PKC介导的心肌细胞T管重构的机制研究

Heart failure is a pathologic state that is primarily characterized by a loss of cardiac contractile function. Protein kinase C (PKC) isozymes contribute to the development of heart failure through dysregulation of Ca2+ handling properties and disruption of contractile function in cardiomyocytes. However, the mechanisms by which PKC activation leads to Ca2+ dysfunction are incompletely understood. The applicant’s established work gave a new insight into the mechanisms by showing that pulsed activation of PKC in cardiomyocytes is sufficient to induce severe and long-lasting remodeling of T-tubule system, the highly regular membrane invagination that provides structural basis for effective Ca2+ induced Ca2+ release (CICR) and in turn contraction of cardiomyocytes. In the present project, we will focus on the molecular mechanisms underlying the PKC induced T-tubule remodeling. PACSIN2, a substrate of PKC, contains a F-BAR domain which senses membrane curvature and deforms membrane into tubules. Though expressed in heart, the cardiac specific function of PACSIN2 has not been studied. We speculate that PACSIN2 may contribute to maintenance of T-tubules in cardiomyocytes. The central hypothesis of this project is that PKC mediated phosphorylation of PACSIN2 at serine 313 decreases PACSIN2’s membrane binding and tubulation capacities, resulting in the loss of an important structural support for T-tubules. In the present project, we will establish inducible PACSIN2 silencing mouse and transgenic mouse strains carrying phospho-mimetic or phospho-resistant PACSIN2 mutants, in combination with high resolution confocal microscopy and cardiac physiology assay, to determine the impact of PKC mediated phosphorylation of PACSIN2 on T-tubule remodeling and cardiac function.

心力衰竭过程中，蛋白激酶C(PKC) 家族激酶通过抑制心肌细胞钙离子瞬变而降低心肌收缩力，诱发心力衰竭。然而，PKC影响心肌细胞钙离子调控的机制仍然有待进一步理解。心肌细胞的收缩依赖于称为钙离子诱导的钙离子释放的机制。该机制的有效执行依赖于心肌细胞的特殊结构T管（T-tubule）。申请人发现，暂时性的提升PKC活性就可以导致T管的重构，进而造成长期的钙离子瞬变障碍。我们将进一步研究其中的机制。PACSIN2是PKC的一个底物， 其膜结合能力受PKC调控。PACSIN2可以诱导细胞膜折叠成为管状，在细胞膜内陷（caveolae）的形成，和内吞中起作用，但在心肌中尚作用不明。鉴于T管是高曲率内陷膜结构， PACSIN2很可能识别并维持T管的结构。本课题的假说是：PKC磷酸化PACSIN2，引起PACSIN2脱离细胞膜，导致T管失去一个重要的结构支持而发生破坏。本课题将验证该假说。

蛋白激酶C(PKC)在心力衰竭中发挥重要功能，该作用机制是通过调控钙离子实现的，钙离子释放依赖于心肌细胞的特殊结构T管。蛋白激酶C如何具体调控T管的重构是研究的一个重点。通过本项目的研究，我们发现PKC在小鼠压力超负荷模型中表达先升高后降低，抑制PKC后减弱心衰程度以及T管损伤程度；短暂激活PKC能长期影响T管的完整性和钙离子的释放。PKC影响T管重构进而导致心肌肥厚的机制之一是通过磷酸化PACSIN2来实现的。PKC活性瞬时升高相伴随的现象是心脏PACSIN2的磷酸化水平升高。PKC抑制剂阻止了PACSIN2的磷酸化。血管紧张素2降低PACSIN2的磷酸化水平，且使PACSIN2从细胞膜上往细胞质中转移。此外，我们还发现PKC活性瞬时升高伴随有actin细胞骨架重排现象。通过药物持续抑制或促进actin细胞骨架的形成都可以减少PKC活化造成的T管损伤，而瞬时抑制或促进actin细胞骨架的形成可以造成显著的T管损伤。我们进一步发现，抑制张力激活通道（Stretch-activated channel， SAC），可以有效抑制细胞骨架过性变构，以及PKC瞬时激活造成的T小管损伤。本项目目前已发表标注本基金号的SCI文章3篇，接收1篇。