Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral fellowship Introduction Phosphoinositide 3-kinase C2α (PI3KC2α) is a ubiquitously expressed class II PI3K isoform, which has been previously shown to be involved in the control of vesicular trafficking. However, its role in the myocardium has not been investigated. The primary aim of this study is to elucidate the mechanism by which mechanism PI3KC2α controls cardiac pathophysiology. Methods Cardiomyocyte-specific PI3KC2α knockout (PI3KC2α KO) animals were generated by crossing mice expressing a tamoxifen-inducible Cre recombinase under the control of the αMHC promoter with PI3KC2αflox/flox mice. Zebrafish embryos were injected with a PI3KC2α (PI3KC2α morphants) or a control morpholino (control morphants) at 1-cell stage. HEK293 with stable overexpression of GFP-tagged β2-AR were generated and transfected with either scramble or PI3KC2α siRNAs. Results Our findings indicate that PI3KC2α KO mice display a reduced cardiac contractility compared to wildtype animals. Similarly, PI3KC2α morphants zebrafish exhibit lower heart rate and fractional shortening compared to controls, both at baseline and after isoproterenol (ISO) stimulation. A similar unresponsiveness to ISO was found in vivo in PI3KC2α KO mice where chronic treatment with the β-adrenergic receptor (β-AR) agonist failed to induce the classical β-AR-mediated remodeling, characterized by an increase of the left ventricular mass and of cardiomyocyte area. These findings suggest that PI3KC2α plays a critical role in the regulation of β-AR signaling. In agreement, cAMP levels failed to increase in response to ISO treatment in PI3KC2α morphants. Furthermore, silencing of PI3KC2α in HEK293-GFP-β2-AR cells resulted in an increased GFP-β2-AR internalization compared to control cells, both at baseline and after ISO stimulation. Interestingly, preliminary findings indicate that overexpression of constitutively active Rab11Q70L induced a redistribution of GFP-β2-AR at the plasma membrane in PI3KC2α-silenced cells. Conclusion Overall, we identify a key role for PI3KC2α in the control of cardiac contractility through the regulation of β2-AR trafficking to the plasma membrane through a Rab11-dependent mechanism.

中文翻译：

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磷酸肌醇 3-激酶 C2alpha 通过调节 β2-肾上腺素能受体再循环来控制心脏收缩力


资金致谢 资金来源类型： 公共赠款 – 仅限国家预算。主要资金来源： 博士生奖学金 简介 磷酸肌醇 3-激酶 C2α （PI3KC2α） 是一种普遍表达的 II 类 PI3K 亚型，先前已被证明参与控制囊泡运输。然而，它在心肌中的作用尚未得到研究。本研究的主要目的是阐明 PI3KC2α 控制心脏病理生理学的机制。方法 通过在 αMHC 启动子的控制下，将表达他莫昔芬诱导型 Cre 重组酶的小鼠与 PI3KC2αflox/flox 小鼠杂交，产生心肌细胞特异性 PI3KC2α 敲除 （PI3KC2α KO） 动物。斑马鱼胚胎在 1 细胞期注射 PI3KC2α （PI3KC2α 形态） 或对照吗啉 （对照形态）。生成稳定过表达 GFP 标记的 β2-AR 的 HEK293，并用 scramble 或 PI3KC2α siRNA 转染。结果 我们的研究结果表明，与野生型动物相比，PI3KC2α KO 小鼠的心脏收缩力降低。同样，与对照组相比，PI3KC2α 形态斑马鱼在基线和异丙肾上腺素 （ISO） 刺激后表现出较低的心率和分数缩短。在 PI3KC2α KO 小鼠体内发现对 ISO 的类似无反应，其中用 β-肾上腺素能受体 （β-AR） 激动剂进行慢性治疗未能诱导经典的 β-AR 介导的重塑，其特征是左心室质量和心肌细胞面积增加。这些发现表明 PI3KC2α 在 β-AR 信号传导的调节中起关键作用。一致，PI3KC2α 形态体中 cAMP 水平响应 ISO 处理后未能增加。 此外，与对照细胞相比，HEK293-GFP-β2-AR 细胞中 PI3KC2α 的沉默导致 GFP-β2-AR 内化增加，无论是在基线还是 ISO 刺激后。有趣的是，初步结果表明，组成型活性 Rab11Q70L 的过表达诱导 GFP-β2-AR 在 PI3KC2α 沉默细胞质膜上的重新分布。结论 总体而言，我们确定了 PI3KC2α 通过调节 β2-AR 转运到质膜中的关键作用 Rab11 依赖性机制。