Umar Sadiq, Zhu Wuqiang, Souza-Neto Fernando, Bender Ingrid, Wu Steven C, Healy Chastity L, O'Connell Timothy D, van Berlo Jop H
Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Oral Biology, University of Illinois Chicago, Chicago, IL 60612, USA.
Cells. 2025 May 1;14(9):664. doi: 10.3390/cells14090664.
RBFOX1 is an RNA-binding protein that regulates alternative splicing and RNA processing in the neurons, skeletal muscle, and heart. We intended to define the role of RBFOX1 in regulating calcium homeostasis to maintain normal cardiac function. We generated cardiomyocyte-specific gene-deletion mice (cKO). The cardiomyocyte-specific deletion of RBFOX1 was confirmed by Western blotting and immunohistochemistry. The cKO mice showed mild hypertrophy and depressed cardiac function under homeostatic conditions, which did not deteriorate with age. Pressure overload by trans-aortic constriction (TAC) caused exaggerated cardiac hypertrophy and accelerated heart failure in cKO compared with wild-type mice. We performed Western blotting to assess the expression of important Ca-handling proteins, which showed alterations in the phosphorylation of PLN and CAMKII and decreased expression of SERCA2. We measured the Ca dynamics and noted significantly delayed Ca reuptake into the sarcoplasmic reticulum. Importantly, the decrease in SERCA2 expression was not due to reduced mRNA expression or altered splicing. To assess the possibility of the post-transcriptional regulation of SERCA2 expression by RBFOX1, we performed RNA immunoprecipitation (RIP), which showed the binding of RBFOX1 protein to mRNA, which was confirmed in luciferase assays with the 3'-untranslated region fused to luciferase. Finally, we performed a puromycin incorporation experiment, which showed that RBFOX1 enhances SERCA2 protein translation. Our results show that RBFOX1 plays a crucial role in regulating the expression of Ca-handling genes to maintain normal cardiac function. We show an important post-transcriptional role of RBFOX1 in regulating SERCA2 expression.
RBFOX1是一种RNA结合蛋白，可调节神经元、骨骼肌和心脏中的可变剪接和RNA加工。我们旨在确定RBFOX1在调节钙稳态以维持正常心脏功能中的作用。我们构建了心肌细胞特异性基因敲除小鼠（cKO）。通过蛋白质免疫印迹法和免疫组织化学法证实了RBFOX1在心肌细胞中的特异性缺失。cKO小鼠在稳态条件下表现出轻度肥大和心脏功能降低，且不会随年龄恶化。与野生型小鼠相比，经主动脉缩窄（TAC）造成的压力过载在cKO小鼠中导致了过度的心脏肥大并加速了心力衰竭。我们进行了蛋白质免疫印迹法以评估重要钙处理蛋白的表达，结果显示受磷蛋白（PLN）和钙/钙调蛋白依赖性蛋白激酶II（CAMKII）的磷酸化发生改变，且肌浆网钙ATP酶2（SERCA2）的表达降低。我们测量了钙动力学，并注意到钙重新摄取到肌浆网中的过程明显延迟。重要的是，SERCA2表达的降低并非由于mRNA表达减少或剪接改变所致。为了评估RBFOX1对SERCA2表达进行转录后调控的可能性，我们进行了RNA免疫沉淀（RIP）实验，结果显示RBFOX1蛋白与mRNA结合，这在荧光素酶实验中通过将3'非翻译区与荧光素酶融合得到了证实。最后，我们进行了嘌呤霉素掺入实验，结果显示RBFOX1增强了SERCA2蛋白的翻译。我们的结果表明，RBFOX1在调节钙处理基因的表达以维持正常心脏功能中起关键作用。我们展示了RBFOX1在调节SERCA2表达中重要的转录后作用。
