Department of Human Anatomy, Histology and Embryology, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China.
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
Theranostics. 2020 May 15;10(14):6182-6200. doi: 10.7150/thno.46553. eCollection 2020.
: Smooth muscle-motility disorders are mainly characterized by impaired contractility and functional intestinal obstruction. Some of these cases are caused by genetic mutations of smooth muscle genes ACTA2, ACTG2, MYH11, MYLK and LMOD1. Still the etiology is complex and multifactorial and the underlying pathology is poorly understood. Integrin interaction protein Kindlin-2 is widely expressed in striated and smooth muscle cells (SMC). However, the function of Kindlin-2 in the smooth muscle remains elusive. : We generated two mouse models using different cre promoter transgenic mice, Kindlin-2 SM22α-cre+ (cKO mice) and Kindlin-2; MYH-cre+ (iKO mice). Embryos and adult tissues were prepared for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay. We investigated ultrastructure changes of mouse smooth muscle using transmission electron microscopy (TEM) and measured smooth muscle contractile force in mounting aortic and intestinal rings using the multiwire myograph system (DMT 620M). In addition, cell traction force microscopy (CTFM) was applied to observe the functional change of primary SMC after Kindlin-2 depletion by RNAi. : Depletion of Kindlin-2 encoding gene Fermt2 in embryonic smooth muscles leads to apoptosis, downregulates the key components of SMC, impairs smooth muscle development, and finally causes embryonic death at E14.5. Tamoxifen-induced Kindlin-2-specific knockout in adult mouse smooth muscle showed decreased blood pressure, intestinal hypoperistalsis, and eventually died of intestinal obstruction. Kindlin-2 depletion also leads to downregulated Myh11, α-SMA, and CNN, shortened myofilament, broken myofibrils, and impaired contractility of the smooth muscles in iKO mice. Mechanistically, loss of Kindlin-2 decreases Ca2 influx in primary vascular smooth muscle cells (PVSMC) by downregulating the expression of calcium-binding protein S100A14 and STIM1. : We demonstrated that Kindlin-2 is essential for maintaining the normal structure and function of smooth muscles. Loss of Kindlin-2 impairs smooth muscle formation during embryonic development by inducing apoptosis and jeopardizes the contraction of adult smooth muscle by blocking Ca influx that leads to intestinal obstruction. Mice with Kindlin-2 depletion in adult smooth muscle could be a potent animal model of intestinal obstruction for disease research, drug treatment and prognosis.
平滑肌运动障碍主要表现为收缩功能障碍和功能性肠梗阻。其中一些病例是由平滑肌基因 ACTA2、ACTA2、MYH11、MYLK 和 LMOD1 的基因突变引起的。然而，病因复杂且多因素，潜在的病理机制仍不清楚。整合素相互作用蛋白 Kindlin-2 在横纹肌和平滑肌细胞（SMC）中广泛表达。然而，Kindlin-2 在平滑肌中的功能仍然难以捉摸。
我们使用不同的 cre 启动子转基因小鼠生成了两种小鼠模型，即 Kindlin-2 SM22α-cre+（cKO 小鼠）和 Kindlin-2；MYH-cre+（iKO 小鼠）。胚胎和成年组织用于苏木精和伊红（H&E）染色、免疫组织化学（IHC）和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）凋亡检测。我们使用透射电子显微镜（TEM）研究了小鼠平滑肌的超微结构变化，并使用多丝肌描记系统（DMT 620M）测量了主动脉和肠环的平滑肌收缩力。此外，应用细胞牵引力显微镜（CTFM）观察 RNAi 耗尽 Kindlin-2 后原代 SMC 的功能变化。
在胚胎平滑肌中耗尽编码 Kindlin-2 的基因 Fermt2 导致细胞凋亡、下调 SMC 的关键成分、损害平滑肌发育，并最终导致 E14.5 时胚胎死亡。在成年小鼠平滑肌中用他莫昔芬诱导的 Kindlin-2 特异性敲除导致血压下降、肠道蠕动减少，最终死于肠梗阻。Kindlin-2 缺失还导致 iKO 小鼠的 Myh11、α-SMA 和 CNN 下调、肌丝缩短、肌原纤维断裂以及平滑肌收缩力受损。在机制上，Kindlin-2 的缺失通过下调钙结合蛋白 S100A14 和 STIM1 减少了血管平滑肌细胞（PVSMC）中的 Ca2+内流。
我们证明了 Kindlin-2 对于维持平滑肌的正常结构和功能是必不可少的。在胚胎发育过程中，Kindlin-2 的缺失通过诱导细胞凋亡而损害平滑肌的形成，并通过阻断 Ca2+内流来危及成年平滑肌的收缩，从而导致肠梗阻。成年平滑肌中 Kindlin-2 缺失的小鼠可能成为一种有效的肠梗阻动物模型，用于疾病研究、药物治疗和预后。
