Herpud1 modulates hypertrophic signals independently of calmodulin nuclear translocation in rat myocardium-derived H9C2 cells
Herpud1 modulates hypertrophic signals independently of calmodulin nuclear translocation in rat myocardium-derived H9C2 cells
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DOI：
10.1016/j.bbrc.2023.02.038
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发表时间：
2023-02-20
影响因子：
3.1
通讯作者：
Yano，Masafumi
中科院分区：
文献类型：
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作者：
Fujioka，Riko;Yamamoto，Takeshi;Yano，Masafumi
本研究通过稳定表达同型半胱氨酸反应性内质网泛素样结构域成员1（Herpud 1）的大鼠心肌细胞H9 C2细胞，观察其对钙调蛋白（CaM）核转位和胞浆Ca ~（2+）水平的影响，探讨Herpud 1基因在心肌细胞肥大中的作用。然后用血管紧张素II（Ang II）或丹曲林（DAN）处理这些细胞，血管紧张素II（Ang II）刺激心脏肥大反应，丹曲林（DAN）阻断细胞内Ca 2+的释放。为了在eGFP荧光存在下观察细胞内Ca 2+，使用Rohd-3Ca 2+传感染料。为了检测抑制Herpud 1表达的效果，将Herpud 1小干扰RNA（siRNA）转染到H9 C2细胞中。为了检查是否由Ang II诱导的肥大可以通过Herpud 1过表达来抑制，将Herpud 1表达载体引入H9 C2细胞中。使用eGFP荧光观察CaM易位。首先，Ang Ⅱ诱导H9 C2细胞肥大，使细胞内钙调素（CaM）核转位，胞浆内Ca ~（2+）升高，而DAN可抑制Ang Ⅱ诱导的H9 C2细胞肥大。我们还发现，Herpud 1过表达抑制血管紧张素II诱导的细胞肥大，而不阻止钙调素的核转位或胞浆钙升高。此外，Herpud 1敲低诱导肥大，而没有钙调素的核转位，这是不抑制DAN治疗。最后，Herpud 1过表达抑制Ang II诱导的NFATc 4核转位，但不抑制Ang II诱导的CaM核转位或HDAC 4核输出。
In this study, we aimed to analyze the role of the Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (Herpud1) gene in the development of cardiomyocyte hypertrophy in association with Calmodulin (CaM) nuclear translocation and cytosolic Ca2+levels.To observe the mobilization of CaM in cardiomyocytes, we stably expressed eGFP-CaM in rat myocardium-derived H9C2 cells. These cells were then treated with Angiotensin II (Ang II), which stimulates a cardiac hypertrophic response, or dantrolene (DAN), which blocks the release of intracellular Ca2+. To observe intracellular Ca2+in the presence of eGFP fluorescence, a Rohd-3 Ca2+sensing dye was used. To examine the effect of suppressing Herpud1 expression, Herpud1 small interfering RNA (siRNA) were transfected into H9C2 cells. To examine whether hypertrophy induced by Ang II could be suppressed by Herpud1 overexpression, a Herpud1-expressing vector was introduced into H9C2 cells. CaM translocation was observed using eGFP fluorescence. Nuclear translocation of Nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) and nuclear export of Histone deacetylase 4 (HDAC4) were also examined.First, Ang II induced H9C2 hypertrophy with nuclear translocation of CaM and elevation of cytosolic Ca2+, which were inhibited by DAN treatment. We also found that Herpud1 overexpression suppressed Ang II-induced cellular hypertrophy without preventing nuclear translocation of CaM or elevation of cytosolic Ca2+. Additionally, Herpud1 knockdown induced hypertrophy without the nuclear translocation of CaM, which was not inhibited by DAN treatment. Finally, Herpud1 overexpression suppressed Ang II-induced NFATc4 nuclear translocation but did not suppress Ang II-induced CaM nuclear translocation or HDAC4 nuclear export.Ultimately, this study lays the groundwork for elucidating the anti-hypertrophic effects of Herpud1 and the underlying mechanism of pathological hypertrophy.