Benzoni P, Nava L, Giannetti F, Guerini G, Gualdoni A, Bazzini C, Milanesi R, Bucchi A, Baruscotti M, Barbuti A
The Cell Physiology MiLAb; Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
The Cell Physiology MiLAb; Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
J Mol Cell Cardiol. 2021 Aug;157:104-112. doi: 10.1016/j.yjmcc.2021.05.001. Epub 2021 May 6.
miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166 SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating I currents; miR1OE SAN-like cells instead have only fast-activating I with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and I alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.
miR-1是心脏中含量最丰富的微小RNA（miRNA），可调节多种转录因子和离子通道的表达。影响心率的情况，如耐力训练和心脏疾病，会伴随miR-1的上调或下调。在此，我们利用小鼠胚胎干细胞（mESC）研究了miR-1过表达在窦房结（SAN）细胞发育和功能中的作用。我们生成了过表达miR-1和绿色荧光蛋白（EGFP）的mESC（miR1OE）或仅过表达EGFP的mESC（EM）。使用CD166标记从分化的mESC中筛选出类SAN细胞。对早期mES来源的心脏祖细胞、类SAN细胞以及过表达miR-1的新生大鼠心室跳动心肌细胞（NRVC）进行了基因表达和电生理分析。与EM细胞相比，miR1OE细胞显著增加了CD166 SAN前体细胞的比例（23%对12%）以及参与SAN发育的转录因子TBX5和TBX18的水平。与EM细胞相比，miR1OE类SAN细胞心动过缓（1.3对2 Hz）。与天然SAN细胞的数据一致，EM类SAN心肌细胞显示出表达慢激活或快激活I电流的两类细胞群体；而miR1OE类SAN细胞只有快激活I电流，其电导显著降低。蛋白质免疫印迹和免疫荧光分析显示，与EM CD166+前体细胞相比，miR-1OE细胞中的HCN4信号减少。这些数据共同表明miR-1对慢激活HCN4亚基有特异性下调作用。重要的是，心率和I电流的改变与miR-1的发育效应无关，在瞬时过表达miR-1的NRVC中情况相似。总之，我们证明了miR-1的双重作用，在发育过程中它控制窦房结前体的正常发育，而在成熟的类SAN细胞中它调节HCN4起搏通道的翻译，从而调节心跳速率。
