Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Japan.
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Japan.
Biochem Biophys Res Commun. 2018 May 23;499(4):954-959. doi: 10.1016/j.bbrc.2018.04.026. Epub 2018 Apr 11.
Excessive increase of cytosolic Ca through the activation of L-type Ca channels (LTCCs) via β adrenergic receptor induces apoptosis of cardiomyocytes. Canstatin, a cleaved fragment of collagen type IV α2 chain, is abundantly expressed in normal heart tissue. We previously reported that canstatin inhibits β adrenergic receptor-stimulated apoptosis in cardiomyoblasts. Here, we tested the hypothesis that canstatin regulates LTCCs activity in ventricular cardiomyocytes. Collagen type IV α2 chain (COL4A2) small interfering (si) RNA (for canstatin suppression) or control siRNA was injected via jugular vein in Wistar rats. Two days after the injection, electrocardiogram (ECG) was recorded and the left ventricular tissue was isolated using Langendorff apparatus. Immunofluorescence staining was performed to clarify the distribution of canstatin in cardiomyocytes. The knockdown efficiency was confirmed by Western blotting. The L-type Ca channel current (I) of ventricular cardiomyocyte was measured by a whole-cell patch clamp technique. In immunofluorescence staining, colocalization of canstatin and α integrin was observed in the isolated ventricular cardiomyocytes. The I of ventricular cardiomyocyte isolated from COL4A2 siRNA-injected rats was significantly enhanced compared with control siRNA-injected rats. Recombinant canstatin (250 ng/ml) significantly reversed it. ECG analysis showed that QT interval tended to be shortened and amplitude of T wave was significantly increased in the COL4A2 siRNA-injected rats. In summary, we for the first time clarified that suppressing canstatin expression increases the basal I in ventricular cardiomyocytes. It is proposed that canstatin might play a role in the stabilization of cardiac function through the modulation of LTCC activity in cardiomyocytes.
通过β肾上腺素能受体激活 L 型钙通道 (LTCCs) 导致细胞浆内 Ca2+ 过度增加，从而诱导心肌细胞凋亡。Canstatin 是胶原类型 IV α2 链的裂解片段，在正常心脏组织中大量表达。我们之前报道过，Canstatin 可抑制心肌细胞中的β肾上腺素能受体刺激的凋亡。在这里，我们测试了 Canstatin 是否调节心室肌细胞中 LTCCs 活性的假说。通过颈静脉向 Wistar 大鼠注射胶原类型 IV α2 链 (COL4A2) 小干扰 (si)RNA（用于抑制 Canstatin）或对照 siRNA。注射后两天，记录心电图 (ECG) 并使用 Langendorff 装置分离左心室组织。进行免疫荧光染色以阐明 Canstatin 在心肌细胞中的分布。通过 Western 印迹确认敲低效率。通过全细胞膜片钳技术测量心室肌细胞的 L 型钙通道电流 (I)。在免疫荧光染色中，在分离的心室肌细胞中观察到 Canstatin 和 α 整合素的共定位。与对照 siRNA 注射大鼠相比，COL4A2 siRNA 注射大鼠的心室肌细胞 I 明显增强。重组 Canstatin (250ng/ml) 显著逆转了这一现象。ECG 分析表明，COL4A2 siRNA 注射大鼠的 QT 间期趋于缩短，T 波幅度明显增加。总之，我们首次阐明抑制 Canstatin 表达会增加心室肌细胞的基础 I。提出 Canstatin 通过调节心肌细胞中的 LTCC 活性在稳定心脏功能方面可能发挥作用。
