Data_Sheet_1_Phosphatidylinositol-4,5-Bisphosphate Binding to Amphiphysin-II Modulates T-Tubule Remodeling: Implications for Heart Failure.docx

BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.

BIN1（肌球蛋白-II）是一种参与T-管（TT）形成和磷脂酰肌醇-4,5-二磷酸（PIP2）定位于肌节膜的结构蛋白。本研究的目的是确定PIP2介导的BIN1靶向肌节膜在心力衰竭（HF）发展过程中是否受损，以及是否导致TT重塑。免疫组化显示，在正常大鼠和人类心室肌细胞中的TTs中，BIN1、Cav1.2、PIP2和磷脂酶-Cβ1（PLCβ1）共定位。与年龄匹配的对照组相比，在心力衰竭进展过程中自发性高血压大鼠的PIP2水平降低。对包括最长（心脏BIN1 #4）和最短（心脏BIN1 #1）两种同型以及人类骨骼BIN1在内的两种原生小鼠心脏特异性BIN1同型进行PIP Strip试验，发现所有同型均与PIP2结合。此外，过表达所有三种BIN1同型导致HL-1细胞形成管状结构。在两个螺旋之间的短环段中的一个三赖氨酸基序发生突变，并被负电荷取代，从而消除了管状结构，这表明PIP2可能除已知的一致性结合位点外，还可能发生相互作用。在大鼠心室肌细胞中进行的药理学PIP2耗竭导致TT丢失，并伴随钙离子释放的改变，这些改变在心力衰竭期间典型地出现在肌细胞中，包括细胞长度方向的释放变异性增加，以及治疗细胞中上升时间、峰值时间和衰减时间的减慢。这些结果表明，PIP2的耗竭可能导致TT破坏，并表明PIP2与心脏BIN1的相互作用对于TT的维持和功能是必需的。