Inhibition of CREB-CBP Signaling Improves Fibroblast Plasticity for Direct Cardiac Reprogramming.
Inhibition of CREB-CBP Signaling Improves Fibroblast Plasticity for Direct Cardiac Reprogramming.
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作者：
Bektik E;Sun Y;Dennis AT;Sakon P;Yang D;Deschênes I;Fu JD
将成纤维细胞直接重新编程为诱导心肌细胞（iCM）是一种很有前景的方法，但在心脏再生中仍然是一个挑战。我们的努力重点是通过了解基本机制来提高效率。一个主要挑战是培养的成纤维细胞的可塑性因批次而异，机制未知。在这里，我们注意到，即使在原代细胞培养物中，部分体外培养的成纤维细胞也已被激活分化为肌成纤维细胞，以 αSMA 的表达为标志。增加 cAMP 水平的毛喉素和 TGFβ 抑制剂 SB431542 都可以有效抑制培养的成纤维细胞的肌成纤维细胞分化。然而，SB431542 有所改善，但毛喉素阻断了感染 Gata4、Mef2c 和 Tbx5 (GMT) 逆转录病毒的成纤维细胞的 iCM 重编程。此外，cAMP下游信号通路的抑制剂PKA或CREB-CBP显着提高了重编程的效率。一致地，抑制 CREB-CBP 的另一个上游调节因子 p38/MAPK 也提高了重编程效率。然后，我们研究了在原代培养的成纤维细胞中抑制这些信号通路是否可以提高其重编程的可塑性，并发现用 CREB-CBP 抑制剂预处理培养的成纤维细胞可显着提高要重编程的成纤维细胞的细胞可塑性，产生的 iCM 比未处理的对照细胞多约 2 倍。总之，抑制 CREB-CBP 信号传导可改善成纤维细胞的可塑性，从而实现直接心脏重编程。
Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a promising approach but remains a challenge in heart regeneration. Efforts have focused on improving the efficiency by understanding fundamental mechanisms. One major challenge is that the plasticity of cultured fibroblast varies batch to batch with unknown mechanisms. Here, we noticed a portion of in vitro cultured fibroblasts have been activated to differentiate into myofibroblasts, marked by the expression of αSMA, even in primary cell cultures. Both forskolin, which increases cAMP levels, and TGFβ inhibitor SB431542 can efficiently suppress myofibroblast differentiation of cultured fibroblasts. However, SB431542 improved but forskolin blocked iCM reprogramming of fibroblasts that were infected with retroviruses of Gata4, Mef2c, and Tbx5 (GMT). Moreover, inhibitors of cAMP downstream signaling pathways, PKA or CREB-CBP, significantly improved the efficiency of reprogramming. Consistently, inhibition of p38/MAPK, another upstream regulator of CREB-CBP, also improved reprogramming efficiency. We then investigated if inhibition of these signaling pathways in primary cultured fibroblasts could improve their plasticity for reprogramming and found that preconditioning of cultured fibroblasts with CREB-CBP inhibitor significantly improved the cellular plasticity of fibroblasts to be reprogrammed, yielding ~2-fold more iCMs than untreated control cells. In conclusion, suppression of CREB-CBP signaling improves fibroblast plasticity for direct cardiac reprogramming.
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影响因子：
3
作者：
Baum J;Duffy HS
通讯作者：
Duffy HS
DOI：
10.1007/978-1-0716-1084-8_3
发表时间：
2021
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Bektik E;Fu JD
通讯作者：
Fu JD
影响因子：
3.7
作者：
Bektik E;Dennis A;Prasanna P;Madabhushi A;Fu JD
通讯作者：
Fu JD
影响因子：
3.8
作者：
Ge, Y. C.;Li, J. N.;Sun, K.
通讯作者：
Sun, K.
影响因子：
5.5
作者：
Kockskämper, J;Erlenkamp, S;Glitsch, HG
通讯作者：
Glitsch, HG