Cyclosporine A Treatment Inhibits Abcc6-Dependent Cardiac Necrosis and Calcification following Coxsackievirus B3 Infection in Mice

Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, PAbcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.

柯萨奇病毒B3型（Coxsackievirus type B3, CVB3）是一种嗜心肌性肠道病毒。该病毒感染可引发心肌细胞坏死与心肌炎症，受损伤的心肌组织会被纤维化或钙化组织替代，进而导致心脏功能永久性改变。CVB3暴露个体的发病程度由宿主遗传背景、病毒毒力与环境因素共同决定。本研究旨在筛选可调控CVB3感染后心肌病理进程的基因。

与129X1亚系小鼠相比，感染CVB3的129S1小鼠出现了更严重的心肌病理损伤，但两组的病毒载量并无差异。连锁分析在7号染色体上鉴定出一个主效位点（LOD值：8.307），并确定ABCC6为该位点的功能基因。基因芯片表达谱分析显示，线粒体相关基因的表达调控存在基因型依赖性差异。电子显微镜观察发现，感染CVB3的Abcc6敲除（Abcc6-/-）小鼠的线粒体基质中存在大量羟基磷灰石样物质沉积，而野生型同窝小鼠则无此现象。

环孢素A（Cyclosporine A, CsA）可通过抑制亲环蛋白D（Cyclophilin D, CypD）来阻断线粒体通透性转换孔的开放。对Abcc6-/-小鼠给予CsA治疗后，其心肌坏死与钙化程度均降低了一半以上。此外，CsA对同时缺失CypD和Abcc6的双敲除小鼠的CVB3诱导表型无显著影响。

综上，本研究证实Abcc6基因突变会使小鼠在CVB3感染后更易出现心脏钙化；同时我们证明，在Abcc6缺陷小鼠中，CypD参与调控心肌坏死与钙化过程，抑制CypD可发挥心脏保护作用。