From the Department of Medicine, Division of Cardiology (A.S., R.H., P.W.B., K.R., J.M.C., H.W., K.I.S., E.M., A.C.S., Y.C., A.E., S.D., P.L., S.M.W., J.C.W.), Institute for Stem Cell Biology and Regenerative Medicine (A.S., R.H., P.W.B., K.R., J.M.C., H.W., K.I.S., E.M., A.C.S., Y.C., A.E., S.D., P.L., S.M.W., J.C.W.), Stanford Cardiovascular Institute (A.S., R.H., P.W.B., K.R., J.M.C., H.W., K.I.S., E.M., A.C.S., Y.C., A.E., S.D., P.L., K.R.-H., S.M.W., J.C.W.), Department of Biology (A.S., R.H., K.R.-H.), Department of Microbiology and Immunology (C.M., J.E.C.), and Department of Radiology, Molecular Imaging Program (J.C.W.), Stanford University School of Medicine, CA.
Circ Res. 2014 Aug 29;115(6):556-66. doi: 10.1161/CIRCRESAHA.115.303810. Epub 2014 Jul 11.
Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection.
This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy.
hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonβ1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonβ1 treatment.
This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
病毒性心肌炎是一种危及生命的疾病，可能导致心力衰竭或心律失常。柯萨奇病毒 B3 株（CVB3）是病毒性心肌炎的主要致病因子，它是一种正链 RNA 肠道病毒。然而，人类心脏组织难以获得足够数量用于研究心脏特异性病毒感染的机制。
本研究旨在探讨人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）是否可用于模拟柯萨奇病毒引起的病毒性心肌炎的发病过程，并筛选抗病毒治疗药物的疗效。
用携带荧光素酶的柯萨奇病毒 B3 株（CVB3-Luc）感染 hiPSC-CMs。采用明场显微镜、免疫荧光和钙成像技术，研究细胞形态和钙处理的变化，以明确病毒感染的 hiPSC-CMs 的特征。采用生物发光成像技术定量检测 hiPSC-CMs 中的病毒增殖。在体外检测干扰素β1、利巴韦林、吡咯烷二硫代氨基甲酸盐和氟西汀等抗病毒化合物对 hiPSC-CMs 中 CVB3-Luc 增殖的抑制作用。这些化合物抑制 CVB3-Luc 增殖的能力与之前研究中报道的药物作用一致。通过 CVB3-Luc 感染的 hiPSC-CMs 的基因表达谱进行机制分析表明，干扰素β1 处理后可激活病毒 RNA 和蛋白清除途径。
本研究表明 hiPSC-CMs 表达柯萨奇病毒和腺病毒受体，易受柯萨奇病毒感染，可用于预测抗病毒药物的疗效。我们的结果表明，hiPSC-CM/CVB3-Luc 测定法是一种灵敏的平台，可高通量筛选新型抗病毒治疗药物的有效性。
