Abstract：
Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3\'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission.
We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4.
We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms.
These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.
We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4.
We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms.
These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.
摘要：
背景：在发育中的心脏中逐渐形成横向小管（t-小管），关键实现心肌细胞Ca2+稳态的成熟。膜弯曲和支架蛋白BIN1（两栖蛋白-2）参与了这一过程。然而,目前尚不清楚所报道的各种BIN1亚型涉及哪些，以及BIN1功能是否受其推定的结合伴侣MTM1（肌管蛋白）调节，磷酸肌醇3'-磷酸酶，和DNM2(dynamin-2)，一种被认为介导膜裂变的GTP酶。
方法：我们研究了BIN1，MTM1和DNM2在发育中的小鼠心肌细胞T管形成中的作用，以及基因修饰的HL-1和人诱导的多能干细胞衍生的心肌细胞。通过共聚焦和Airyscan显微镜对感兴趣的T小管和蛋白质进行成像，通过RT-qPCR和Western印迹检查表达模式。使用Fluo-4记录Ca2+释放。
结果：我们观察到，在出生后的小鼠心脏中，BIN1从早期发育阶段沿Z线定位，与t小管初始萌芽和支架的作用一致。T管增殖和组织与4种检测到的BIN1亚型的逐步和平行增加有关。观察到所有同种型在心肌细胞中诱导管形,但产生具有不同几何形状的t-小管。BIN1诱导的管状包含L型Ca2通道，与caveolin-3和ryanodine受体共定位，并有效触发Ca2+释放。发育过程中BIN1的上调与MTM1表达的增加平行。尽管MTM1和小鼠心脏BIN1亚型之间没有直接结合，缺乏外显子11，高MTM1水平是BIN1诱导的插管所必需的，表明磷酸肌醇稳态的中心作用。相比之下,发育中的心脏显示DNM2水平下降。的确,我们观察到高水平的DNM2对t管形成有抑制作用，尽管这种蛋白质与BIN1沿Z线定位，并结合所有4种同工型。
结论：这些发现表明，BIN1，MTM1和DNM2在控制心肌细胞t-小管生长方面具有平衡和协同作用。
方法：我们研究了BIN1，MTM1和DNM2在发育中的小鼠心肌细胞T管形成中的作用，以及基因修饰的HL-1和人诱导的多能干细胞衍生的心肌细胞。通过共聚焦和Airyscan显微镜对感兴趣的T小管和蛋白质进行成像，通过RT-qPCR和Western印迹检查表达模式。使用Fluo-4记录Ca2+释放。
结果：我们观察到，在出生后的小鼠心脏中，BIN1从早期发育阶段沿Z线定位，与t小管初始萌芽和支架的作用一致。T管增殖和组织与4种检测到的BIN1亚型的逐步和平行增加有关。观察到所有同种型在心肌细胞中诱导管形,但产生具有不同几何形状的t-小管。BIN1诱导的管状包含L型Ca2通道，与caveolin-3和ryanodine受体共定位，并有效触发Ca2+释放。发育过程中BIN1的上调与MTM1表达的增加平行。尽管MTM1和小鼠心脏BIN1亚型之间没有直接结合，缺乏外显子11，高MTM1水平是BIN1诱导的插管所必需的，表明磷酸肌醇稳态的中心作用。相比之下,发育中的心脏显示DNM2水平下降。的确,我们观察到高水平的DNM2对t管形成有抑制作用，尽管这种蛋白质与BIN1沿Z线定位，并结合所有4种同工型。
结论：这些发现表明，BIN1，MTM1和DNM2在控制心肌细胞t-小管生长方面具有平衡和协同作用。
