼠血压急剧升高，但 G6PDS188F 大鼠的血压却没有升高。最后，我们的结果表明：1）K + 通道蛋白表达增加导致平滑肌静息膜电位降低，2）平滑肌中电压门控 Ca2+ 通道活性降低有助于降低 L-NAME 和 G6PDS188F 突变体与 WT 大鼠相比高脂肪饮食引起的高血压和动脉僵硬度。总之，导致 G6PD（PPP 中的限速酶）中单个氨基酸（S188F）被替换的突变将血管平滑肌的特性赋予了血管平滑肌，可以保护生物体免受与血管疾病相关的危险因素的影响。
Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, non-synonymous single nucleotide polymorphism (SNP; S188F), in glucose-6-phosphate dehydrogenase (G6pd) are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean SNP (G6PDS188F) using CRISPR-Cas9 genome editing. In rats homozygous for the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway (PPP) and other ancillary metabolic pathways connected to the PPP were reduced (P<0.05) in the arteries of G6PDS188F versus WT rats. Intriguingly, G6PDS188F mutants, as compared to WT rats, developed less large arterial stiffness and hypertension evoked by high fat diet and nitric oxide synthase inhibition with L-NG-nitroarginine methyl ester (L-NAME). Intravenous injection of a voltage-gated L-type Ca2+ channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PDS188F rats. Finally, our results suggested that: 1) lower resting membrane potential of smooth muscle caused by increased expression of K+ channel proteins and 2) decreased voltage-gated Ca2+ channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-NAME and high fat diet to G6PDS188F mutants as compared to WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the PPP, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.