Salhi Hussam E, Hassel Nathan C, Siddiqui Jalal K, Brundage Elizabeth A, Ziolo Mark T, Janssen Paul M L, Davis Jonathan P, Biesiadecki Brandon J
Department of Physiology and Cell Biology and Davis Heart and Lung Research Institute, Ohio State University Columbus, OH, USA.
Front Physiol. 2016 Dec 15;7:567. doi: 10.3389/fphys.2016.00567. eCollection 2016.
Troponin I (TnI) is a major regulator of cardiac muscle contraction and relaxation. During physiological and pathological stress, TnI is differentially phosphorylated at multiple residues through different signaling pathways to match cardiac function to demand. The combination of these TnI phosphorylations can exhibit an expected or unexpected functional integration, whereby the function of two phosphorylations are different than that predicted from the combined function of each individual phosphorylation alone. We have shown that TnI Ser-23/24 and Ser-150 phosphorylation exhibit functional integration and are simultaneously increased in response to cardiac stress. In the current study, we investigated the functional integration of TnI Ser-23/24 and Ser-150 to alter cardiac contraction. We hypothesized that Ser-23/24 and Ser-150 phosphorylation each utilize distinct molecular mechanisms to alter the TnI binding affinity within the thin filament. Mathematical modeling predicts that Ser-23/24 and Ser-150 phosphorylation affect different TnI affinities within the thin filament to distinctly alter the Ca-binding properties of troponin. Protein binding experiments validate this assertion by demonstrating pseudo-phosphorylated Ser-150 decreases the affinity of isolated TnI for actin, whereas Ser-23/24 pseudo-phosphorylation is not different from unphosphorylated. Thus, our data supports that TnI Ser-23/24 affects TnI-TnC binding, while Ser-150 phosphorylation alters TnI-actin binding. By measuring force development in troponin-exchanged skinned myocytes, we demonstrate that the Ca sensitivity of force is directly related to the amount of phosphate present on TnI. Furthermore, we demonstrate that Ser-150 pseudo-phosphorylation blunts Ser-23/24-mediated decreased Ca-sensitive force development whether on the same or different TnI molecule. Therefore, TnI phosphorylations can integrate across troponins along the myofilament. These data demonstrate that TnI Ser-23/24 and Ser-150 phosphorylation regulates muscle contraction in part by modulating different TnI interactions in the thin filament and it is the combination of these differential mechanisms that provides understanding of their functional integration.
肌钙蛋白I（TnI）是心肌收缩和舒张的主要调节因子。在生理和病理应激期间，TnI通过不同的信号通路在多个残基处发生差异磷酸化，以使心脏功能与需求相匹配。这些TnI磷酸化的组合可以表现出预期或意外的功能整合，即两种磷酸化的功能不同于单独每个磷酸化的组合功能所预测的功能。我们已经表明，TnI丝氨酸-23/24和丝氨酸-150磷酸化表现出功能整合，并且在心脏应激反应中同时增加。在本研究中，我们研究了TnI丝氨酸-23/24和丝氨酸-150的功能整合以改变心脏收缩。我们假设丝氨酸-23/24和丝氨酸-150磷酸化各自利用不同的分子机制来改变细肌丝内TnI的结合亲和力。数学模型预测，丝氨酸-23/24和丝氨酸-150磷酸化影响细肌丝内不同的TnI亲和力，从而明显改变肌钙蛋白的钙结合特性。蛋白质结合实验通过证明假磷酸化丝氨酸-150降低分离的TnI对肌动蛋白的亲和力来验证这一断言，而丝氨酸-23/24假磷酸化与未磷酸化没有差异。因此，我们的数据支持TnI丝氨酸-23/24影响TnI-TnC结合，而丝氨酸-150磷酸化改变TnI-肌动蛋白结合。通过测量肌钙蛋白交换的去皮心肌细胞中的力发展，我们证明力的钙敏感性与TnI上存在的磷酸盐量直接相关。此外，我们证明，无论在相同还是不同的TnI分子上，丝氨酸-150假磷酸化都会减弱丝氨酸-23/24介导的钙敏力发展的降低。因此，TnI磷酸化可以沿着肌丝在肌钙蛋白之间整合。这些数据表明，TnI丝氨酸-２３/２４和丝氨酸-１５０磷酸化部分地通过调节细肌丝中不同的TnI相互作用来调节肌肉收缩，正是这些不同机制的组合提供了对它们功能整合的理解。
