Interdisciplinary Stem Cell Institute, Department of Pediatrics, Leonard M. Miller School of Medicine, University of Miami, FL (X.L., J.L., E.C.M., C.L.P., K.H., F.R., H.T., M.S.K.).
Departments of Ophthalmology and Medicine, Stanford Cardiovascular Institute, Stanford University, Palo Alto, CA (J.L., Y.L., Q.Y., H.T., M.S.K.).
Circulation. 2020 Sep 8;142(10):948-962. doi: 10.1161/CIRCULATIONAHA.119.044893. Epub 2020 Jul 2.
The Ca/calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the β isoform of the calcineurin catalytic A-subunit (CaNAβ) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. It is unclear how the pleiotropic second messenger Ca drives excitation-contraction coupling while not stimulating hypertrophy by calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectivity in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease.
Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Förster resonance energy transfer reporters were used to assay Ca and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus-mediated gene delivery in the mouse were used to study calcineurin signaling after transverse aortic constriction in vivo.
CIP4 (Cdc42-interacting protein 4)/TRIP10 (thyroid hormone receptor interactor 10) was identified as a new polyproline domain-dependent scaffold for CaNAβ2 by yeast 2-hybrid screen. Cardiac myocyte-specific gene deletion in mice attenuated pressure overload-induced pathological cardiac remodeling and heart failure. Blockade of CaNAβ polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes; disruption of anchoring in vivo using an adeno-associated virus gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell Förster resonance energy transfer biosensor imaging of cultured myocytes revealed that Ca levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca levels and calcineurin activity detected by nonlocalized Förster resonance energy transfer sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca and calcineurin signal transduction.
These results support a structural model for Ca and CaNAβ compartmentation in cells based on an isoform-specific mechanism for calcineurin protein-protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAβ in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAβ polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAβ-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further preclinical investigation.
钙/钙调蛋白依赖性磷酸酶钙调神经磷酸酶是疾病中心肌细胞肥大的关键调节剂。一个尚未解释的悖论是，尽管在相同的细胞中表达钙调神经磷酸酶 A 亚基（CaNAβ）的α 同工型，但β 同工型的钙调神经磷酸酶是诱导病理性心肌肥大所必需的。尚不清楚多效第二信使 Ca 如何在正常心脏中不通过钙调神经磷酸酶刺激肥大而驱动兴奋-收缩偶联。阐明钙调神经磷酸酶信号传导中赋予这种选择性的机制应该揭示针对疾病中磷酸酶的新策略。
研究了原代成年大鼠心室肌细胞的形态和细胞内信号转导。使用新的Förster 共振能量转移报告器来检测活细胞中的 Ca 和钙调神经磷酸酶活性。在体内通过横主动脉缩窄研究了小鼠中的条件基因缺失和腺相关病毒介导的基因传递。
通过酵母 2 杂交筛选鉴定 CIP4（CDC42 相互作用蛋白 4）/TRIP10（甲状腺激素受体相互作用蛋白 10）为 CaNAβ2 的新多脯氨酸结构域依赖性支架。在小鼠中，心肌细胞特异性基因缺失可减弱压力超负荷诱导的病理性心脏重塑和心力衰竭。使用竞争肽抑制培养的心肌细胞中浓缩性肥大的 CaNAβ 多脯氨酸依赖性锚定阻断；使用腺相关病毒基因治疗载体在体内破坏锚定可抑制压力超负荷后的心肌肥大并改善收缩功能。培养的心肌细胞的活细胞 Förster 共振能量转移生物传感器成像显示，神经激素刺激会增加与 CIP4 隔室相关的 Ca 水平和钙调神经磷酸酶活性，但起搏作用最小。相反，通过非局部化 Förster 共振能量转移传感器检测到的 Ca 水平和钙调神经磷酸酶活性是由起搏引起的，而由神经激素刺激引起的则最小，这为 Ca 和钙调神经磷酸酶信号转导的细胞内隔室化提供了功能证据。
这些结果支持基于钙调神经磷酸酶蛋白-蛋白相互作用和定位的同工型特异性机制的细胞内 Ca 和 CaNAβ 隔室化的结构模型。该机制解释了 CaNAβ 在肥大中的特定作用及其在病理应激条件下的选择性激活。破坏 CaNAβ 多脯氨酸依赖性锚定构成了针对负责心血管疾病病理性心脏重塑的 CaNAβ 特异性信号的合理治疗靶向策略，值得进一步进行临床前研究。
